International Journal of Medical Sciences and Health Care

Vol-1 Issue-7 (Ijmshc-705)

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Vol-1 Issue-7 (Ijmshc-705)

ISCHEMIA REPERFUSION IN THE RATS KIDNEY MODULATE THE PHYSIOLOGICAL FUNCTION OF SOME DISTAL ORGANS OF THE BODY
*Dallatu, Muhammad Kabiru; Bisong, Dickson, Rizvi, Yasmin 3, Erhabor Osaro 4
1. 2. 3. 4. Department of Chemical Pathology, Faculty of Medical Laboratory Science Usmanu Danfodiyo University, Sokoto-Nigeria. Department of Laboratory Services, Ben Taub Hospital Texas Medical Center, Houston TX USA Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern University, Houston TX USA Department of Haematology Faculty of Medical Laboratory Science Usmanu Danfodiyo University, Sokoto-Nigeria

All Correspondence to: Dr Dallatu, Muhammad Kabiru - Department of Chemical Pathology, Faculty of Medical Laboratory Science Usmanu Danfodiyo University, Sokoto-Nigeria

Abstract:
Background: Body of literature are becoming pronounced that pathological condition in one organ of the body might have an effect on other distal organs owing to the fact, that the entire body metabolism is orchestrated centrally. Pathological events occurring in an organ are likely to be extended to other organs. Pretreatment that minimize these events are presumed to be beneficial to the extended organs. Methods: Following 30 min of ischemia and 48 h of reperfusion in the kidney, rats under anesthesia were sacrificed and blood sample collected through cardiac puncture. Serum level of troponin I, and activities of total creatine kinase (CK), mass creatine kinase (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) were estimated spectrophotometrically. Results: Serum troponin I increased to 0.031 0.001 ng/ml in the ischemic group, and following pretreatment with Lmm (600mg/kg), serum level of troponin I decreased significantly to 0.021 0.001 ng/ml (P<.05). Serum activities of creatine kinase (CK) and creatine kinase-MB (CK-MB) in the ischemic rats was 144.6 1.0 u/l, and 56.0 2.01 u/l, but following pretreatment with L-mm, the activities decreased significantly to 97.95 0.55 u/l and 23.15 0.98 u/l respectively. Serum activities of liver enzymes in the ischemic group was AST; 27.6 1.02 u/l, ALT; 50.1 2.3 u/l and GGT; 59.20 0.6 u/l, but following pretreatment with L-mm, AST activity decreased to 21.1 0.24 u/l (P <.05), ALT to 39.6 1.21 u/l (P <.05) and GGT to 38.2 0.35 u/l (P <.05). Conclusion: Our finding indicates ischemia and reperfusion in the kidney may likely affect other organs and predisposed these patients to other cardiovascular risk events.

Key Words: Ischemia, reperfusion, kidney, liver, heart.

