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reperfusion in rats
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A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats Mahmoud I. Youssef, Amr A.A. Mahmoud *, Rasha H. Abdelghany Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2015 Accepted 16 April 2015 Available online xxx
Acute kidney injury (AKI) is associated with high mortality resulting from extra-renal organ damage, particularly the heart. The present study aimed to investigate the protective effect of sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, against renal and remote cardiac damage induced by ischemia/ reperfusion (IR), a leading cause of AKI. In this attempt, we compared the effects of sitagliptin to furosemide, a loop diuretic. Furosemide is commonly used clinically in AKI however, there is a lack of evidence regarding its beneficial effects in AKI. In addition, the combined administration of both drugs was also investigated. Ischemia was induced in anesthetized male Wistar rats by occluding both renal pedicles for 30 min followed by reperfusion for 24 h. Sitagliptin (5 mg kg1), furosemide (245 mg kg1) or their combination were administered orally at 5 h post-IR and 2 h before euthanasia. Administration of sitagliptin or furosemide ameliorated renal and cardiac deterioration induced by renal IR. This was manifested as significant reduction of serum creatinine, urea, cystatin c, creatine kinase-MB, cardiac troponin-I and lactate dehydrogenase (P < 0.05). Drug treatment significantly inhibited IR-induced elevation of TNF-a, NF-kB and caspase-3 (P < 0.05) in kidney and heart tissue. In addition, they significantly suppressed malondialdehyde, NO and iNOS content, whereas they increased glutathione and antioxidative enzymes activity (P < 0.05) in both tissues. Interestingly, a superior protection was observed with the combination compared to the individual drugs. We assume that this combination represents a promising regimen for managing AKI, particularly with the poor clinical outcome obtained with furosemide alone. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Sitagliptin Furosemide Myocardial injury Ischemia/reperfusion Inflammation
1. Introduction Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality rates. It has been estimated that death in 20% of hospitalized patients and up to 50% of patients admitted to the intensive care unit is attributed to AKI [1]. In addition, AKI plays a pivotal role in the development and progression of chronic kidney disease and end-stage renal disease [2]. Moreover, it is associated with high treatment costs regarding the need for transplantation and renal replacement therapy [3].
* Corresponding author. Tel.: +20 111 099 2406; fax: +20 552 303 266. E-mail addresses: [email protected] (M.I. Youssef), [email protected] (Amr A.A. Mahmoud), [email protected] (R.H. Abdelghany).
Renal ischemia/reperfusion (IR) injury is a leading cause of AKI in native and transplanted kidneys [4,5]. It results from the reduction of renal blood flow that eventually leads to impairment of oxygen delivery to renal cells [6]. Different causes have been suggested for the reduction in renal blood flow including shock, sepsis, hepatorenal syndrome, decreased effective intravascular volume and use of some medications [7]. The mechanism of IR-induced AKI is complex involving interactions between renal tubular injury, vascular injury, and inflammation. On one hand, hypoxic damage to the renal tubular cells stimulates the release of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-a), interleukin (IL)-6, and other chemotactic cytokines which aid in the recruitment of immune cells [8]. On the other hand, ischemia-induced endothelial injury plays an important role in the adhesion of leukocytes via release of adhesion molecules such as selectins and intercellular adhesion molecule-1 (ICAM-1) [9]. The interaction between endothelium
http://dx.doi.org/10.1016/j.bcp.2015.04.010 0006-2952/ß 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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Handling at Zagazig University (ECAHZU), at the Faculty of Pharmacy, Zagazig University, Egypt in accordance with the recommendations of the Weatherall report. Every effort was done to minimize the number of animals used and their suffering during experiments.
and leukocytes results in activation of leukocytes, obstruction of capillaries, production of cytokines, and aggravation of inflammation [10]. Other possible mechanisms have been suggested including increased oxidative stress [11], impairment of nitric oxide (NO) pathway [12] and apoptosis [13]. Despite being associated with high mortality, AKI is not usually the direct cause of death [14]. However, AKI-induced systemic inflammatory response and progression of damage to remote organs have been implicated in the high patient mortality. A causal link has been suggested to exist between AKI and dysfunction of distant extra-renal organs such as the heart [15], the liver [16] and the lung [17]. It has been demonstrated that cardiac failure in AKI patients is one of the leading causes of death [18,19]. Similarly, another study of AKI patients showed that death for cardiac failure was the greatest among other AKI-induced organs failure [20]. It has been suggested that cardiac injury induced by AKI is attributed to increased systemic and cardiac TNF-a, IL-1, and ICAM-1 expression that can lead to neutrophil infiltration and myocyte apoptosis [15]. Even with increased availability and widespread application of renal replacement therapy, outcomes are not improved [21]. Therefore, identifying new therapies that can ameliorate cardiac injury during AKI is critical in limiting the high mortality and improving the outcome. Glucagon-like peptide-1 (GLP-1), which belongs to incretins, can reduce blood glucose through stimulation of insulin secretion from b-cells [22]. Different studies showed that GLP-1 has favorable effects in different experimental models of ischemia. For example, this peptide has been shown to exert cardioprotective effects on IR-injured hearts or cardiomyocytes [23]. In addition, exenatide, an analogue of GLP-1, has the potential to attenuate AKI induced by IR in rats [24]. The biological activity of GLP-1 can be extended via inhibiting dipeptidyl peptidase-4 (DPP4), the enzyme system responsible for its degradation. Indeed, different DPP4 inhibitors such as sitagliptin, saxagliptin and linagliptin are currently used for the treatment of type 2 diabetes based on the reduced cleavage of GLP-1 [25,26]. Previous studies showed that sitagliptin could ameliorate the deleterious effects induced by either renal IR injury [27] or cardiac IR injury [28]. The present study was conducted to investigate the effect of sitagliptin on the remote myocardial damage induced by renal IR injury in rats. In addition, we attempted to identify the underlying mechanism of action. In our attempt, we compared the effects of sitagliptin to furosemide and investigated the effect of their combination as well. Using of furosemide is based on the widespread clinical application of diuretics during AKI [29,30]. To our knowledge, this is the first report discussing not only the local effects of these drugs on renal IR injury, but also its effects on the ensuing distant cardiac complications.
Dose of sitagliptin was chosen based on previous studies [27,31]. Dose of furosemide was chosen to simulate the oral highdose (35 mg kg1 day1) used in the clinical settings for managing AKI [32]. The equivalent rat dose was interpolated from the mentioned human dose using approximate dose conversion factors described by Freireich et al. [33]. The time points for drug administration were chosen based on the results of Williams et al. [34]. In this study, the authors demonstrated that injury might be initiated 4 h following IR procedure. Therefore, they suggested that therapeutic interventions within 6 h following IR procedure would be the most effective in ameliorating injury. In addition, their results revealed that peak renal damage occurred 24 h following IR; therefore, this time point would be helpful in monitoring the protective effects of investigated agents. Depending on these findings, the administration of drugs was started 5 h following IR procedure (i.e., within the first 6 h) in order to ensure that kidney injury has been already established. The second dose was administered 2 h before euthanasia at 24 h following IR procedure, where peak damage occurs.
2. Materials and methods
2.6. Methods
2.1. Experimental animals
2.6.1. Induction of IR injury Rats were fasted overnight but had free access to water. At the day of surgery, animals were anaesthetized with an intraperitoneal injection of thiopental sodium (EIPICO Pharmaceuticals, 10th of Ramadan City, Egypt) at a dose of 120 mg kg1 [35], and then placed on a heating pad to keep the body temperature constant at approximately 37 8C. Left and right kidneys were exposed by flank incisions on both sides, respectively. Bilateral ischemia was induced by occluding both renal pedicles using non-traumatic clamps (Dieffenbach Bulldog Clamps, Harvard Apparatus Ltd., Kent, UK) for 30 min according to the method described by Kelly et al. [36]. Complete ischemia was confirmed by blanching of kidneys. After the indicated ischemic period, clamps were released and reperfusion was started for 24 h. The kidneys were observed for 2–5 min to ensure normal blood reflow, which is indicated by
Adult male Wistar rats (180–250 g) were used in the current study. Animals were obtained from the Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Rats were acclimatized for one week prior to experiments. The animals were housed in stainless steel cages (three rats/cage) and kept at controlled temperature (23 2 8C), humidity (60 10%) and light/dark (12/ 12 h) cycle. Rats were supplied with commercially available normal chow diet and water ad libitum. 2.2. Ethical statement Experimental design and animal handling procedures were approved by the local authorities, Ethical Committee for Animal
2.3. Drugs Sitagliptin (Januvia1) was obtained from Merck, Sharp & Dohme (Cairo, Egypt), while furosemide was supplied from Amoun Pharmaceutical Co. S.A.E. (Obour City, Egypt). All other chemicals were of analytical grade. Drugs were dissolved in distilled water immediately before administration. 2.4. Experimental design Rats were randomly divided into five experimental groups (n = 6 each). Group 1 (sham-operated), group 2 (IR injury only), group 3 (rats received sitagliptin 5 mg kg1 orally at 5 h post-IR and 2 h before euthanasia), group 4 (rats received furosemide 245 mg kg1 orally at 5 h post-IR and 2 h before euthanasia), and group 5 (rats received a combination of sitagliptin 5 mg kg1 orally plus furosemide 245 mg kg1 orally at 5 h post-IR and 2 h before euthanasia). Rats of group 1 (sham-operated) and group 2 (IR injury only) did not receive any drug treatment, but rather vehicle only. 2.5. Rationale of drug dosing
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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the change of kidney color to red again. There is a time lag of about 1–1.5 min between clamping of the right and left kidney. Therefore, the ischemic time for each kidney was recorded separately to ensure that both kidneys receive the same duration of ischemia. Incisions were closed by continuous stitches using vicryl sutures. Animals were kept on the heating pad and monitored until they become fully conscious before being returned to the cages [37]. Sham-operated rats underwent identical surgical procedure with the exception of application of clamps.
2.6.7. Determination of lipid peroxides content in kidney and heart Lipid peroxidation was assessed by measuring the content of malondialdehyde (MDA), a byproduct of lipid peroxidation process. The measurement was performed using a kit supplied by Diamond Diagnostics (Cairo, Egypt), following the manufacturer’s instructions. This assay depends on the reaction of MDA in samples with thiobarbituric acid (TBA) forming a red MDA-TBA adduct that can be quantified colorimetrically at wavelength 532 nm.
