Journal of Forensic Science and Medicine

: 2018  |  Volume : 4  |  Issue : 1  |  Page : 1--6

An investigation of postmortem urotensin II receptor levels in brain and kidney tissues in a rat model of cardiac ischemia

Mustafa Talip Sener1, Erol Akpinar2, Elif Cadirci2, Zekai Halici2, Irfan Cinar2, Ahmet Nezih Kok1,  
1 Department of Forensic Medicine, Faculty of Medicine, Ataturk University, Erzurum 25240, Turkey
2 Department of Pharmacology, Faculty of Medicine, Ataturk University, Erzurum 25240, Turkey

Correspondence Address:
Erol Akpinar
Department of Pharmacology, Faculty of Medicine, Ataturk University, Erzurum 25240


This study aimed to investigate changes in postmortem urotensin II receptor (UTR) levels in brain and kidney tissues in a rat model of cardiac ischemia. The rats were divided into two groups: a control group and a cardiac ischemia-induced group. Cardiac ischemia was created by an intraperitoneal injection of a single lethal dose of isoproterenol (ISO; 850 mg/kg). Plasma UT, blood urea nitrogen, and creatinine levels were determined 0 h postmortem. Brain and kidney UTR mRNA expression levels were determined 0, 1, 3, 6, 12, 24, 48, and 72 h postmortem. The histopathological appearance of brain and kidney tissues was also evaluated. Plasma UT and plasma creatinine levels were increased in the cardiac ischemia-induced group as compared with those in the control group (P < 0.001). Ischemia resulted in histopathological changes in brain and cerebellum tissue. The morphological evaluation revealed Purkinje cell degeneration (P = 0.037) and dark neurons (P = 0.004). The UTR expression level decreased after 1 h postmortem in the brain and after 3 h postmortem in the kidneys in the cardiac ischemia-induced group as compared with that in the control group (P < 0.001). The observed changes in UTR expression levels may be valuable in clinical practice in the field of forensic medicine. These changes may be used as a marker in postmortem evaluations of sudden death caused by ischemia-induced cardiac shock.

How to cite this article:
Sener MT, Akpinar E, Cadirci E, Halici Z, Cinar I, Kok AN. An investigation of postmortem urotensin II receptor levels in brain and kidney tissues in a rat model of cardiac ischemia.J Forensic Sci Med 2018;4:1-6

How to cite this URL:
Sener MT, Akpinar E, Cadirci E, Halici Z, Cinar I, Kok AN. An investigation of postmortem urotensin II receptor levels in brain and kidney tissues in a rat model of cardiac ischemia. J Forensic Sci Med [serial online] 2018 [cited 2022 Dec 8 ];4:1-6
Available from:

Full Text


Ischemic heart disease is a leading cause of death, and 90% of unexpected adult deaths are due to atherosclerotic heart disease. [1] Acute myocardial infarctions (MIs) result in ischemic, mechanical, arrhythmic, embolic, and inflammatory complications. Deaths due to acute MIs are primarily caused by arrhythmic complications. Autopsy findings in deaths from acute MIs vary, depending on the duration and degree of the thromboembolism. If a specific time has not been established between the beginning of the disease and death, a pathological diagnosis of MI cannot be made. [2],[3],[4] Diagnosing the cause of death is often difficult in postmortem examinations of cases where there is no significant stenosis in the coronary arteries and where no biochemical markers are found in routine tests carried out in postmortem examinations of patients who died as a result of acute MIs. [5]

Urotensin II (UT) is a strong vasoconstrictor, which affects cardiac, renal, and vascular tissues. UT also has a vasodilatory effect on some veins and arteries (e.g., renal and mesenteric arteries). [6],[7] UT is expressed in several organs, such as heart, lung, liver, brain, and kidney. [8] Previous research reported that the plasma concentration of UT was low in healthy individuals but high in those with pathological conditions, such as acute MIs, coronary failure, cardiac shock, hypertension, atherosclerosis, kidney failure, diabetes, and liver diseases. [8],[9],[10],[11] Research also demonstrated that the level of this endogenous substance significantly increased in cardiac ischemia, as the pericardium was affected. [12],[13]