Introduction Ischemia derived from Greek ischein to restrain and haima blood. The main metabolic function of cardiovascular system is the delivery of oxygen and other nutrients to the tissues of the body and to dramatically adjust the supply to meet the current metabolic demand of such tissues 1. Ischemia occurs when the blood supply to a tissue is inadequate to meet the tissue metabolic demands and is followed by 3 principal biological processes; hypoxia, insufficiency of metabolic substrates and accumulation of metabolic wastes. It is a great insult to cells and tissues than hypoxia alone1. In certain surgical procedures, tissues are subjected to purposeful ischemia to regulate and prevent wastage of blood. Evidence has accumulated, that even with sophisticated techniques, prolonged ischemic periods may cause unwanted outcomes, because of complex mechanism called reperfusion injury. Clamping and unclamping of arteries is associated with serious haemodynamic and homeostatic disturbances in virtually all biochemical systems, because of the decrease in blood flow to the affected organ in particular and distal effect to others 2. Restoration of blood flow after the occlusion, results in tissue damage due to rapid reoxygenation, changes in PH or intracellular ion transport 3. Activated neutrophils release agents (Reactive oxygen species and proteinases) which aggravate injury to endothelial cells already subjected to anoxia and reoxygenation. Endothelial damage results in a decreased vasodilatory response to hypoxia which may result into further decrease in blood flow 4. Hypoxia-inducible factor 1 alpha (HIF-1) is a transcription factor at the center of cellular adaptation to hypoxic insults, owing to its ability to induce the expression of variety of genes in a coordinated and physiologic manner to salvage cellular contents from ischemia-induced cellular injury 5. Stability of HIF-1, however, is under the enzymatic activity of prolyl hydroxylase domain (PHD) enzyme, which hydroxylates specific prolyl residues on HIF-1 for subsequent rapid degradation of HIF-1 by proteasomes 6. It becomes evident, that maneuvers to stabilize and activate HIF-1 through PHD inhibition in one of the promising ways to prevent injury and ultimately treat ischemic insults.
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Unpublished data from our laboratory 7 shows a promising therapeutic effect of treatment with Lmimosine, an inhibitor of PHD in the prevention of rats kidney against ischemia/reperfusion. However, body of literature are becoming pronounced that pathological condition in one organ of the body might have an effect on other distal organs 8, owing to the fact, that the entire body metabolism is orchestrated centrally. Distal organ effect is becoming an important factor to be considered when trying to understand the pathological mechanism of organ failure. It is an understudied field and it calls for closer subspecialty interaction to achieve progress in this area 8. A decade ago, acute kidney injury (AKI) was thought to be a benign entity that could be managed easily with supportive care and dialysis 9. Although, epidemiologic data clearly demonstrated that AKI is independently associated with increased mortality, the mechanism by which AKI causes death remains unclear. One possible explanation for the increased mortality is that AKI causes deleterious systemic effects including injury to other organs 9. Katra et al 10, reported that even minor renal dysfunction has now been established as an independent risk for cardiovascular diseases (CVD). The mechanism responsible for the association thus remains unclear. Additionally, the risk for CVD and prevalence in patients with less severe renal impairment (such as AKI) are poorly described, partly because this population is less captive than those requiring dialysis or transplantation management. Ironically, it is this population that CVD prevention strategies are likely to have greatest benefit 11. Greater effort is needed to reduce this therapeutic gap. This could provide a platform to deduce the hypothesis that I/R-induced renal disease is a risk factor to other distal organ diseases. The current study was therefore designed to evaluate the effect of acute ischemia/reperfusion injury in rat kidney, liver and cardiac functions. Materials and methods Unless otherwise specified in the text, all chemicals used were obtained from Sigma-Aldrich (St. Louis, MO, USA) and Siemens Healthcare Diagnostics Inc. Newart, DE. USA, and all are of analytical grade. Animals: Adult male Sprague-Dawley rats (450-500g) obtained from Harlan Sprague-Dawley (Houston, Texas, USA) were used for the study. The animals were maintained in a room with lighting that was adjusted to produce a normal day-night cycle. They were maintained on a standard diet of Purina chow and allowed ad libitum access to water and food at least 3 days to become acclimatized to the housing condition before use in experiments. All protocols were approved by the Institutional Animal Care Facility Committee, Texas Southern University, Houston-USA. The rats ware randomly divided into three groups. These include sham operated controls, ischemia/reperfusion group and Lmimosine treated ischemia/reperfusion group. Administration of L-mimosine: L-mimosine, 600 mg/kg dissolved in 10% NaHCO3 adjusted to pH 7.4 was administered to rats by oral gavages, 6 hours before induction of ischemia. Sham
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operated rats were placed on the vehicle in the same manner. Induction of Ischemia/reperfusion: Rats were anaesthetized with ketamine (100mg/kg, i.p.) and a left lateral flank incision was made. The kidneys were exposed and the renal artery and vein were identified and occluded for 30 min with a non-traumatic artery clamp 12. Ischemia was confirmed visually by blanching of the kidney. The incision site was covered with gauze soaked with normal saline to prevent evaporation. At the end of the ischemia induction, reperfusion was allowed for 3 hrs and scanning repeated. Animals were placed on a thermostatically controlled warming table to maintain the animals body temperature at 37C. At the end of the occlusion period, the clamp was removed and reperfusion allowed. The incision was closed with muscular layer with 4.0 mononylon absorbable sutures and skin layer with silk (4.0). Animals were returned to metabolic cages for 24 hrs urine collection. In sham-operated controls, the rats were treated identically except that the kidneys were only exposed but not clamped. Measurement of Biochemical analytes : Serum level of troponin I, and activities of total creatine kinase (CK), mass creatine kinase (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) were estimated with kits obtained from Siemens Healthcare Diagnostics Inc. Newart, DE. USA, and all are based on antigen antibody recognition chemistry. STATISTICAL ANALYSIS Data were analyzed using Prism statistical soft ware and group comparisons were made using ANOVA followed by Bonferroni post hoc test. In all cases, P < .05 was considered as significant. RESULTS Following 30 minutes of ischemia and 48 hours of reperfusion to the kidney, serum troponin I levels increased significantly (P<.05) to 0.031 0.001 ng/ml in the ischemic group, compared to the concentration of 0.011 0.001 ng/ml in the controls. But in the group pretreated with L-mm (600mg/kg), serum level of troponin I decreased significantly to 0.021 0.001 ng/ml (Figure 1). Mean serum activity of CK in the ischemic rats was 144.6 1.0 u/l, and this was significantly decreased (P<.05) to 97.95 0.55 u/l in the group pretreated with L-mm as shown in figure 2.In figure 3 of the results, mean serum activity of CK-MB in the ischemic rats was 56.0 2.01 u/l which significantly decreased to 23.15 0.96 u/l in the group pretreated with L-mm (P<.05). Mean serum activities of liver enzymes AST, ALT and GGT shown in figures 4, 5 and 6 increased significantly following ischemic injury to the rats kidney. Mean serum AST activities in the ischemic group was 27.6 1.02 u/l, ALT 50.1 2.3 u/l and GGT 59.20 0.6 u/l . Following pretreatment with L-mm, there were significant decrease in the serum activities of the all the studied enzymes: AST activity decreasing to 21.1 0.24 u/l (P<.05), ALT to 39.6 1.21 u/l (P<.05) and GGT to 38.2 0.35 u/l (P<.05).
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Discussion End stage renal disease is strongly associated with risk of cardiovascular diseases and the risk of subsequent cardiovascular events is higher among patients with chronic kidney disease than among persons with normal renal function 13. It is neither clear whether a similar association exists for patients with less severely impaired renal function 14 nor by which mechanisms any such association would be mediated. This question is important because the incidence of renal functional impairment is increasing rapidly 15. In the current study, we attempted to explore the effect of ischemia/reperfusion, an acute type of injury in the kidney, on distal organs such as liver and heart. Even minor renal dysfunction has now been established as an independent risk for cardiovascular diseases 10. Freda et al 16, reported that patients with renal insufficiency may have increased serum troponins even in the absence of clinically suspected acute myocardial ischemia. This might signify a transition stage to overt cardiovascular events these patients are likely to