2.6.2. Blood sampling and serum preparation At the end of reperfusion period (24 h), blood samples were collected immediately from the retro orbital sinus of rats using heparinized microcapillary tubes. Serum was prepared by centrifugation (Hermle Z230, Gosheim, Germany) of blood tubes at 3000 rpm for 30 min. Serum was stored at 20 8C and thawed just before use for the determination of different biochemical parameters.
2.6.8. Determination of reduced glutathione (GSH) content in kidney and heart Glutathione (GSH) content was determined colorimetrically using a diagnostic kit supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. The assay is based on the reduction of 5,50 dithiobis (2-nitrobenzoic acid) with GSH in the sample to produce a yellow product, whose absorbance can be measured at wavelength 405 nm.
2.6.3. Tissue sampling Rats were euthanized by cervical dislocation under anesthesia with thiopental sodium (120 mg kg1) for tissue specimen collection. Kidney and heart were removed and rinsed thoroughly with saline and divided into two equal parts. The first part was kept in 10% phosphate-buffered formalin at room temperature for histopathological examination, while the other part was immersed immediately in liquid nitrogen and kept at 80 8C. Tissue homogenates were prepared using Polytron PT1200E Disperser (Kinematica AG, Luzern, Switzerland) in ice-cold phosphatebuffered saline (PBS, pH 7.2). Homogenates were centrifuged at 12,000 g for 10 min, and supernatants were used for measurements.
2.6.9. Determination of glutathione-s-transferase (GST) activity in kidney and heart Glutathione-s-transferase (GST) activity was measured using an assay kit supplied by Sigma-Aldrich (St. Louis, MO, USA). In this method, GST catalyzes the conjugation of L-glutathione to 1Chloro-2,4-dinitrobenzene (CDNB) forming GS-DNB conjugate, which absorbs at wavelength 340 nm. The rate of increase in its absorption is directly proportional to the GST activity.
2.6.4. Assessment of kidney function Serum creatinine and serum urea were determined by colorimetric methods using kits supplied by Diamond Diagnostics (Cairo, Egypt), following the manufacturer’s instructions. Absorbance of the final products were read using Jenway Genova spectrophotometer (Bibby Scientific, Staffordshire, UK). Serum cystatin c level was measured using a quantitative sandwich enzyme immunoassay with the aid of Quantikine ELISA kit, specific for mouse and rat, supplied by R&D Systems (Minneapolis, MN, USA). 2.6.5. Determination of biochemical markers of myocardial injury Serum creatine kinase MB (CK-MB) isoenzyme and cardiac troponin I (cTnI) were measured by enzyme-linked immunosorbent assay (ELISA) technique using kits supplied by Shanghai Crystal day Biotech Co. (Shanghai, China) and Kamiya Biomedical Company (Seattle, WA, USA), respectively, following the manufacturer’s instructions. Serum lactate dehydrogenase (LDH) activity was measured by pyruvate kinetic liquid reaction as described by Pesce [38], using a diagnostic kit supplied by Spinreact (Girona, Spain). The method is based on the ability of LDH to catalyze the reduction of pyruvate by NADH forming lactate and NAD+. The rate of decrease in the concentration of NADH, measured spectrophotometrically at wavelength 340 nm, is proportional to the catalytic concentration of LDH in the sample. 2.6.6. Determination of TNF-a and nuclear factor-kappa B (NF-kB) content in kidney and heart Tumor necrosis factor-alpha (TNF-a) and nuclear factor-kappa B (NF-kB) contents were measured by ELISA technique using ELISA kits provided by R&D Systems (Minneapolis, MN, USA) and EIAab (Wuhan, China), respectively, following the manufacturer’s instructions.
2.6.10. Determination of superoxide dismutase (SOD) activity in kidney and heart Superoxide dismutase (SOD) activity was assessed using a kit supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. This assay depends on the generation of superoxide ions from the conversion of xanthine and O2 to uric acid and H2O2 by xanthine oxidase (XO). The superoxide anion then coverts a tetrazolium salt into a formazan dye. Addition of SOD to this reaction reduces superoxide ion levels, thereby lowering the rate of formazan dye formation. The enzyme activity is then measured as the percent inhibition of the rate of formazan dye formation. 2.6.11. Determination of catalase activity and nitric oxide (NO) content in kidney and heart Catalase activity and NO content were measured colorimetrically using kits supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. 2.6.12. Determination of inducible nitric oxide synthase (iNOS) activity and caspase-3 content in kidney and heart Inducible nitric oxide synthase (iNOS) and caspase-3 were measured using ELISA kits supplied by Shanghai Crystal day Biotech Co., Ltd (Shanghai, China) and USCN Life Science Inc. (Wuhan, China), respectively, following the manufacturer’s instructions. 2.6.13. Histopathological examination Specimens of the kidney and the heart from different groups were fixed in 10% phosphate-buffered formalin solution at room temperature. Specimens were dehydrated in graded ethanol (70– 100%), cleared in xylene and embedded in paraffin. Paraffinembedded tissue sections (5 mm thick) were prepared, mounted on slides and kept at room temperature. Thereafter, slides were dewaxed in xylene; hydrated using graded ethanol, and stained by hematoxylin and eosin (HE) dyes. The sections were examined under light microscope and photographed with a digital camera (Canon, Japan).
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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2.7. Statistical analysis All data were expressed as mean standard error of the mean (SEM). Statistical analysis was performed using Graphpad prism software v.5 (GraphPad Software Inc., La Jolla, CA, USA). The statistical significance of differences between groups was tested using one-way analysis of variance (ANOVA) followed by Tukey’s Post-test. A significant difference was assumed for values of P < 0.05.
these parameters with furosemide was significantly higher compared to sitagliptin group. Furthermore, the combined administration of sitagliptin and furosemide, not only significantly reduced urea, creatinine and cystatin c levels compared to IR, sitagliptin alone or furosemide alone groups, but also restored their values to normal levels seen with sham-operated rats. 3.2. Effect on biomarkers of myocardial injury
As depicted in Fig. 1, bilateral renal IR significantly elevated serum urea (59 1.2 vs. 25 0.89 mg/dl, P < 0.001), cystatin c (140 1.7 vs. 30 1.2 pg/ml, P < 0.001) and creatinine (1.3 0.09 vs. 0.6 0.05 mg/dl, P < 0.001) levels compared to sham-operated rats. Administration of either sitagliptin (5 mg kg1) or furosemide (245 mg kg1) to rats subjected to renal IR injury significantly reduced serum urea (23.7% and 42.4%, respectively, P < 0.001), cystatin c (54.2% and 67.1%, respectively, P < 0.001) and creatinine (38.5% and 53.9%, respectively, P < 0.001) levels compared to untreated rats subjected to IR injury only. The reduction observed in
Rats underwent IR injury showed significantly elevated serum CK-MB (16 0.48 vs. 1.9 0.04 ng/ml, P < 0.001), cTnI (11 0.26 vs. 1.2 0.02 ng/ml, P < 0.001) levels and LDH (308 3.3 vs. 129 2 U/ml, P < 0.001) activity compared to sham-operated rats. Treatment of rats subjected to renal IR injury with sitagliptin or furosemide significantly decreased serum CK-MB (57.5% and 76.9%, respectively, P < 0.001), cTnI (62.7% and 78.2%, respectively, P < 0.001) levels and LDH (44.8% and 49.7%, respectively, P < 0.001) activity compared to untreated rats subjected to IR injury only. In addition, furosemide resulted in a significantly higher degree of reduction of CK-MB and cTnI compared to rats received sitagliptin alone (Fig. 2). The concomitant administration of sitagliptin with furosemide successfully brought back the values of these markers close to normal levels of shamoperated rats (P > 0.05).
Fig. 1. Changes in serum urea level (A, plotted on left axis), serum cystatin c level (A, plotted on right axis) and serum creatinine level (B) at 24 h after ischemia/ reperfusion (IR) injury (n = 5–6). Sham, sham-operated rats; IR, ischemia/ reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, ** P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
Fig. 2. Changes in serum creatine kinase-MB isoenzyme (CK-MB) level (A, plotted on left axis), serum cardiac troponin I (cTnI) level (A, plotted on right axis) and serum lactate dehydrogenase (LDH) activity (B) at 24 h after ischemia-reperfusion (IR) injury (n = 6). Sham, sham-operated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
3. Results 3.1. Effect on kidney function
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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3.3. Effect on tissue TNF-a and NF-kB content As shown in Fig. 3, renal IR injury resulted in a significant increase in TNF-a (about 4-fold, P < 0.001) and NF-kB (about 3fold, P < 0.001) content of kidney and heart tissue compared to sham-operated rats. Similar to what described before, administration of sitagliptin or furosemide significantly decreased TNF-a and NF-kB content of kidney and heart tissue compared to untreated rats in IR group. Both sitagliptin and furosemide, when administered together, significantly inhibited the elevation of these parameters that their values in kidney and heart tissue were comparable to normal levels of sham-operated animals. 3.4. Effect on tissue oxidative stress markers and antioxidative enzymes activity Ischemia/reperfusion resulted in significant elevation of kidney and heart content of MDA (P < 0.001), NO (P < 0.001) and activity of iNOS (P < 0.001), whereas it resulted in significant reduction of GSH content (P < 0.001), GST (P < 0.001), SOD (P < 0.001) and catalase (P < 0.001) activity compared to sham-operated rats. Treatment of rats with sitagliptin (5 mg kg1) or furosemide (245 mg kg1) significantly diminished the abnormal alterations in these parameters; however, the effect exerted by furosemide was
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significantly higher compared to sitagliptin. In addition, a combination of both sitagliptin and furosemide normalized the levels of these parameters to values similar to those observed in sham-operated rats (Table 1). 3.5. Effect on tissue caspase-3 activity As depicted in Fig. 4, rats underwent IR injury had significantly higher levels of caspse-3 content in kidney (60 1.4 vs. 7.7 0.37 nmol/g tissue, P < 0.001) and heart (22 0.61 vs. 2.9 0.11 nmol/g tissue, P < 0.001) compared to sham-operated rats. Administration of sitagliptin or furosemide resulted in significant decrease of caspase-3 content in kidney (76.6% and 84.3%, respectively, P < 0.001) and heart (77.7% and 87.2%, respectively, P < 0.001) tissue compared to IR rats. Rats received furosemide showed significantly lower levels of caspase-3 in kidney (P < 0.01) and heart (P < 0.01) tissue compared to rats received sitagliptin. Simultaneous administration of sitagliptin plus furosemide totally and significantly inhibited the increase of capase-3 content in both kidney and heart tissue. The combined administration of sitagliptin plus furosemide resulted in significant reduction of caspase-3 activity in kidney tissue compared to either sitagliptin or furosemide alone (P < 0.05). Furthermore, combined drugs significantly lowered caspase-3 activity in heart tissue compared to sitagliptin alone (P < 0.05). 3.6. Histopathological examination of kidney and heart tissue
Fig. 3. Changes in tissue content of tumor necrosis factor-alpha (TNF-a) (A) and nuclear factor-kappa B (NF-kB) (B) of kidney (plotted on left axis) and heart (plotted on right axis) at 24 h after ischemia/reperfusion (IR) injury (n = 6). Sham, shamoperated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
Sham-operated rats showed normal kidneys with normal glomeruli, proximal convoluted, distal convoluted and collecting tubules (Fig. 5a). Additionally, they showed normal myocardium with striated muscle fibers and centrally located nuclei (Fig. 6a). Occasionally, slight congestion in blood capillaries in the heart and increased eosinophilia of the renal tubular epithelium in the kidneys were observed. On the other hand, rats that underwent IR injury showed diffuse cortical and medullary ischemic necrosis with extensive hemorrhage at the medulla and corticomedullary junction of the kidney (Fig. 5b). Heart specimens revealed focal areas of degeneration and myonecrosis in the cardiac muscle fibers with loss of its striation. Their nuclei were pyknotic or completely absent. The sarcoplasm became contracted and more eosinophilic. Severe congestion, edema and hemorrhage were visualized around the blood vessels. Numerous inflammatory cells, mostly neutrophils, were observed among the affected cardiac muscle fibers (Fig. 6b). Kidney tissues from IR rats treated with sitagliptin (5 mg kg1) showed necrosis in a group of cortical tubules of both proximal and distal convoluted tubules. Such necrosis was represented as pyknosis and karyolysis (Fig. 5c). On the other hand, the heart specimen showed focal areas of degeneration and myonecrosis with pyknosis and karyolysis. Severe congestion of some cardiac blood vessels was also noticed (Fig. 6c). Kidneys from IR rats that received furosemide (245 mg kg1) showed necrosis in few scattered renal tubules (Fig. 5d). The heart was mostly normal with small focal area of degeneration and myonecrosis and no evidence of edema or hemorrhage (Fig. 6d). Administration of a combination of sitagliptin and furosemide resulted in a marked amelioration in the myonecrosis of the heart with few cardiac muscles revealing pyknotic nuclei particularly around blood vessels (Fig. 6e). The endothelium of the coronary blood vessels was intact. The kidneys were normal with individual cell necrosis, represented as pyknosis and karyolysis. Some nuclei of renal epithelium revealed mitosis (Fig. 5e). A scoring of the histopathological findings is summarized in Table 2.