Although several studies have focused on various aspects of the UT and its receptor (UTR), the aforementioned have not been examined in postmortem studies of patients with pathological conditions, such as hypertension, atherosclerosis, kidney failure, diabetes, and liver diseases. In our previous study, we analyzed changes in the expression level of the UTR in postmortem heart tissue in cases of sudden deaths due to cardiac ischemia. [14] The UTR expression level was significantly high in the cardiac ischemia-related deaths as compared with that of a control group at both morgue (+4°C) and ambient (+20°C) temperatures in the first 48 h postmortem in heart tissue. [14] Information is lacking on UTR expression levels in tissues other than heart tissue in cases of deaths resulting from cardiac ischemia. UT is not specific to the heart in healthy individuals. It is not known how changes in UTR expression levels affect tissues other than the heart in cases of cardiac ischemia. In the present study, we aimed to determine changes in UTR expression levels in brain and kidney tissues during the postmortem process in cases of death from cardiac ischemia and to determine whether UTR expression levels can be employed as a marker in such cases.


Experimental animals and groups

In total, 96 male albino Wistar rats (220-230 g) were obtained from the Experimental Animal Laboratory of Medicinal and Experimental Application and Research Center of Ataturk University, Turkey. The rats were kept on sawdust bedding in standard plastic cages in a well-ventilated room at 22°C under a 14/10 h light/dark cycle.

The rats were randomly divided into two groups: A cardiac ischemia-induced group (n = 48) and a control group (n = 48). UTR levels in each group were determined 0, 1, 3, 6, 12, 24, 48, and 72 h postmortem (n = 6 at each time point). To induce cardiac ischemia, isoproterenol (ISO) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) was dissolved in normal saline solution and administered as a single intraperitoneal dose of 850 mg/kg. [14] Following the ISO injection, all the rats died within approximately 30 min. The rats in the control group received only an injection of saline (0.9%) and were sacrificed using the cervical dislocation method.

At the point of death (0 h), blood samples were collected. At all time points (0, 1, 3, 6, 12, 24, 48, and 72 h), brain and kidney samples were collected and stored at −80°C for biochemical and molecular analyses and in 10% formalin for histopathological analysis. After death, all the experimental animals were maintained in a closed cabin, with relative humidity of 40 ± 3% and a stable temperature of 20°C.

Histopathological analysis

Brain, cerebellum, and kidney tissues were fixed in a 10% buffered formalin solution, following routine tissue processing. Then, 4 mm-thick slices were cut from paraffin blocks and stained with hematoxylin and eosin. The stained sections were microscopically evaluated using a light microscope, with a camera attachment (Nikon Eclipse Ni). All lesions were photographed. In the morphological analysis, acute neuronal damage (dark neurons) in brain tissue and Purkinje cell reactions in cerebellar tissue were recorded. In kidney tissue, the glomerulus structure and cells, as well as the tube structure and epithelial cells, were examined. Numbers of dark neurons and Purkinje cells in the parenchyma were counted under Χ10 magnification.

Molecular analyses of kidney and brain tissues

Total RNA extraction and cDNA synthesis

Kidney and brain tissues (20 mg) were stabilized in an RNA stabilization reagent (RNA later, Qiagen, Hilden, Germany) and then disrupted using TissueLyser II (Qiagen) (2 Χ 2 min for kidney; 2 Χ 5 min). Total RNA was purified using an RNeasy Mini Kit (Qiagen) in a QIAcube (Qiagen), according to the manufacturer's instructions. The RNA samples were then reverse transcribed into complementary DNA using a high-capacity cDNA reverse transcription (RT) kit (Applied Biosystems, Foster city, CA). Then, 10 ml of total RNA was treated with 2 ml of 10X RT buffer, 0.8 ml of 25X dNTPs mix, 2 ml of 10X RT random primers, 1 ml of multiscribe RT, and 4.2 ml of DEPC-H 2 O (ultrapure, 0.1% diethylpyrocarbonate). RT was carried out at 25°C for 10 min, followed by 120 min at 37°C and finally 5 min at 85°C, using a Veriti 96-well thermal cycler (Applied Biosystems). The concentration and quality of the cDNA were assessed and quantified using an epoch spectrophotometer system and take 3 Plate (BioTek Inc., Winooski, VT).