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experience. Renal IRI is associated with the release of inflammatory agents that are cytotoxic to renal cells. These include neutrophils and macrophages which beside their inherent effects to the kidney also activate the released cytokines. These cells in circulation can migrate to distal organs such as the heart and initiate similar injury. According to Alpert et al 17, troponins are released because of necrosis. Early release is thought to be attributable to the cytosolic pool, and later release to the structural pool. Here we demonstrated for the first time that significant activation of HIF-1 through L-mm inhibition of PHD occurs after 48 h of I/R in the kidney and partially might be responsible for decreased generation of cytotoxic cells in the renal vasculature and possible distal effect they have on the heart. HIF-1 is the main transcription factor involved in the regulation of transcriptional responses to hypoxia. HIF-1 levels accumulate and trigger an increase in expression of genes involved in glycolysis, glucose metabolism, mitochondrial function, cell survival and resistance to

oxidative stress 18.

Figure1. Serum troponin I level in the rat. Serum troponin was measured with RXL spectrophotometer (Siemens Healthcare Diagnostics Inc. Newart, DE. USA,) in control, ischemic (I/R+ Vehicle) and in ischemic rats treated with L-mm *p < 0.05 in the L-mm group when compared to vehicle. Serum activities of creatine kinase (CK) as shown in figure 2 elevates following 48 h of IRI to the kidney. It is well known that levels of creatine kinase (CK), creatine kinase-MB (CK-MB) and myoglobin are altered in patients with uremia 19. Their sources may be from silent myocardial injuries or micro infarcts. Micro injury in these patients may result from conditions like silent ischemia, cardio toxicity from changes in osmolality/ion fluxes, increased preload and myocardial stretch, and nonischemic myocardial injury due to uremic toxins 20, 21. Generation of oxidative stress and tissue hypoxia in the kidney may translocate to the heart culminating in irreversible tissue damage and aggravate organ ischemia 22. By restoring renal function through HIF-1 activation, possible causes of cardiac enzymes leakage as seen in uremic milieu can be eliminated. Treatment with L-mm in the current study might activate HIF-1 and subsequent downstream protective actions.