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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Table 1 Changes in oxidative stress markers and antioxidative enzyme activity in kidney and heart at 24 h after ischemia/reperfusion (IR) injury. Sham
IR
IR + SIT
IR + FUR a
IR + SIT/FUR ab
MDA (nmol/g tis.)
K H
7.1 0.42 3.8 0.05
48 1.0* 23 0.66*
13 0.71 5.9 0.27a
8.3 0.61 3.9 0.07ab
6.3 0.33ab 3.1 0.09ab
GSH (mmol/g tis.)
K H
3.5 0.1 1.7 0.06
0.9 0.01* 0.5 0.01*
1.8 0.04a 1.1 0.04a
2.6 0.16ab 1.5 0.01ab
3.8 0.08abc 2.0 0.08abc
GST (mmol/g tis.)
K H
4.4 0.09 2.1 0.07
1.17 0.02* 0.59 0.02*
2.5 0.15a 1.5 0.04a
3.2 0.03ab 1.9 0.01ab
3.9 0.1abc 2.4 0.03abc
SOD (U/g tis.)
K H
10.7 0.28 4.7 0.07
2.2 0.05* 1.3 0.02*
5.9 0.14a 2.7 0.01a
9.1 0.2ab 3.6 0.06ab
10.8 0.11abc 4.7 0.05abc
Catalase (U/g tis.)
K H
11.8 0.23 5 0.17
3.2 0.07* 1.3 0.02*
6.9 0.41a 3.0 0.07a
10.1 0.2ab 4.0 0.21ab
11.9 0.35abc 5.1 0.07abc
NO (mmol/g tis.)
K H
19 0.39 6.15 0.07
75 0.97* 28 0.62*
28 0.41a 10.7 0.16a
21 0.99ab 6.9 0.2ab
17 0.42abc 5.4 0.13abc
iNOS (ng/g tis.)
K H
24 0.30 12 0.21
90 1.0* 33 0.65*
36 0.61a 16 0.21a
28 0.49ab 13 0.38ab
20 0.35abc 9.3 0.14abc
Values are expressed as mean SEM (n = 6) in kidney (K) and heart (H). IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). MDA, malondialdehyde; GSH, reduced glutathione; GST, glutathione-s-transferase; SOD, superoxide dismutase; NO, nitric oxide; iNOS, inducible nitric oxide; g tis., gram tissue. Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, aP < 0.05 vs. IR, bP < 0.05 vs. IR + SIT, cP < 0.05 vs. IR + FUR.
4. Discussion It has become obvious that clinically much of the high patient mortality associated with AKI occurs as a result of systemic inflammatory response and progression to multiple organ failure [39]. Therefore, the present study was designed to evaluate the protective effect of sitagliptin, furosemide or their combination in the settings of acute AKI in rats and the associated remote cardiac damage. We were interested particularly in this target because cardiac dysfunction in AKI patients, among other distant organs failure, has been recognized as a leading cause of death [18,20,40]. In our study, AKI was induced in rats by using the bilateral ischemic model. We used this model because it is very close to pathological conditions observed in human where blood supply of both kidneys is normally reduced [41–43]. Bilateral renal ischemia for 30 min followed by reperfusion resulted in marked deterioration of kidney function at 24 h after IR procedure. This was evident from the significant elevation of
Fig. 4. Changes in tissue content of caspase-3 of kidney (plotted on left axis) and heart (plotted on right axis) at 24 h after ischemia/reperfusion (IR) injury (n = 6). Sham, sham-operated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
serum creatinine (Fig. 1b) and urea (Fig. 1a) levels, which are widely used to assess kidney function, compared to shamoperated animals that underwent the same surgical procedure without IR injury. In addition, IR injury resulted in significant increase of serum cystatin c level. Although an increase of serum creatinine is used clinically to identify AKI [44], however, cystatin c has been shown by different studies to be superior than creatinine, especially to detect minor reduction in glomerular filtration rate (GFR) [45,46]. The elevation of these markers is attributed to the sudden decline in GFR induced by AKI. Histopathological examination revealed that the deterioration of kidney function was associated with renal structural damage manifested by necrosis and extensive hemorrhage at the medulla and corticomedullary junction of the kidney (Fig. 5b). Taken together, these parameters confirm the proper induction of AKI in our experiments. Furthermore, our results show that renal IR injury did not only affect the kidneys, but also resulted in distant myocardial injury as evidenced by the significant elevation of serum CK-MB, cTnI levels and LDH activity (Fig. 2) compared to sham group. These biomarkers have been widely applied for the identification of acute myocardial injury [47]. In addition, histopathological examination showed focal areas of degeneration and myonecrosis in the cardiac muscle fibers with severe congestion, edema and hemorrhage around the blood vessels (Fig. 6b). This finding illustrates that AKI rarely occurs in isolation; however, it causes complications in other distant non-renal organs. The deterioration of kidney function and associated myocardial injury were significantly suppressed by treatment with sitagliptin, furosemide or their combination. In addition, drug treatment reduced the structural damage caused by IR injury in kidney and heart tissue. In this regard, combined administration of both drugs resulted in marked amelioration in the myonecrosis of the heart (Fig. 6e). Similarly, the kidneys appeared normal with minor cell necrosis (Fig. 5e). Different mechanisms of acute renal IR injury have been proposed including the activation of inflammatory response, inflammatory cell infiltration, generation of reactive oxygen species (ROS), and apoptosis [48–50]. Therefore, in order to elucidate the mechanisms by which these drugs reduced IR-induced myocardial injury, we investigated their effects on inflammatory cytokines, oxidative stress and apoptosis in kidney and heart tissue.
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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Fig. 5. Histological examination of kidney tissue at 24 h after ischemia/reperfusion (IR) procedure. Representative light micrograph are depicted from (a) sham-operated rats: showing normal structure with slight eosinophils at the epithelial lining of the proximal convoluted tubules (arrows), (b) rats underwent IR only: showing diffuse cortical and medullary necrosis (arrows) with extensive hemorrhage at the medulla and at the corticomedullary junction (arrowheads), (c) rats underwent IR procedure and received sitagliptin (5 mg kg1): showing necrosis in a group of cortical tubules with pyknosis and karyolysis (arrows), (d) rats underwent IR procedure and received furosemide (245 mg kg1): showing necrosis in few scattered renal tubules (arrows) and contracted glomerular tufts (arrowheads) and (e) rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1): showing individual cell necrosis with pyknosis and karyolysis (arrows) and intact glomerular tufts (arrowhead), with some nuclei of the renal epithelium revealing mitosis. Tissue specimens were stained with HE dyes (scale bar = 50 mM).
Our results showed that renal IR procedure significantly increased TNF-a and NF-kB content in kidney and heart compared to sham rats (Fig. 3), which is consistent with previous studies [15,24]. It is apparent that ischemia-induced damage to the renal
tubular cells stimulates the release TNF-a [8], which consequently activates the canonical pathway of NF-kB leading to its translocation to the nucleus [51]. In turn, NF-kB enhances the expression of other important mediators including cytokines, chemokines, iNOS,
Fig. 6. Histological examination of heart tissue at 24 h after ischemia/reperfusion (IR) procedure. Representative light micrograph are depicted from (a) sham-operated rats: showing normal muscle fibers with slight eosinophils at the sarcoplasm (arrowheads) and slight congestion (arrows), (b) rats underwent IR only: showing diffuse myonecrosis (arrows), congestion (c), edema and hemorrhage (arrowhead), (c) rats underwent IR procedure and received sitagliptin (5 mg kg1): showing myonecrosis (arrows) and severe congestion (arrowhead), (d) rats underwent IR procedure and received furosemide (245 mg kg1): showing focal small areas of myonecrosis (arrows) and (e) rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1): showing normal myocardium with rare myonecrosis (arrows) and intact endothelium of coronary blood vessels (arrowhead). Tissue specimens were stained with HE dyes (scale bar = 50 mM).