Relative quantification of gene expression by the real-time reverse transcriptase-polymerase chain reaction

UTR expression in synthesized cDNA from rat kidney and brain RNA was analyzed using a step one plus real-time polymerase chain reaction (PCR) system (Applied Biosystems). A quantitative PCR was run using a TaqMan probe mix based on TaqMan probe-based technology (Applied Biosystems). The real-time PCR was performed using primers generated for rat UTR and rat b-actin. [15] The results were expressed as relative fold, compared with control animals. The expression data for b-actin in each tissue were used as an endogenous control. For each tissue, triplicate determinations were performed in a 96-well optical plate for targets using 9 ml of cDNA (100 ng), 1 ml of primer perfect probe mix, and 10 ml of QuantiTect Probe PCR master mix (Qiagen, Hilden, Germany) in each 20 ml reaction. The plates were heated for 2 min at 50°C and then for 10 min at 95°C. Subsequently, 40 cycles of 15 s at 94°C and cycles of 60 s at 60°C were performed. All data are expressed as the fold change in expression compared to the expression in the control group, using the 2−DDCt method. [16] The sequences of the primers used for the real-time PCR are given in [Table 1].{Table 1}

Biochemical analysis of urotensin levels

Plasma UT level was measured with a rat-UT enzyme-linked immunosorbent assay kit (SUNRED, Shanghai, PRC). All the reagents, samples, and standards were prepared by the addition of 50 ml of standard and 40 ml of sample to individual wells. Samples were then added to all the wells, except for the blank and standards to the 10 ml of UT antibody. They were added to all the wells, except for the 50 ml blank in streptavidin horseradish peroxidase. The wells were then covered and incubated at 37°C for 60 min. The wells were aspirated and cleaned 5 times. Chromogen A (50 ml) and Chromogen B (50 ml) were added to each well and incubated in the dark at 37°C for 10 min. The Chroma of the color and concentration of rat UT of the sample were positively correlated. After incubation, 50 ml of stop solution was added, and absorbance of the sample was taken at 450 nm.

Statistical analysis

GraphPad Prism; version 5.00 (San Diego, CA, USA) and the Statistical Package for the Social Sciences (SPSS), version 20.0 (IBM, SPSS, Inc., Chicago, IL, USA), programs were used for the statistical analysis. Analysis of the distribution and homogeneity of variances in each group showed that the data were normally distributed. UTR expression levels between groups were compared using a repeated-measures analysis of variance. Dark neurons and Purkinje cells in the parenchyma were counted under a Χ10 objective. The Mann-Whitney U-test was performed for comparisons of mean values of histopathological findings, comparisons of mean values of biochemical parameters, and comparisons of differences between averages. P < 0.05 was considered statistically significant.


Biochemical findings

Although there was no significant between-group difference in plasma blood urea nitrogen (BUN) values 0 h postmortem, creatinine levels were 3 times higher in the cardiac ischemia-induced group as compared with those in the control group [Table 2]. In the control group, the plasma UT level was too low to measure 0 h postmortem, whereas it was increased 7 folds in the cardiac ischemia group [Figure 1].{Figure 1}{Table 2}

Urotensin II receptor expression level

In brain tissue, the UTR expression level was relatively low in the cardiac ischemia-induced group as compared with that in the control group [Figure 2]. As shown in [Table 3], there was a statistically significant difference in UTR expression levels 1, 3, 6, 24, 48, and 72 h postmortem (P < 0.001). In kidney tissue, the UTR expression level was low at all time points in the cardiac ischemia-induced group [Figure 3], but there was a statistically significant difference in the expression level 6 and 12 h postmortem (P < 0.001), as shown in [Table 4].{Figure 2}{Figure 3}{Table 3}{Table 4}