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Figure 2. Serum activity of creatine kinase (CK). Creatine kinase was measured with RXL spectrophotometer (Siemens Healthcare Diagnostics Inc. Newart, DE. USA,) in control, ischemic (I/R+ Vehicle) and ischemic rats treated with L-mm. *p <0.05 in the L-mm group when compared with vehicle.

Figure 3. Serum activity of creatine kinase MB in the rat. CK-MB was measured using RXL spectrophotometer (Siemens Healthcare Diagnostics Inc. Newart, DE. USA,) in the control, ischemic (I/R+V ehicle) and in ischemic rats treated with L-mm. *p < 0.05 when compared to vehicle. Kim et al 21, reported that acute kidney injury may lead to severe hepatic and intestinal injury with periportal hepatocyte vacuolization, small intestinal necrosis, apoptosis and proinflammatory up regulation. Body of evidences accumulate implicating these agents to ischemic injury to the kidney and possible distal organ effects to the liver. Sustained presence of iNOS-derived NO might become detrimental by increasing toxic reactive oxygen species leading to liver injury 23. Activation of HIF-1 through treatment with L-mm might in part, contribute to the observed protection. HIF-1 act through repertoire of HIF-1 responsive genes. These include genes involved in angiogenesis, glucose metabolism vasomotor control, and erythropoiesis, many of which are involved in either the delivery of oxygen and nutrients to cells or controlling cellular utilization of these substrates 24.

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Figure 4. Serum activity of gamma glutamyl transferase in the rat . GGT level was measured using RXL spectrophotometer (Siemens Healthcare Diagnostics Inc. Newart, DE. USA,) in control, ischemic (I/R+ Vehicle) and in ischemic rats treated with L-mm. *p < 0.05 in L-mm when compared with vehicle.

Figure 5. Serum activity of aspartate amino transferase in rats. AST level was measured with RXL spectrophotometer (Siemens Healthcare Diagonistics Inc. Newart, DE. USA,) in control, ischemic (I/R+ Vehicle) and in ischemic rats treated with L-mm. *p <0.05 in L-mm when compared to vehicle.