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Table 2 Histopathology scoring of kidney and heart tissue at 24 h after ischemia/reperfusion (IR) procedure. Groups
Kidney
Heart
Deg. and Nec.
Cong.
Ed.
Hg.
Deg. and Nec.
Cong.
Ed.
Hg.
Sham IR IR + SIT IR + FUR IR+ SIT/FUR
++++ ++ +
+++ ++ + +
++ ++ ++ +
++ + +
++++ ++ +
+++ ++ + +
+++ ++ +
+++ + +
IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1 ). Deg. and Nec., degeneration and necrosis; Cong., congestion; Ed., edema; Hg., hemorrhage. () nil, (+) mild, () nil to mild, (++) moderate, (+++) severe, (++++) more severe.
and adhesion molecules such as E-selectin and ICAM-1 [52,53]. In addition, the inflammatory response mediated by NF-kB at the vascular endothelium leads to migration and adhesion of leukocytes, which is enhanced by over-expression of adhesion molecules [9,54]. The subsequent activation of leukocytes results in obstruction of capillaries, further production of cytokines, and aggravation of the inflammatory response [10]. Indeed, we detected neutrophils infiltrating kidney and heart after IR procedure at 24 h (Fig. 6b). These inflammatory markers were significantly reduced in rats treated with sitagliptin, furosemide or their combination. The combination of both sitagliptin and furosemide totally inhibited the elevation of theses markers. Our results demonstrated that renal IR injury generated oxidative stress and increased the production of ROS in the kidney and the heart compared to sham rats. This was manifested as increased formation of MDA, which is a widely accepted marker of oxidative stress [55]. Different sources have been suggested for ROS production including XO enzyme, mitochondria, and activation of leukocyte NADPH oxidase [56–58]. In addition, IR rats had significantly high content of NO in kidney and heart tissue compared to sham rats. Although NO has an important role in renal hemodynamic regulation, however, many authors suggested its implication in ischemia-induced renal tubular injury [59–62]. The deleterious effect of NO is suggested to occur via its reaction with superoxide anion forming peroxynitrite, which is a highly toxic oxidant [63]. It has been suggested that the increased production of NO is attributed to IR-induced upregulation of iNOS [62]. We could observe that iNOS was significantly increased in kidney and heart tissue from IR rats compared to sham rats. In addition, our results are in harmony with previous studies, which demonstrated the crucial role of oxidative and nitrosative stress in acute renal ischemia [61,64–67]. Furthermore, our results revealed that IR injury impaired the non-enzymatic and enzymatic anti-oxidant protective mechanisms in kidney and heart as manifested by the significant reduction of GSH, GST, SOD and catalase activity in IR rats compared to sham rats. Under physiological conditions, they can scavenge and detoxify ROS, however, during ischemia supplies of these endogenous scavengers and enzymes are overwhelmed by ROS leading to cellular injury. This hypothesis is supported by the findings of other studies showing reduction of cellular stores of these scavengers during brain [68] and heart [69] ischemia. Increased oxidative stress has detrimental consequences, such as impairment of membrane function, induced by lipid peroxidation, and oxidative DNA damage [70]. The ultimate result is cellular death via necrosis or apoptosis [71]. This explains our findings of increased caspase-3 content (Fig. 4), reflecting increased apoptotic cell death, and necrosis (Table 2) in kidney and heart after IR injury procedure. Administration of sitagliptin, furosemide or their combination significantly reduced oxidative stress induced by renal IR injury and induced the activity of the anti-oxidative enzymes in kidney
and heart. This, in part, had a positive impact on improving the kidney function and reducing associated myocardial injury. This effect can be attributed to the reduction of leukocyte infiltration in heart and kidney tissue as shown in Figs. 5 and 6. In this regard, our results agree with Vaghasiya et al. [27], who reported that sitagliptin reduced oxidative stress in renal tissue after IR via reduction of leukocyte infiltration, as well as decreasing xanthine oxidase (XO) activity. One of the interesting findings of the present study was the observation that furosemide afforded rats greater protection against IR injury and the associated myocardial injury than sitagliptin. Presumably, this can be attributed to particular renal actions of furosemide including increased renal blood flow and subsequent increase in GFR, mediated via inhibition of prostaglandin dehydrogenase and subsequent increase of prostaglandin (PG) E2, a potent renal vasodilator [72,73]. Indeed, furosemide has been shown to increase GFR in healthy volunteers [74,75]. In addition, furosemide reduces oxygen consumption of the renal tubular cells via inhibition of the sodium/potassium chloride co-transporter [76,77]. Increased renal blood flow and amelioration of hypoxic insult, besides its anti-apoptotic effect [78], allows furosemide to further diminish the inflammatory cascade induced by renal ischemia and the myocardial injury that ensues better than sitagliptin. Although, the effects of GLP-1 receptor agonists and sitagliptin on GFR are controversial [79–81], we cannot rule out the possibility that the smaller effect of sitagliptin compared to furosemide might be attributed to the small dose of sitagliptin used in this study. Another interesting finding in our study is the nearly complete blockade of IR-induced damage in kidney and heart produced by combined administration of both sitagliptin and furosemide. The protection exerted by the combination was superior to each drug administered alone. This was manifested as normalization of the measured parameters. It seems that this is a result of additive effects of both drugs by affecting different pathways. Despite being used widely by most clinicians for managing AKI, evidence for beneficial effects, concerning outcome and mortality, of loop diuretics still lacking even with experimental results suggesting a therapeutic potential. Clinically, diuretics have been shown to increase the risk of AKI or lack therapeutic benefit in different settings [82–86]. Considering this discrepancy of diuretic effect in managing AKI, it is urgently required to identify agents with possible protective effect in the clinical settings. Like previous studies, we could show therapeutic potential for sitagliptin. However, our study still offers some points of novelty, unlike other reports. Previous studies investigated the effect of sitagliptin on either renal ischemia [24,27] or cardiac ischemia [28,87, 88] only, however, none described the effect on distant myocardial damage induced by renal IR injury. Our study for the first time, to our knowledge, provides an experimental evidence for beneficial effect of sitagliptin to protect not only against renal IR injury, but also against the associated remote myocardial damage, which is
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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recognized as a leading cause of mortality during AKI. Second, the dose of sitagliptin used in our study is either smaller or used for shorter duration compared to other studies. Vaghasiya et al. [27] used sitagliptin at a dose of 5 mg kg1 day1, orally during 2 weeks starting 11 days before renal IR injury. On the other hand, Chen et al. [24] used sitagliptin at a dose of 600 mg kg1 at 1, 24, and 48 h post-IR. In the present study we used sitagliptin twice post-IR at a dose of only 5 mg kg1. We assume that such small dose would be safe enough to be tried clinically in the settings of AKI, despite the necessity of dose reduction with deteriorated kidney function, where it is advised to reduce the dose of sitagliptin from 100 mg day1 to 25 mg day1 with creatinine clearance less than 30 mL/ min [89]. However, higher doses of sitagliptin still represent other possible alternatives, particularly, in patients with creatinine clearance over 50 mL/min. Third, the remarkable protection observed with the combination of sitagliptin and furosemide suggests it as a beneficial therapeutic regimen in AKI post-IR injury and qualifies it as a potential candidate for clinical trials. This seems promising in improving the total outcome and reducing mortality, particularly with the failure of diuretics alone to offer reasonable therapeutic benefit. However, this needs further clinical investigations to ascertain or rule out the experimental data observed in our study. The study has a few limitations. First, higher doses of sitagliptin have not been tried, which would allow for a better comparison of its effects with furosemide. Second, even though dosing schedule has been selected carefully, different dosing schedules would be helpful for better optimization of the drug treatment. In summary, sitagliptin could significantly suppress IR-induced inflammatory cascade, oxidative stress and apoptosis, thus protecting from kidney damage and associated distant cardiac injury. Superior protection was offered by a combination of sitagliptin and furosemide. Therefore, we assume that this combination represents a promising regimen during AKI, particularly that caused by IR injury. This might be due to the extra protection from myocardial damage occurring during AKI, which is an important cause of death in those patients. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments The authors acknowledge Dr. Mohamed Hamed Mohamed, Professor of Pathology, Faculty of Veterinary Medicine, Zagazig University for his great effort in the hispathological examination. References [1] J.P. Lafrance, D.R. Miller, Acute kidney injury associates with increased long-term mortality, J. Am. Soc. Nephrol.: JASN 21 (2010) 345–352. [2] S.G. Coca, B. Yusuf, M.G. Shlipak, A.X. Garg, C.R. Parikh, Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis, Am. J. Kidney Dis.: Off. J. Natl. Kidney Found. 53 (2009) 961–973. [3] S.M. Bagshaw, Short- and long-term survival after acute kidney injury, Nephrol. Dial. Transpl. 23 (2008) 2126–2128. [4] F. Liano, J. Pascual, C. Gamez, A. Gallego, M.A. Bajo, L.S. Sicilia, et al., Epidemiology of acute renal failure: a prospective, multicenter, community-based study, Kidney Int. 50 (1996) 811–818. [5] C. Rippe, A. Rippe, A. Larsson, D. Asgeirsson, B. Rippe, Nature of glomerular capillary permeability changes following acute renal ischemia-reperfusion injury in rats, Am. J. Physiol.-Renal 291 (2006), F1362-F8. [6] M. Le Dorze, M. Legrand, D. Payen, C. Ince, The role of the microcirculation in acute kidney injury, Curr. Opin. Crit. Care 15 (2009) 503–508. [7] J.V. Bonventre, L. Yang, Cellular pathophysiology of ischemic acute kidney injury, J. Clin. Invest. 121 (2011) 4210–4221. [8] M.J. Burne-Taney, J. Kofler, N. Yokota, M. Weisfeldt, R.J. Traystman, H. Rabb, Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury, Am. J. Physiol. Renal Physiol. 285 (2003) F87–F94.