Histopathological findings

In the histopathological analysis, all hours were analyzed. Findings 0 h postmortem are shown in the figures. As compared with the control group, an increase in dark neuron structures in the brain and Purkinje cell degeneration in cerebellum tissues were observed in the cardiac ischemia-induced group [Figure 4]a, b and [Figure 5]a, b. [Table 5] presents the histomorphological differences in the brain and cerebellum cells. In brain and cerebellum tissue, increased degeneration of Purkinje cells (P = 0.037) and dark neurons (P = 0.004) were observed in the cardiac ischemia-induced group. In kidney tissues, no histomorphological differences were observed in either the cardiac ischemia-induced group or control group [Figure 6]a and b. In brain tissue, autolytic changes, such as deterioration of fibril morphology, general loss of the nucleus of neurons, and vacuolization, were observed postmortem. Deterioration of the morphology of the proximal and distal tubular epithelium and glomerular capillary network, in addition to vacuolization in kidney tissue, were evident at all postmortem hours in both groups.{Figure 4}{Figure 5}{Figure 6}{Table 5}


This study investigated whether brain and kidney UTR mRNA expression levels changed in the postmortem process after sudden death due to cardiac ischemia. As is well known, the temperature of the environment, pH, proteolytic enzymes, and bacteria tissues influence tissues and peptide receptor stability postmortem. The results of the present study showed that peptide receptors remained stable for at least 16 h postmortem. Of the tissue types analyzed, the stability of peptide receptors in brain tissues was the most sensitive. [17] In the present study, we analyzed differences in UTR expression in brain and kidney tissues at different hours postmortem. It is possible that autolysis may have affected our results. However, as little autolytic destruction occurs during the early stages of the postmortem process, we believe that the impact of autolysis was minimal.

Previous studies showed that plasma UT levels were elevated in cardiovascular disorders, such as ischemic or nonischemic heart failure, and that increases in plasma UT levels played a significant role in changing blood pressure. [12],[18],[19] Contrary to these studies, other studies found decreased plasma UT concentrations in patients with acute MIs and suggested that such decreases may be associated with more severe injury of the myocardium. [20],[21] In this study, we observed a marked increase in the plasma UT level in the cardiac ischemia group immediately after death. The increased level of UT in the systemic circulation may be a compensatory mechanism to protect vascular tonus during ischemia induced.cardiogenic shock just before death.

The kidney is a major site of UT in mammals. The parent compound pre-pro UT is highly expressed in human kidneys. [22] UT helps to regulate kidney function through reflex control of the glomerular filtration rate, and it exerts effects on blood pressure by acting as a vasodilator and natriuretic in the kidneys. [23] It The plasma UT level is elevated in renal failure, congestive heart failure, diabetes mellitus, systemic hypertension, and portal hypertension caused by liver cirrhosis. According to previous research, UT may have a cardioprotective effect and play an important role in cardiovascular regulation. [24] The effects of UT on the cardiovascular system are controlled by UT in the central nervous system. [25] UT is found in many organs other than the kidney (e.g., hippocampus, thalamus, hypothalamus, pineal gland, pituitary gland, pons, medulla, and spinal cord). UT also plays an important role as a neuromodulator or neurotransmitter in the brain. In the present study, although plasma UT levels increased postmortem, there were no differences in UTR expression levels from 1 h to 3 h postmortem in the brain and kidney tissues of either group. This finding may be due to the short response time to increases in plasma UT levels. After 3 h postmortem, brain and kidney UTR expression levels decreased, and plasma UT levels increased, leading to downregulation and lysis of the receptor. [20]

In the present study, the histomorphological analysis of brain tissue revealed significant ischemic-induced changes. These changes can be explained by the high sensitivity of brain tissue to ischemia. [26] In contrast, the histomorphological analysis of kidney tissue revealed no ischemic-related changes in the early stages of the postmortem process. The latter finding can be explained by the resistance of kidney tissue to ischemia. [27] An increase in plasma UT levels may cause renal vasodilation, leading to kidney perfusion. [28],[29] A sudden loss in acute kidney functions can be identified by increased BUN and serum creatinine levels. According to RISK (risk, injury, failure, loss of kidney function, and end-stage kidney disease) criteria, a 50% increase in serum creatinine levels carries some risk of acute kidney failure. [30] We observed no ischemia-induced changes in kidney tissues in the histopathological analysis. The increase detected in plasma creatinine was evaluated, as it was not enough protected besides compensatory mechanisms of the kidneys in response to cardiogenic shock.

