Vol 1,issue 7 paper (5) page 26-31

International Journal of Medical Sciences and Health Care Vol-1 Issue-7 (Ijmshc-705)
http://www.ijmshc.com Page 30

ISCHEMIA REPERFUSION IN THE RATS KIDNEY MODULATE THE
PHYSIOLOGICAL FUNCTION OF SOME DISTAL ORGANS OF THE BODY
*Dallatu, Muhammad Kabiru¹; Bisong, Dickson², Rizvi, Yasmin3
, Erhabor Osaro 4
1. Department of Chemical Pathology, Faculty of Medical Laboratory Science Usmanu Danfodiyo University, Sokoto-Nigeria.
2. Department of Laboratory Services, Ben Taub Hospital Texas Medical Center, Houston TX USA
3. Center for Cardiovascular Diseases, College of Pharmacy and Health Sciences, Texas Southern University, Houston TX USA
4. 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 L-
mm (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 re-
oxygenation, 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 re-
oxygenation. 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-1α) 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-1α,
however, is under the enzymatic activity of prolyl
hydroxylase domain (PHD) enzyme, which hydroxylates
specific prolyl residues on HIF-1α for subsequent rapid
degradation of HIF-1α by proteasomes 6.
It becomes evident, that maneuvers to stabilize and
activate HIF-1α through PHD inhibition in one of the
promising ways to prevent injury and ultimately treat
ischemic insults.

Unpublished data from our laboratory 7
shows a
promising therapeutic effect of treatment with L-
mimosine, an inhibitor of PHD in the prevention of rat‟s
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 L-
mimosine 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
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 animal‟s body
temperature at 37ºC. 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
rat‟s 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).

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
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-1α 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-1α is
the main transcription factor involved in the regulation
of transcriptional responses to hypoxia. HIF-1α 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-1α 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-1α and subsequent downstream
protective actions.

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-1α through treatment with L-mm might in part, contribute to the observed protection. HIF-1α act
through repertoire of HIF-1α 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.

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.
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-1α activation
could decrease or even reverse progression of

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): 80-
85.
2. Hines RL, Marschall KE. Stoelting‟s 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 ischemia–reperfusion injury.
Cardiovasc Res 2005; 67 (4): 594-603.
5. Nangaku M, Tanaka T. Drug discovery for
overcoming chronic kidney disease (CKD): Prolyl-
hydroxylase 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 ischemia-
reperfusion 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.
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: 1486-
1494.
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 insufficiency-
review 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: 959-
969.
18. Loor G, Schumacker PT. Role of hypoxia-inducible
factor in cell survival during myocardial ischemia-
reperfusion. Cell Death Differ 2008; 15: 686-690.
19. Bennett AE, Bertholf RL. Discordant results of CK-
MB 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, D‟Agati 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 70-
year-old men : a community – based cohort study.
Circulation 2006; 113: 1071-8.

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