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A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats Mahmoud I. Youssef, Amr A.A. Mahmoud *, Rasha H. Abdelghany Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Zagazig 44519, Egypt
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 February 2015 Accepted 16 April 2015 Available online xxx
Acute kidney injury (AKI) is associated with high mortality resulting from extra-renal organ damage, particularly the heart. The present study aimed to investigate the protective effect of sitagliptin, a dipeptidyl peptidase-4 (DPP4) inhibitor, against renal and remote cardiac damage induced by ischemia/ reperfusion (IR), a leading cause of AKI. In this attempt, we compared the effects of sitagliptin to furosemide, a loop diuretic. Furosemide is commonly used clinically in AKI however, there is a lack of evidence regarding its beneficial effects in AKI. In addition, the combined administration of both drugs was also investigated. Ischemia was induced in anesthetized male Wistar rats by occluding both renal pedicles for 30 min followed by reperfusion for 24 h. Sitagliptin (5 mg kg1), furosemide (245 mg kg1) or their combination were administered orally at 5 h post-IR and 2 h before euthanasia. Administration of sitagliptin or furosemide ameliorated renal and cardiac deterioration induced by renal IR. This was manifested as significant reduction of serum creatinine, urea, cystatin c, creatine kinase-MB, cardiac troponin-I and lactate dehydrogenase (P < 0.05). Drug treatment significantly inhibited IR-induced elevation of TNF-a, NF-kB and caspase-3 (P < 0.05) in kidney and heart tissue. In addition, they significantly suppressed malondialdehyde, NO and iNOS content, whereas they increased glutathione and antioxidative enzymes activity (P < 0.05) in both tissues. Interestingly, a superior protection was observed with the combination compared to the individual drugs. We assume that this combination represents a promising regimen for managing AKI, particularly with the poor clinical outcome obtained with furosemide alone. ß 2015 Elsevier Inc. All rights reserved.
Keywords: Sitagliptin Furosemide Myocardial injury Ischemia/reperfusion Inflammation
1. Introduction Acute kidney injury (AKI) is a major clinical problem associated with high morbidity and mortality rates. It has been estimated that death in 20% of hospitalized patients and up to 50% of patients admitted to the intensive care unit is attributed to AKI [1]. In addition, AKI plays a pivotal role in the development and progression of chronic kidney disease and end-stage renal disease [2]. Moreover, it is associated with high treatment costs regarding the need for transplantation and renal replacement therapy [3].
* Corresponding author. Tel.: +20 111 099 2406; fax: +20 552 303 266. E-mail addresses: [email protected] (M.I. Youssef), [email protected] (Amr A.A. Mahmoud), [email protected] (R.H. Abdelghany).
Renal ischemia/reperfusion (IR) injury is a leading cause of AKI in native and transplanted kidneys [4,5]. It results from the reduction of renal blood flow that eventually leads to impairment of oxygen delivery to renal cells [6]. Different causes have been suggested for the reduction in renal blood flow including shock, sepsis, hepatorenal syndrome, decreased effective intravascular volume and use of some medications [7]. The mechanism of IR-induced AKI is complex involving interactions between renal tubular injury, vascular injury, and inflammation. On one hand, hypoxic damage to the renal tubular cells stimulates the release of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-a), interleukin (IL)-6, and other chemotactic cytokines which aid in the recruitment of immune cells [8]. On the other hand, ischemia-induced endothelial injury plays an important role in the adhesion of leukocytes via release of adhesion molecules such as selectins and intercellular adhesion molecule-1 (ICAM-1) [9]. The interaction between endothelium
http://dx.doi.org/10.1016/j.bcp.2015.04.010 0006-2952/ß 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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Handling at Zagazig University (ECAHZU), at the Faculty of Pharmacy, Zagazig University, Egypt in accordance with the recommendations of the Weatherall report. Every effort was done to minimize the number of animals used and their suffering during experiments.
and leukocytes results in activation of leukocytes, obstruction of capillaries, production of cytokines, and aggravation of inflammation [10]. Other possible mechanisms have been suggested including increased oxidative stress [11], impairment of nitric oxide (NO) pathway [12] and apoptosis [13]. Despite being associated with high mortality, AKI is not usually the direct cause of death [14]. However, AKI-induced systemic inflammatory response and progression of damage to remote organs have been implicated in the high patient mortality. A causal link has been suggested to exist between AKI and dysfunction of distant extra-renal organs such as the heart [15], the liver [16] and the lung [17]. It has been demonstrated that cardiac failure in AKI patients is one of the leading causes of death [18,19]. Similarly, another study of AKI patients showed that death for cardiac failure was the greatest among other AKI-induced organs failure [20]. It has been suggested that cardiac injury induced by AKI is attributed to increased systemic and cardiac TNF-a, IL-1, and ICAM-1 expression that can lead to neutrophil infiltration and myocyte apoptosis [15]. Even with increased availability and widespread application of renal replacement therapy, outcomes are not improved [21]. Therefore, identifying new therapies that can ameliorate cardiac injury during AKI is critical in limiting the high mortality and improving the outcome. Glucagon-like peptide-1 (GLP-1), which belongs to incretins, can reduce blood glucose through stimulation of insulin secretion from b-cells [22]. Different studies showed that GLP-1 has favorable effects in different experimental models of ischemia. For example, this peptide has been shown to exert cardioprotective effects on IR-injured hearts or cardiomyocytes [23]. In addition, exenatide, an analogue of GLP-1, has the potential to attenuate AKI induced by IR in rats [24]. The biological activity of GLP-1 can be extended via inhibiting dipeptidyl peptidase-4 (DPP4), the enzyme system responsible for its degradation. Indeed, different DPP4 inhibitors such as sitagliptin, saxagliptin and linagliptin are currently used for the treatment of type 2 diabetes based on the reduced cleavage of GLP-1 [25,26]. Previous studies showed that sitagliptin could ameliorate the deleterious effects induced by either renal IR injury [27] or cardiac IR injury [28]. The present study was conducted to investigate the effect of sitagliptin on the remote myocardial damage induced by renal IR injury in rats. In addition, we attempted to identify the underlying mechanism of action. In our attempt, we compared the effects of sitagliptin to furosemide and investigated the effect of their combination as well. Using of furosemide is based on the widespread clinical application of diuretics during AKI [29,30]. To our knowledge, this is the first report discussing not only the local effects of these drugs on renal IR injury, but also its effects on the ensuing distant cardiac complications.
Dose of sitagliptin was chosen based on previous studies [27,31]. Dose of furosemide was chosen to simulate the oral highdose (35 mg kg1 day1) used in the clinical settings for managing AKI [32]. The equivalent rat dose was interpolated from the mentioned human dose using approximate dose conversion factors described by Freireich et al. [33]. The time points for drug administration were chosen based on the results of Williams et al. [34]. In this study, the authors demonstrated that injury might be initiated 4 h following IR procedure. Therefore, they suggested that therapeutic interventions within 6 h following IR procedure would be the most effective in ameliorating injury. In addition, their results revealed that peak renal damage occurred 24 h following IR; therefore, this time point would be helpful in monitoring the protective effects of investigated agents. Depending on these findings, the administration of drugs was started 5 h following IR procedure (i.e., within the first 6 h) in order to ensure that kidney injury has been already established. The second dose was administered 2 h before euthanasia at 24 h following IR procedure, where peak damage occurs.
2. Materials and methods
2.6. Methods
2.1. Experimental animals
2.6.1. Induction of IR injury Rats were fasted overnight but had free access to water. At the day of surgery, animals were anaesthetized with an intraperitoneal injection of thiopental sodium (EIPICO Pharmaceuticals, 10th of Ramadan City, Egypt) at a dose of 120 mg kg1 [35], and then placed on a heating pad to keep the body temperature constant at approximately 37 8C. Left and right kidneys were exposed by flank incisions on both sides, respectively. Bilateral ischemia was induced by occluding both renal pedicles using non-traumatic clamps (Dieffenbach Bulldog Clamps, Harvard Apparatus Ltd., Kent, UK) for 30 min according to the method described by Kelly et al. [36]. Complete ischemia was confirmed by blanching of kidneys. After the indicated ischemic period, clamps were released and reperfusion was started for 24 h. The kidneys were observed for 2–5 min to ensure normal blood reflow, which is indicated by
Adult male Wistar rats (180–250 g) were used in the current study. Animals were obtained from the Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Rats were acclimatized for one week prior to experiments. The animals were housed in stainless steel cages (three rats/cage) and kept at controlled temperature (23 2 8C), humidity (60 10%) and light/dark (12/ 12 h) cycle. Rats were supplied with commercially available normal chow diet and water ad libitum. 2.2. Ethical statement Experimental design and animal handling procedures were approved by the local authorities, Ethical Committee for Animal
2.3. Drugs Sitagliptin (Januvia1) was obtained from Merck, Sharp & Dohme (Cairo, Egypt), while furosemide was supplied from Amoun Pharmaceutical Co. S.A.E. (Obour City, Egypt). All other chemicals were of analytical grade. Drugs were dissolved in distilled water immediately before administration. 2.4. Experimental design Rats were randomly divided into five experimental groups (n = 6 each). Group 1 (sham-operated), group 2 (IR injury only), group 3 (rats received sitagliptin 5 mg kg1 orally at 5 h post-IR and 2 h before euthanasia), group 4 (rats received furosemide 245 mg kg1 orally at 5 h post-IR and 2 h before euthanasia), and group 5 (rats received a combination of sitagliptin 5 mg kg1 orally plus furosemide 245 mg kg1 orally at 5 h post-IR and 2 h before euthanasia). Rats of group 1 (sham-operated) and group 2 (IR injury only) did not receive any drug treatment, but rather vehicle only. 2.5. Rationale of drug dosing
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the change of kidney color to red again. There is a time lag of about 1–1.5 min between clamping of the right and left kidney. Therefore, the ischemic time for each kidney was recorded separately to ensure that both kidneys receive the same duration of ischemia. Incisions were closed by continuous stitches using vicryl sutures. Animals were kept on the heating pad and monitored until they become fully conscious before being returned to the cages [37]. Sham-operated rats underwent identical surgical procedure with the exception of application of clamps.
2.6.7. Determination of lipid peroxides content in kidney and heart Lipid peroxidation was assessed by measuring the content of malondialdehyde (MDA), a byproduct of lipid peroxidation process. The measurement was performed using a kit supplied by Diamond Diagnostics (Cairo, Egypt), following the manufacturer’s instructions. This assay depends on the reaction of MDA in samples with thiobarbituric acid (TBA) forming a red MDA-TBA adduct that can be quantified colorimetrically at wavelength 532 nm.
2.6.2. Blood sampling and serum preparation At the end of reperfusion period (24 h), blood samples were collected immediately from the retro orbital sinus of rats using heparinized microcapillary tubes. Serum was prepared by centrifugation (Hermle Z230, Gosheim, Germany) of blood tubes at 3000 rpm for 30 min. Serum was stored at 20 8C and thawed just before use for the determination of different biochemical parameters.