A previous study reported that the UTR expression level was high in heart tissue in a postmortem cardiac ischemia model in the first 48 h, at both +4°C and +20°C. [14] In the present study, we found no increase in the UTR expression level in brain and kidney tissues, and it remained low. Although UT is not specific to the heart, these findings indicate that the UTR expression level may be a significant marker of sudden death due to cardiac ischemia.


Plasma UT and plasma creatinine levels increased in rats in the postmortem period following sudden death due to cardiac ischemia, and plasma UT expression level decreased in other organs, such as the brain and kidney. Changes in the UTR expression level may serve as a marker of sudden death due to cardiac ischemia.

Compliance with ethical standards

Ethics approval: All applicable international guidelines for the care and use of animals were followed. The animal experiments were approved by the local animal care committee (No. 93/26.05.2016) of Ataturk University.


This study was part of a dissertation titled "Investigation of postmortem UTR and endothelin 1 levels in brain and kidney tissues in rats that died of ISO toxicity," which was supported by the Ataturk University Scientific Research Project (BAP 2014/036). The authors gratefully acknowledge financial support from the Ataturk University Scientific Research Council.

Financial support and sponsorship

This study was financially supported by the Ataturk University Scientific Research Council with project number "BAP 2014/036.

Conflicts of interest

There are no conflicts of interest.