Figure 6. Serum activity of alanine amino transferase in the rat. ALT activity was measured with RXL spectrophotometer (Siemens Healthcare Diagnostics Inc. Newart, DE. USA,) in control, ischemic (I/R+ Vehicle) and in ischemic rats treated with L-mm. * p < 0.05 in L-mm group when compared to vehicle. In summary, our data highlighted the relevance of acute kidney injury to markers that may translate to future of CVD events. Subjects with mild renal injury are associated with CVD risk factors and are more prone to CVD events when compared with subjects with normal renal function. Despite all the controversy in the usefulness of some of these markers, increasing body of evidence suggest that any detectable troponin, even at levels below diagnostic cutoffs, might be associated with adverse prognosis 25.
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In the current study, significant differences exist between the ischemic and ischemic groups treated with L-mm, but all are within the reported reference values. These might signify a silent pathology that can be easily missed and progress to overt pathology. Patients with mild renal insufficiency may likely suffer CVD episodes, and that restoration of renal functions through HIF-1 activation could decrease or even reverse progression of
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cardiovascular disease in patients with acute renal disease. Acknowledgements The authors want to acknowledge the assistance of the Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern University Houston for granting the research visit and the management of Usmanu Danfodiyo University, Sokoto as well as the Nigerian Education Trust Fund (ETF) for sponsorship. Ethical approval All authors hereby declare that Principles of laboratory animal care (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiemts have been examined and approved by the appropriate ethics committee. References 1.Bhalodia Y, Kanzariya , Patel N, Vaghasiya J, Jivani N, Raval H. Nephroprotective activity of benincasa cerifera fruit extract on ischemia/reperfusion-induced renal damage in Rat. Iranian J Kid Dis 2009; 3 (2): 8085. 2. Hines RL, Marschall KE. Stoeltings anesthesia and co-existing diseases. 5th edition. Churchill Livingstone. Philadelphia, USA 2008. 3. Bojakowski K, Abramczyk P, Bojakowska M, Zwolinska A, Przybylski J, Gaciong Z. Fucoidan improves sthe renal blood flow in the early stage of renal ischemia/reperfusion injury in the rat. J Physiol Pharmacol 2001; 52 (1): 137-143. 4. Ajay k, Kirstan K, Meijing W, Ben M, Jeffrey M, Daniel R. Cellular and molecular mechanisms of sex differences in renal ischemiareperfusion injury. Cardiovasc Res 2005; 67 (4): 594-603. 5. Nangaku M, Tanaka T. Drug discovery for overcoming chronic kidney disease (CKD): Prolylhydroxylase inhibitors to activate hypoxia-inducible factor (HIF) as a novel therapeutic approach in CKD. J Pharmacol Sci 2009; 109: 24-31. 6. Nangaku M, Nishi H, Miyata T. Role of chronic hypoxia and hypoxia inducible factors in kidney diseases. Chinese Med J 2008; 121(3): 257-265. 7. Dallatu MK, Oyekan AO. The contribution of Prolylhydroxylase domain (PHDs) to ischemiareperfusion injury in the rat kidney. (Unpublished). 8. Floegel J, Uhling S. Kidney calling lung and call back: how organs talk to each other. Nephrol Dial Transplant 2010; 25:32-34. 9. Faubel S. Acute kidney injury and multiple organ dysfunction syndrome. Minerva Urol Neprol 2009; 61 (3): 171-88. 10. Katra V, Mahjan S, Agarwal KS, Tiwari SC. Cardiorenal disease: A clinical intersection. Intern Urol Nephrol 2005; 37 (1) :175-184. 11.Culleton BF, Larson MG, Wilson PWF, Evans JC, Parfrey PS, Levy D. Cardiovascular disease and mortality in a community-based cohort with mild renal insufficiency. Kid Internl 2006; 56: 2214-2219. 12. Chatterjee P K, Thiemermann C. An invivo model of ischemia/reperfusion and inflammation of the kidneys of the rat. Methods Mol Biol 2003; 225:223-237. 13. Wright RS, Reeder GS, Herzog CA. Acute myocardial infarction and renal dysfunction: a high-risk combination. Ann Intern Med 2002; 137: 563-70.
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14. Garg AX, Clark FC, Haynes RB. Moderate renal insufficiency and the risk of cardiovascular mortality: Results from the NHANES I. Kid Intern 2002; 61: 14861494. 15.Henry RMA, Kostense PJ, Bos G, Decker JM, Nijpels G, Heine RJ, Bouter LM, Stehouwer CDA. Mild renal insufficiency is associated with increased cardiovascular mortality: The Hoorn Study. Kid Intern 2002; 62: 1402-1407. 16. Freda BJ, Tang WH, VanLente F, Peacock WF, Francis GS. Cardiac troponins in renal insufficiencyreview and clinical implications. J Am Coll Cardiol 2002; 40: 2065-2071. 17. Alpert J S, Thygesen K, Antman E, Bass JP. Myocardial infarction redefined-a consensus document of the Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction. J Am Coll Cardiol 2000; 36: 959969. 18. Loor G, Schumacker PT. Role of hypoxia-inducible factor in cell survival during myocardial ischemiareperfusion. Cell Death Differ 2008; 15: 686-690. 19. Bennett AE, Bertholf RL. Discordant results of CKMB and troponin I measurements: a review of 14 cases. Ann Clin Lab Sci 2000; 30:167-173. 20. Bozbas H, Yildirir A, Muderrisoglu H. Cardiac enzymes, renal failure and renal transplantation. Clin Med Res 2006; 4 (1): 79-84. 21. Kim M, Park SW, Kim M, DAgati VD, Lee HT. Isoflurane activates intestinal sphingosine kinase to protect against bilateral nephrectomy induced liver and intestinal dysfunction. Anesthesiology 2006; 114 (2): 363-373. 22. William C, Ditting T, Yeelken R, Jacobl J. Cardiorenal connection: The role of Hypoxia and Oxidative Stress. In Studies on renal disorders, oxidative stress in applied basic research and clinical practices. First edition. Huntana press.2011. 23. Vardanian AJ, Busuttil RW, Kupiec-Weglinski JW. Molecular mediators of liver ischemia and reperfusion injury: a brief review. Mol Med 2008; 14: 337-345. 24. Semenza GL. Hypoxia-inducible factor 1: control of oxygen homeostasis in health and disease. Pediatr Res 2001; 49: 614-617. 25. Zethelius B, Johnson N, Venge P. Troponin I as a predictor of coronary heart disease and mortality in 70year-old men : a community based cohort study. Circulation 2006; 113: 1071-8.

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