2.6.8. Determination of reduced glutathione (GSH) content in kidney and heart Glutathione (GSH) content was determined colorimetrically using a diagnostic kit supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. The assay is based on the reduction of 5,50 dithiobis (2-nitrobenzoic acid) with GSH in the sample to produce a yellow product, whose absorbance can be measured at wavelength 405 nm.
2.6.3. Tissue sampling Rats were euthanized by cervical dislocation under anesthesia with thiopental sodium (120 mg kg1) for tissue specimen collection. Kidney and heart were removed and rinsed thoroughly with saline and divided into two equal parts. The first part was kept in 10% phosphate-buffered formalin at room temperature for histopathological examination, while the other part was immersed immediately in liquid nitrogen and kept at 80 8C. Tissue homogenates were prepared using Polytron PT1200E Disperser (Kinematica AG, Luzern, Switzerland) in ice-cold phosphatebuffered saline (PBS, pH 7.2). Homogenates were centrifuged at 12,000 g for 10 min, and supernatants were used for measurements.
2.6.9. Determination of glutathione-s-transferase (GST) activity in kidney and heart Glutathione-s-transferase (GST) activity was measured using an assay kit supplied by Sigma-Aldrich (St. Louis, MO, USA). In this method, GST catalyzes the conjugation of L-glutathione to 1Chloro-2,4-dinitrobenzene (CDNB) forming GS-DNB conjugate, which absorbs at wavelength 340 nm. The rate of increase in its absorption is directly proportional to the GST activity.
2.6.4. Assessment of kidney function Serum creatinine and serum urea were determined by colorimetric methods using kits supplied by Diamond Diagnostics (Cairo, Egypt), following the manufacturer’s instructions. Absorbance of the final products were read using Jenway Genova spectrophotometer (Bibby Scientific, Staffordshire, UK). Serum cystatin c level was measured using a quantitative sandwich enzyme immunoassay with the aid of Quantikine ELISA kit, specific for mouse and rat, supplied by R&D Systems (Minneapolis, MN, USA). 2.6.5. Determination of biochemical markers of myocardial injury Serum creatine kinase MB (CK-MB) isoenzyme and cardiac troponin I (cTnI) were measured by enzyme-linked immunosorbent assay (ELISA) technique using kits supplied by Shanghai Crystal day Biotech Co. (Shanghai, China) and Kamiya Biomedical Company (Seattle, WA, USA), respectively, following the manufacturer’s instructions. Serum lactate dehydrogenase (LDH) activity was measured by pyruvate kinetic liquid reaction as described by Pesce [38], using a diagnostic kit supplied by Spinreact (Girona, Spain). The method is based on the ability of LDH to catalyze the reduction of pyruvate by NADH forming lactate and NAD+. The rate of decrease in the concentration of NADH, measured spectrophotometrically at wavelength 340 nm, is proportional to the catalytic concentration of LDH in the sample. 2.6.6. Determination of TNF-a and nuclear factor-kappa B (NF-kB) content in kidney and heart Tumor necrosis factor-alpha (TNF-a) and nuclear factor-kappa B (NF-kB) contents were measured by ELISA technique using ELISA kits provided by R&D Systems (Minneapolis, MN, USA) and EIAab (Wuhan, China), respectively, following the manufacturer’s instructions.
2.6.10. Determination of superoxide dismutase (SOD) activity in kidney and heart Superoxide dismutase (SOD) activity was assessed using a kit supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. This assay depends on the generation of superoxide ions from the conversion of xanthine and O2 to uric acid and H2O2 by xanthine oxidase (XO). The superoxide anion then coverts a tetrazolium salt into a formazan dye. Addition of SOD to this reaction reduces superoxide ion levels, thereby lowering the rate of formazan dye formation. The enzyme activity is then measured as the percent inhibition of the rate of formazan dye formation. 2.6.11. Determination of catalase activity and nitric oxide (NO) content in kidney and heart Catalase activity and NO content were measured colorimetrically using kits supplied by Biodiagnostic (Giza, Egypt), following the manufacturer’s instructions. 2.6.12. Determination of inducible nitric oxide synthase (iNOS) activity and caspase-3 content in kidney and heart Inducible nitric oxide synthase (iNOS) and caspase-3 were measured using ELISA kits supplied by Shanghai Crystal day Biotech Co., Ltd (Shanghai, China) and USCN Life Science Inc. (Wuhan, China), respectively, following the manufacturer’s instructions. 2.6.13. Histopathological examination Specimens of the kidney and the heart from different groups were fixed in 10% phosphate-buffered formalin solution at room temperature. Specimens were dehydrated in graded ethanol (70– 100%), cleared in xylene and embedded in paraffin. Paraffinembedded tissue sections (5 mm thick) were prepared, mounted on slides and kept at room temperature. Thereafter, slides were dewaxed in xylene; hydrated using graded ethanol, and stained by hematoxylin and eosin (HE) dyes. The sections were examined under light microscope and photographed with a digital camera (Canon, Japan).
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2.7. Statistical analysis All data were expressed as mean standard error of the mean (SEM). Statistical analysis was performed using Graphpad prism software v.5 (GraphPad Software Inc., La Jolla, CA, USA). The statistical significance of differences between groups was tested using one-way analysis of variance (ANOVA) followed by Tukey’s Post-test. A significant difference was assumed for values of P < 0.05.
these parameters with furosemide was significantly higher compared to sitagliptin group. Furthermore, the combined administration of sitagliptin and furosemide, not only significantly reduced urea, creatinine and cystatin c levels compared to IR, sitagliptin alone or furosemide alone groups, but also restored their values to normal levels seen with sham-operated rats. 3.2. Effect on biomarkers of myocardial injury
As depicted in Fig. 1, bilateral renal IR significantly elevated serum urea (59 1.2 vs. 25 0.89 mg/dl, P < 0.001), cystatin c (140 1.7 vs. 30 1.2 pg/ml, P < 0.001) and creatinine (1.3 0.09 vs. 0.6 0.05 mg/dl, P < 0.001) levels compared to sham-operated rats. Administration of either sitagliptin (5 mg kg1) or furosemide (245 mg kg1) to rats subjected to renal IR injury significantly reduced serum urea (23.7% and 42.4%, respectively, P < 0.001), cystatin c (54.2% and 67.1%, respectively, P < 0.001) and creatinine (38.5% and 53.9%, respectively, P < 0.001) levels compared to untreated rats subjected to IR injury only. The reduction observed in
Rats underwent IR injury showed significantly elevated serum CK-MB (16 0.48 vs. 1.9 0.04 ng/ml, P < 0.001), cTnI (11 0.26 vs. 1.2 0.02 ng/ml, P < 0.001) levels and LDH (308 3.3 vs. 129 2 U/ml, P < 0.001) activity compared to sham-operated rats. Treatment of rats subjected to renal IR injury with sitagliptin or furosemide significantly decreased serum CK-MB (57.5% and 76.9%, respectively, P < 0.001), cTnI (62.7% and 78.2%, respectively, P < 0.001) levels and LDH (44.8% and 49.7%, respectively, P < 0.001) activity compared to untreated rats subjected to IR injury only. In addition, furosemide resulted in a significantly higher degree of reduction of CK-MB and cTnI compared to rats received sitagliptin alone (Fig. 2). The concomitant administration of sitagliptin with furosemide successfully brought back the values of these markers close to normal levels of shamoperated rats (P > 0.05).
Fig. 1. Changes in serum urea level (A, plotted on left axis), serum cystatin c level (A, plotted on right axis) and serum creatinine level (B) at 24 h after ischemia/ reperfusion (IR) injury (n = 5–6). Sham, sham-operated rats; IR, ischemia/ reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, ** P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
Fig. 2. Changes in serum creatine kinase-MB isoenzyme (CK-MB) level (A, plotted on left axis), serum cardiac troponin I (cTnI) level (A, plotted on right axis) and serum lactate dehydrogenase (LDH) activity (B) at 24 h after ischemia-reperfusion (IR) injury (n = 6). Sham, sham-operated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
3. Results 3.1. Effect on kidney function
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3.3. Effect on tissue TNF-a and NF-kB content As shown in Fig. 3, renal IR injury resulted in a significant increase in TNF-a (about 4-fold, P < 0.001) and NF-kB (about 3fold, P < 0.001) content of kidney and heart tissue compared to sham-operated rats. Similar to what described before, administration of sitagliptin or furosemide significantly decreased TNF-a and NF-kB content of kidney and heart tissue compared to untreated rats in IR group. Both sitagliptin and furosemide, when administered together, significantly inhibited the elevation of these parameters that their values in kidney and heart tissue were comparable to normal levels of sham-operated animals. 3.4. Effect on tissue oxidative stress markers and antioxidative enzymes activity Ischemia/reperfusion resulted in significant elevation of kidney and heart content of MDA (P < 0.001), NO (P < 0.001) and activity of iNOS (P < 0.001), whereas it resulted in significant reduction of GSH content (P < 0.001), GST (P < 0.001), SOD (P < 0.001) and catalase (P < 0.001) activity compared to sham-operated rats. Treatment of rats with sitagliptin (5 mg kg1) or furosemide (245 mg kg1) significantly diminished the abnormal alterations in these parameters; however, the effect exerted by furosemide was
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significantly higher compared to sitagliptin. In addition, a combination of both sitagliptin and furosemide normalized the levels of these parameters to values similar to those observed in sham-operated rats (Table 1). 3.5. Effect on tissue caspase-3 activity As depicted in Fig. 4, rats underwent IR injury had significantly higher levels of caspse-3 content in kidney (60 1.4 vs. 7.7 0.37 nmol/g tissue, P < 0.001) and heart (22 0.61 vs. 2.9 0.11 nmol/g tissue, P < 0.001) compared to sham-operated rats. Administration of sitagliptin or furosemide resulted in significant decrease of caspase-3 content in kidney (76.6% and 84.3%, respectively, P < 0.001) and heart (77.7% and 87.2%, respectively, P < 0.001) tissue compared to IR rats. Rats received furosemide showed significantly lower levels of caspase-3 in kidney (P < 0.01) and heart (P < 0.01) tissue compared to rats received sitagliptin. Simultaneous administration of sitagliptin plus furosemide totally and significantly inhibited the increase of capase-3 content in both kidney and heart tissue. The combined administration of sitagliptin plus furosemide resulted in significant reduction of caspase-3 activity in kidney tissue compared to either sitagliptin or furosemide alone (P < 0.05). Furthermore, combined drugs significantly lowered caspase-3 activity in heart tissue compared to sitagliptin alone (P < 0.05). 3.6. Histopathological examination of kidney and heart tissue
Fig. 3. Changes in tissue content of tumor necrosis factor-alpha (TNF-a) (A) and nuclear factor-kappa B (NF-kB) (B) of kidney (plotted on left axis) and heart (plotted on right axis) at 24 h after ischemia/reperfusion (IR) injury (n = 6). Sham, shamoperated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
Sham-operated rats showed normal kidneys with normal glomeruli, proximal convoluted, distal convoluted and collecting tubules (Fig. 5a). Additionally, they showed normal myocardium with striated muscle fibers and centrally located nuclei (Fig. 6a). Occasionally, slight congestion in blood capillaries in the heart and increased eosinophilia of the renal tubular epithelium in the kidneys were observed. On the other hand, rats that underwent IR injury showed diffuse cortical and medullary ischemic necrosis with extensive hemorrhage at the medulla and corticomedullary junction of the kidney (Fig. 5b). Heart specimens revealed focal areas of degeneration and myonecrosis in the cardiac muscle fibers with loss of its striation. Their nuclei were pyknotic or completely absent. The sarcoplasm became contracted and more eosinophilic. Severe congestion, edema and hemorrhage were visualized around the blood vessels. Numerous inflammatory cells, mostly neutrophils, were observed among the affected cardiac muscle fibers (Fig. 6b). Kidney tissues from IR rats treated with sitagliptin (5 mg kg1) showed necrosis in a group of cortical tubules of both proximal and distal convoluted tubules. Such necrosis was represented as pyknosis and karyolysis (Fig. 5c). On the other hand, the heart specimen showed focal areas of degeneration and myonecrosis with pyknosis and karyolysis. Severe congestion of some cardiac blood vessels was also noticed (Fig. 6c). Kidneys from IR rats that received furosemide (245 mg kg1) showed necrosis in few scattered renal tubules (Fig. 5d). The heart was mostly normal with small focal area of degeneration and myonecrosis and no evidence of edema or hemorrhage (Fig. 6d). Administration of a combination of sitagliptin and furosemide resulted in a marked amelioration in the myonecrosis of the heart with few cardiac muscles revealing pyknotic nuclei particularly around blood vessels (Fig. 6e). The endothelium of the coronary blood vessels was intact. The kidneys were normal with individual cell necrosis, represented as pyknosis and karyolysis. Some nuclei of renal epithelium revealed mitosis (Fig. 5e). A scoring of the histopathological findings is summarized in Table 2.