1Fragkouli K, Vougiouklakis T. Sudden cardiac death: An 11-year postmortem analysis in the region of Epirus, Greece. Pathol Res Pract 2010;206:690-4.
2Chappex N, Schlaepfer J, Fellmann F, Bhuiyan ZA, Wilhelm M, Michaud K, et al. Sudden cardiac death among general population and sport related population in forensic experience. J Forensic Leg Med 2015;35:62-8.
3Polacco M, Sedati P, Arena V, Pascali VL, Zobel BB, Oliva A, et al. Visualization of myocardial infarction by post-mortem single-organ coronary computed tomography: A feasibility study. Int J Legal Med 2015;129:517-24.
4Schwendener N, Jackowski C, Persson A, Warntjes MJ, Schuster F, Riva F, et al. Detection and differentiation of early acute and following age stages of myocardial infarction with quantitative post-mortem cardiac 1.5T MR. Forensic Sci Int 2017;270:248-54.
5Campobasso CP, Dell'Erba AS, Addante A, Zotti F, Marzullo A, Colonna MF, et al. Sudden cardiac death and myocardial ischemia indicators: A comparative study of four immunohistochemical markers. Am J Forensic Med Pathol 2008;29:154-61.
6Wu F, Chen G, Zhang A, Yu Y, Fan M, Tang C, et al. Renal urotensin II system plays roles in the regulation of blood pressure in dahl salt-resistant rat. Int J Hypertens 2016;2016:9146870.
7Balat A, Büyükçelik M. Urotensin-II: More than a mediator for kidney. Int J Nephrol 2012;2012:249790.
8Ross B, McKendy K, Giaid A. Role of urotensin II in health and disease. Am J Physiol Regul Integr Comp Physiol 2010;298:R1156-72.
9Zhu YC, Zhu YZ, Moore PK. The role of urotensin II in cardiovascular and renal physiology and diseases. Br J Pharmacol 2006;148:884-901.
10He WY, Chen GJ, Lai X, Wu F, Tang CS, Zhang AH, et al. Expression levels of urotensin II are associated with endoplasmic reticulum stress in patients with severe preeclampsia. J Hum Hypertens 2016;30:129-35.
11Porras-González C, Ureña J, Egea-Guerrero JJ, Gordillo-Escobar E, Murillo-Cabezas F, González-Montelongo Mdel C, et al. Contractile responses to rat urotensin II in resting and depolarized basilar arteries. J Physiol Biochem 2014;70:193-9.
12Chuquet J, Lecrux C, Chatenet D, Leprince J, Chazalviel L, Roussel S, et al. Effects of urotensin-II on cerebral blood flow and ischemia in anesthetized rats. Exp Neurol 2008;210:577-84.
13Tian L, Li Y, Hua W, Jia Y, Zhou M, Gu Y, et al. Expression of urotensin II during focal cerebral ischemic in diabetic rats. Can J Neurol Sci 2014;41:498-503.
14Sener MT, Karakus E, Halici Z, Akpinar E, Topcu A, Kok AN, et al. Can early myocardial infarction-related deaths be diagnosed using postmortem urotensin receptor expression levels? Forensic Sci Med Pathol 2014;10:395-400.
15Cadirci E, Halici Z, Yayla M, Toktay E, Bayir Y, Karakus E, et al. Blocking of urotensin receptors as new target for treatment of carrageenan induced inflammation in rats. Peptides 2016;82:35-43.
16Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402-8.
17Reynolds LM, Reynolds GP. Differential regional N-acetylaspartate deficits in postmortem brain in schizophrenia, bipolar disorder and major depressive disorder. J Psychiatr Res 2011;45:54-9.
18Ashton N. Renal and vascular actions of urotensin II. Kidney Int 2006;70:624-9.
19Song W, Abdel-Razik AE, Lu W, Ao Z, Johns DG, Douglas SA, et al. Urotensin II and renal function in the rat. Kidney Int 2006;69:1360-8.
20Joyal D, Huynh T, Aiyar N, Guida B, Douglas S, Giaid A, et al. Urotensin-II levels in acute coronary syndromes. Int J Cardiol 2006;108:31-5.
21Babiñska M, Holecki M, Prochaczek F, Owczarek A, Kokociñska D, Chudek J, et al. Is plasma urotensin II concentration an indicator of myocardial damage in patients with acute coronary syndrome? Arch Med Sci 2012;8:449-54.
22Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N, et al. Co-expression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 2001;19:2185-90.
23Abdel-Razik AE, Balment RJ, Ashton N. Enhanced renal sensitivity of the spontaneously hypertensive rat to urotensin II. Am J Physiol Renal Physiol 2008;295:F1239-47.
24Ong KL, Lam KS, Cheung BM. Urotensin II: Its function in health and its role in disease. Cardiovasc Drugs Ther 2005;19:65-75.
25Vaudry H, Leprince J, Chatenet D, Fournier A, Lambert DG, Le Mével JC, et al. International union of basic and clinical pharmacology. XCII. Urotensin II, urotensin II-related peptide, and their receptor: From structure to function. Pharmacol Rev 2015;67:214-58.
26Sanderson TH, Reynolds CA, Kumar R, Przyklenk K, Hüttemann M. Molecular mechanisms of ischemia-reperfusion injury in brain: Pivotal role of the mitochondrial membrane potential in reactive oxygen species generation. Mol Neurobiol 2013;47:9-23.
27Rodriguez F, Bonacasa B, Fenoy FJ, Salom MG. Reactive oxygen and nitrogen species in the renal ischemia/reperfusion injury. Curr Pharm Des 2013;19:2776-94.
28Zhang AY, Chen YF, Zhang DX, Yi FX, Qi J, Andrade-Gordon P, et al. Urotensin II is a nitric oxide-dependent vasodilator and natriuretic peptide in the rat kidney. Am J Physiol Renal Physiol 2003;285:F792-8.
29Gardiner SM, March JE, Kemp PA, Bennett T. Bolus injection of human UII in conscious rats evokes a biphasic haemodynamic response. Br J Pharmacol 2004;143:422-30.
30Van Biesen W, Vanholder R, Lameire N. Defining acute renal failure: RIFLE and beyond. Clin J Am Soc Nephrol 2006;1:1314-9.