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Table 1 Changes in oxidative stress markers and antioxidative enzyme activity in kidney and heart at 24 h after ischemia/reperfusion (IR) injury. Sham
IR
IR + SIT
IR + FUR a
IR + SIT/FUR ab
MDA (nmol/g tis.)
K H
7.1 0.42 3.8 0.05
48 1.0* 23 0.66*
13 0.71 5.9 0.27a
8.3 0.61 3.9 0.07ab
6.3 0.33ab 3.1 0.09ab
GSH (mmol/g tis.)
K H
3.5 0.1 1.7 0.06
0.9 0.01* 0.5 0.01*
1.8 0.04a 1.1 0.04a
2.6 0.16ab 1.5 0.01ab
3.8 0.08abc 2.0 0.08abc
GST (mmol/g tis.)
K H
4.4 0.09 2.1 0.07
1.17 0.02* 0.59 0.02*
2.5 0.15a 1.5 0.04a
3.2 0.03ab 1.9 0.01ab
3.9 0.1abc 2.4 0.03abc
SOD (U/g tis.)
K H
10.7 0.28 4.7 0.07
2.2 0.05* 1.3 0.02*
5.9 0.14a 2.7 0.01a
9.1 0.2ab 3.6 0.06ab
10.8 0.11abc 4.7 0.05abc
Catalase (U/g tis.)
K H
11.8 0.23 5 0.17
3.2 0.07* 1.3 0.02*
6.9 0.41a 3.0 0.07a
10.1 0.2ab 4.0 0.21ab
11.9 0.35abc 5.1 0.07abc
NO (mmol/g tis.)
K H
19 0.39 6.15 0.07
75 0.97* 28 0.62*
28 0.41a 10.7 0.16a
21 0.99ab 6.9 0.2ab
17 0.42abc 5.4 0.13abc
iNOS (ng/g tis.)
K H
24 0.30 12 0.21
90 1.0* 33 0.65*
36 0.61a 16 0.21a
28 0.49ab 13 0.38ab
20 0.35abc 9.3 0.14abc
Values are expressed as mean SEM (n = 6) in kidney (K) and heart (H). IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). MDA, malondialdehyde; GSH, reduced glutathione; GST, glutathione-s-transferase; SOD, superoxide dismutase; NO, nitric oxide; iNOS, inducible nitric oxide; g tis., gram tissue. Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, aP < 0.05 vs. IR, bP < 0.05 vs. IR + SIT, cP < 0.05 vs. IR + FUR.
4. Discussion It has become obvious that clinically much of the high patient mortality associated with AKI occurs as a result of systemic inflammatory response and progression to multiple organ failure [39]. Therefore, the present study was designed to evaluate the protective effect of sitagliptin, furosemide or their combination in the settings of acute AKI in rats and the associated remote cardiac damage. We were interested particularly in this target because cardiac dysfunction in AKI patients, among other distant organs failure, has been recognized as a leading cause of death [18,20,40]. In our study, AKI was induced in rats by using the bilateral ischemic model. We used this model because it is very close to pathological conditions observed in human where blood supply of both kidneys is normally reduced [41–43]. Bilateral renal ischemia for 30 min followed by reperfusion resulted in marked deterioration of kidney function at 24 h after IR procedure. This was evident from the significant elevation of
Fig. 4. Changes in tissue content of caspase-3 of kidney (plotted on left axis) and heart (plotted on right axis) at 24 h after ischemia/reperfusion (IR) injury (n = 6). Sham, sham-operated rats; IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1). Statistical analysis using one-way ANOVA, followed by Tukey’s Post-test. *P < 0.05 vs. sham, **P < 0.05 vs. IR, ***P < 0.05 vs. IR + SIT, @P < 0.05 vs. IR + FUR.
serum creatinine (Fig. 1b) and urea (Fig. 1a) levels, which are widely used to assess kidney function, compared to shamoperated animals that underwent the same surgical procedure without IR injury. In addition, IR injury resulted in significant increase of serum cystatin c level. Although an increase of serum creatinine is used clinically to identify AKI [44], however, cystatin c has been shown by different studies to be superior than creatinine, especially to detect minor reduction in glomerular filtration rate (GFR) [45,46]. The elevation of these markers is attributed to the sudden decline in GFR induced by AKI. Histopathological examination revealed that the deterioration of kidney function was associated with renal structural damage manifested by necrosis and extensive hemorrhage at the medulla and corticomedullary junction of the kidney (Fig. 5b). Taken together, these parameters confirm the proper induction of AKI in our experiments. Furthermore, our results show that renal IR injury did not only affect the kidneys, but also resulted in distant myocardial injury as evidenced by the significant elevation of serum CK-MB, cTnI levels and LDH activity (Fig. 2) compared to sham group. These biomarkers have been widely applied for the identification of acute myocardial injury [47]. In addition, histopathological examination showed focal areas of degeneration and myonecrosis in the cardiac muscle fibers with severe congestion, edema and hemorrhage around the blood vessels (Fig. 6b). This finding illustrates that AKI rarely occurs in isolation; however, it causes complications in other distant non-renal organs. The deterioration of kidney function and associated myocardial injury were significantly suppressed by treatment with sitagliptin, furosemide or their combination. In addition, drug treatment reduced the structural damage caused by IR injury in kidney and heart tissue. In this regard, combined administration of both drugs resulted in marked amelioration in the myonecrosis of the heart (Fig. 6e). Similarly, the kidneys appeared normal with minor cell necrosis (Fig. 5e). Different mechanisms of acute renal IR injury have been proposed including the activation of inflammatory response, inflammatory cell infiltration, generation of reactive oxygen species (ROS), and apoptosis [48–50]. Therefore, in order to elucidate the mechanisms by which these drugs reduced IR-induced myocardial injury, we investigated their effects on inflammatory cytokines, oxidative stress and apoptosis in kidney and heart tissue.
Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010
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Fig. 5. Histological examination of kidney tissue at 24 h after ischemia/reperfusion (IR) procedure. Representative light micrograph are depicted from (a) sham-operated rats: showing normal structure with slight eosinophils at the epithelial lining of the proximal convoluted tubules (arrows), (b) rats underwent IR only: showing diffuse cortical and medullary necrosis (arrows) with extensive hemorrhage at the medulla and at the corticomedullary junction (arrowheads), (c) rats underwent IR procedure and received sitagliptin (5 mg kg1): showing necrosis in a group of cortical tubules with pyknosis and karyolysis (arrows), (d) rats underwent IR procedure and received furosemide (245 mg kg1): showing necrosis in few scattered renal tubules (arrows) and contracted glomerular tufts (arrowheads) and (e) rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1): showing individual cell necrosis with pyknosis and karyolysis (arrows) and intact glomerular tufts (arrowhead), with some nuclei of the renal epithelium revealing mitosis. Tissue specimens were stained with HE dyes (scale bar = 50 mM).
Our results showed that renal IR procedure significantly increased TNF-a and NF-kB content in kidney and heart compared to sham rats (Fig. 3), which is consistent with previous studies [15,24]. It is apparent that ischemia-induced damage to the renal
tubular cells stimulates the release TNF-a [8], which consequently activates the canonical pathway of NF-kB leading to its translocation to the nucleus [51]. In turn, NF-kB enhances the expression of other important mediators including cytokines, chemokines, iNOS,
Fig. 6. Histological examination of heart tissue at 24 h after ischemia/reperfusion (IR) procedure. Representative light micrograph are depicted from (a) sham-operated rats: showing normal muscle fibers with slight eosinophils at the sarcoplasm (arrowheads) and slight congestion (arrows), (b) rats underwent IR only: showing diffuse myonecrosis (arrows), congestion (c), edema and hemorrhage (arrowhead), (c) rats underwent IR procedure and received sitagliptin (5 mg kg1): showing myonecrosis (arrows) and severe congestion (arrowhead), (d) rats underwent IR procedure and received furosemide (245 mg kg1): showing focal small areas of myonecrosis (arrows) and (e) rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1): showing normal myocardium with rare myonecrosis (arrows) and intact endothelium of coronary blood vessels (arrowhead). Tissue specimens were stained with HE dyes (scale bar = 50 mM).
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Table 2 Histopathology scoring of kidney and heart tissue at 24 h after ischemia/reperfusion (IR) procedure. Groups
Kidney
Heart
Deg. and Nec.
Cong.
Ed.
Hg.
Deg. and Nec.
Cong.
Ed.
Hg.
Sham IR IR + SIT IR + FUR IR+ SIT/FUR
++++ ++ +
+++ ++ + +
++ ++ ++ +
++ + +
++++ ++ +
+++ ++ + +
+++ ++ +
+++ + +
IR, ischemia/reperfusion only group; IR + SIT, rats underwent IR procedure and received sitagliptin (5 mg kg1); IR + FUR, rats underwent IR procedure and received furosemide (245 mg kg1); IR + SIT/FUR, rats underwent IR procedure and received both sitagliptin (5 mg kg1) and furosemide (245 mg kg1 ). Deg. and Nec., degeneration and necrosis; Cong., congestion; Ed., edema; Hg., hemorrhage. () nil, (+) mild, () nil to mild, (++) moderate, (+++) severe, (++++) more severe.
and adhesion molecules such as E-selectin and ICAM-1 [52,53]. In addition, the inflammatory response mediated by NF-kB at the vascular endothelium leads to migration and adhesion of leukocytes, which is enhanced by over-expression of adhesion molecules [9,54]. The subsequent activation of leukocytes results in obstruction of capillaries, further production of cytokines, and aggravation of the inflammatory response [10]. Indeed, we detected neutrophils infiltrating kidney and heart after IR procedure at 24 h (Fig. 6b). These inflammatory markers were significantly reduced in rats treated with sitagliptin, furosemide or their combination. The combination of both sitagliptin and furosemide totally inhibited the elevation of theses markers. Our results demonstrated that renal IR injury generated oxidative stress and increased the production of ROS in the kidney and the heart compared to sham rats. This was manifested as increased formation of MDA, which is a widely accepted marker of oxidative stress [55]. Different sources have been suggested for ROS production including XO enzyme, mitochondria, and activation of leukocyte NADPH oxidase [56–58]. In addition, IR rats had significantly high content of NO in kidney and heart tissue compared to sham rats. Although NO has an important role in renal hemodynamic regulation, however, many authors suggested its implication in ischemia-induced renal tubular injury [59–62]. The deleterious effect of NO is suggested to occur via its reaction with superoxide anion forming peroxynitrite, which is a highly toxic oxidant [63]. It has been suggested that the increased production of NO is attributed to IR-induced upregulation of iNOS [62]. We could observe that iNOS was significantly increased in kidney and heart tissue from IR rats compared to sham rats. In addition, our results are in harmony with previous studies, which demonstrated the crucial role of oxidative and nitrosative stress in acute renal ischemia [61,64–67]. Furthermore, our results revealed that IR injury impaired the non-enzymatic and enzymatic anti-oxidant protective mechanisms in kidney and heart as manifested by the significant reduction of GSH, GST, SOD and catalase activity in IR rats compared to sham rats. Under physiological conditions, they can scavenge and detoxify ROS, however, during ischemia supplies of these endogenous scavengers and enzymes are overwhelmed by ROS leading to cellular injury. This hypothesis is supported by the findings of other studies showing reduction of cellular stores of these scavengers during brain [68] and heart [69] ischemia. Increased oxidative stress has detrimental consequences, such as impairment of membrane function, induced by lipid peroxidation, and oxidative DNA damage [70]. The ultimate result is cellular death via necrosis or apoptosis [71]. This explains our findings of increased caspase-3 content (Fig. 4), reflecting increased apoptotic cell death, and necrosis (Table 2) in kidney and heart after IR injury procedure. Administration of sitagliptin, furosemide or their combination significantly reduced oxidative stress induced by renal IR injury and induced the activity of the anti-oxidative enzymes in kidney
and heart. This, in part, had a positive impact on improving the kidney function and reducing associated myocardial injury. This effect can be attributed to the reduction of leukocyte infiltration in heart and kidney tissue as shown in Figs. 5 and 6. In this regard, our results agree with Vaghasiya et al. [27], who reported that sitagliptin reduced oxidative stress in renal tissue after IR via reduction of leukocyte infiltration, as well as decreasing xanthine oxidase (XO) activity. One of the interesting findings of the present study was the observation that furosemide afforded rats greater protection against IR injury and the associated myocardial injury than sitagliptin. Presumably, this can be attributed to particular renal actions of furosemide including increased renal blood flow and subsequent increase in GFR, mediated via inhibition of prostaglandin dehydrogenase and subsequent increase of prostaglandin (PG) E2, a potent renal vasodilator [72,73]. Indeed, furosemide has been shown to increase GFR in healthy volunteers [74,75]. In addition, furosemide reduces oxygen consumption of the renal tubular cells via inhibition of the sodium/potassium chloride co-transporter [76,77]. Increased renal blood flow and amelioration of hypoxic insult, besides its anti-apoptotic effect [78], allows furosemide to further diminish the inflammatory cascade induced by renal ischemia and the myocardial injury that ensues better than sitagliptin. Although, the effects of GLP-1 receptor agonists and sitagliptin on GFR are controversial [79–81], we cannot rule out the possibility that the smaller effect of sitagliptin compared to furosemide might be attributed to the small dose of sitagliptin used in this study. Another interesting finding in our study is the nearly complete blockade of IR-induced damage in kidney and heart produced by combined administration of both sitagliptin and furosemide. The protection exerted by the combination was superior to each drug administered alone. This was manifested as normalization of the measured parameters. It seems that this is a result of additive effects of both drugs by affecting different pathways. Despite being used widely by most clinicians for managing AKI, evidence for beneficial effects, concerning outcome and mortality, of loop diuretics still lacking even with experimental results suggesting a therapeutic potential. Clinically, diuretics have been shown to increase the risk of AKI or lack therapeutic benefit in different settings [82–86]. Considering this discrepancy of diuretic effect in managing AKI, it is urgently required to identify agents with possible protective effect in the clinical settings. Like previous studies, we could show therapeutic potential for sitagliptin. However, our study still offers some points of novelty, unlike other reports. Previous studies investigated the effect of sitagliptin on either renal ischemia [24,27] or cardiac ischemia [28,87, 88] only, however, none described the effect on distant myocardial damage induced by renal IR injury. Our study for the first time, to our knowledge, provides an experimental evidence for beneficial effect of sitagliptin to protect not only against renal IR injury, but also against the associated remote myocardial damage, which is
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recognized as a leading cause of mortality during AKI. Second, the dose of sitagliptin used in our study is either smaller or used for shorter duration compared to other studies. Vaghasiya et al. [27] used sitagliptin at a dose of 5 mg kg1 day1, orally during 2 weeks starting 11 days before renal IR injury. On the other hand, Chen et al. [24] used sitagliptin at a dose of 600 mg kg1 at 1, 24, and 48 h post-IR. In the present study we used sitagliptin twice post-IR at a dose of only 5 mg kg1. We assume that such small dose would be safe enough to be tried clinically in the settings of AKI, despite the necessity of dose reduction with deteriorated kidney function, where it is advised to reduce the dose of sitagliptin from 100 mg day1 to 25 mg day1 with creatinine clearance less than 30 mL/ min [89]. However, higher doses of sitagliptin still represent other possible alternatives, particularly, in patients with creatinine clearance over 50 mL/min. Third, the remarkable protection observed with the combination of sitagliptin and furosemide suggests it as a beneficial therapeutic regimen in AKI post-IR injury and qualifies it as a potential candidate for clinical trials. This seems promising in improving the total outcome and reducing mortality, particularly with the failure of diuretics alone to offer reasonable therapeutic benefit. However, this needs further clinical investigations to ascertain or rule out the experimental data observed in our study. The study has a few limitations. First, higher doses of sitagliptin have not been tried, which would allow for a better comparison of its effects with furosemide. Second, even though dosing schedule has been selected carefully, different dosing schedules would be helpful for better optimization of the drug treatment. In summary, sitagliptin could significantly suppress IR-induced inflammatory cascade, oxidative stress and apoptosis, thus protecting from kidney damage and associated distant cardiac injury. Superior protection was offered by a combination of sitagliptin and furosemide. Therefore, we assume that this combination represents a promising regimen during AKI, particularly that caused by IR injury. This might be due to the extra protection from myocardial damage occurring during AKI, which is an important cause of death in those patients. Conflicts of interest The authors declare no conflicts of interest. Acknowledgments The authors acknowledge Dr. Mohamed Hamed Mohamed, Professor of Pathology, Faculty of Veterinary Medicine, Zagazig University for his great effort in the hispathological examination. References [1] J.P. Lafrance, D.R. Miller, Acute kidney injury associates with increased long-term mortality, J. Am. Soc. Nephrol.: JASN 21 (2010) 345–352. [2] S.G. Coca, B. Yusuf, M.G. Shlipak, A.X. Garg, C.R. Parikh, Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis, Am. J. Kidney Dis.: Off. J. Natl. Kidney Found. 53 (2009) 961–973. [3] S.M. Bagshaw, Short- and long-term survival after acute kidney injury, Nephrol. Dial. Transpl. 23 (2008) 2126–2128. [4] F. Liano, J. Pascual, C. Gamez, A. Gallego, M.A. Bajo, L.S. Sicilia, et al., Epidemiology of acute renal failure: a prospective, multicenter, community-based study, Kidney Int. 50 (1996) 811–818. [5] C. Rippe, A. Rippe, A. Larsson, D. Asgeirsson, B. Rippe, Nature of glomerular capillary permeability changes following acute renal ischemia-reperfusion injury in rats, Am. J. Physiol.-Renal 291 (2006), F1362-F8. [6] M. Le Dorze, M. Legrand, D. Payen, C. Ince, The role of the microcirculation in acute kidney injury, Curr. Opin. Crit. Care 15 (2009) 503–508. [7] J.V. Bonventre, L. Yang, Cellular pathophysiology of ischemic acute kidney injury, J. Clin. Invest. 121 (2011) 4210–4221. [8] M.J. Burne-Taney, J. Kofler, N. Yokota, M. Weisfeldt, R.J. Traystman, H. Rabb, Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury, Am. J. Physiol. Renal Physiol. 285 (2003) F87–F94.
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Please cite this article in press as: Youssef MI, et al. A new combination of sitagliptin and furosemide protects against remote myocardial injury induced by renal ischemia/reperfusion in rats. Biochem Pharmacol (2015), http://dx.doi.org/10.1016/j.bcp.2015.04.010