Atorvastatin Protects Kidney from Remote Reperfusion Injury

Accepted Manuscript Atorvastatin Protect Kidney From Remote Reperfusion Injury Carlos Henrique Marques dos Santos, Doroty Mesquita Dourado, Baldomero Antonio Kato da Silva, Henrique Budib Dorsa Pontes, Euler de Azevedo Neto, Giovanna Serra da Cruz Vendas, Ian de Oliveira Chaves, João Victor Cunha Miranda, João Victor Durães Gomes Oliva, Letícia do Espirito Santo Dias, Murillo Henrique Martins de Almeida, Trícia Luna Sampaio

PII:

S0890-5096(17)30944-5

DOI:

10.1016/j.avsg.2017.07.031

Reference:

AVSG 3529

To appear in:

Annals of Vascular Surgery

Received Date: 9 May 2017 Accepted Date: 22 July 2017

Please cite this article as: Marques dos Santos CH, Dourado DM, Kato da Silva BA, Dorsa Pontes HB, de Azevedo Neto E, Serra da Cruz Vendas G, de Oliveira Chaves I, Cunha Miranda JV, Durães Gomes Oliva JV, Dias LdES, Martins de Almeida MH, Sampaio TL, Atorvastatin Protect Kidney From Remote Reperfusion Injury, Annals of Vascular Surgery (2017), doi: 10.1016/j.avsg.2017.07.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Atorvastatin protect kidney from remote reperfusion injury.

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Carlos Henrique Marques dos Santos1, Doroty Mesquita Dourado1, Baldomero

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Antonio Kato da Silva2, Henrique Budib Dorsa Pontes1, Euler de Azevedo Neto1,

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Giovanna Serra da Cruz Vendas1, Ian de Oliveira Chaves1, João Victor Cunha

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Miranda1, João Victor Durães Gomes Oliva1, Letícia do Espirito Santo Dias1, Murillo

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Henrique Martins de Almeida1, Trícia Luna Sampaio1

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Grande-MS-Brazil, 79003-026

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– Anhanguera-Uniderp University – Rua Nova Era, 480 - Itanhangá Park, Campo

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– Federal University of Piauí – Av. São Sebastião, 2819, Parnaiba-PI-Brazil-

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Corresponding author

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Carlos Henrique Marques dos Santos

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Rua Nova Era, 480, Campo Grande-MS-Brazil- CEP79003026

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Tel 55-67-33488200

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[email protected]

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ACCEPTED MANUSCRIPT ABSTRACT

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BACKGROUND: There is a need to find an effective treatment against reperfusion

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injury. The aim of the present study was to evaluate the capacity of the ischemic

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postconditioning and statin to prevent renal reperfusion injury.

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DESIGN AND SETTING: An experimental study developed at Universidade

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Anhanguera-Uniderp.

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METHODS: A total of 41 Wistar rats were distributed into 5 groups: ischemia and

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reperfusion (I/R), ischemic postconditioning (IPC), postconditioning + statin (IPC+S),

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statin (S) and Sham. In the Sham group the infra-renal abdominal aorta was

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dissected and isolated; all others were submitted to aortic clamping for 70 minutes

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(ischemia) and posterior removal of the clamp (reperfusion, 70 minutes). In the IPC

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and IPC+S groups, postconditioning was performed in ischemia and reperfusion

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phases by four cycles of reperfusion and ischemia lasting 30 seconds each. In the

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IPC+S and S groups, preceding the surgical procedure, atorvastatin was

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administered 3.4mg/day for seven days by gavage. After the procedure, the left

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kidney was removed for histological study.

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RESULTS: The mean renal lesion was 4 in the I/R group, 2.44 in the IPC group, 1.22

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in the IPC+S group, 1.11 in the S group, and 1 in the Sham group. The I/R group had

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a higher degree of tissue injury when compared to the others (p<0.001) and the

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IPC+S and S groups improved protection against IPC alone (p<0.05).

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CONCLUSION: Ischemic postconditioning and atorvastatin can minimize renal

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remote reperfusion injury.

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Keywords: ischemia; reperfusion; ischemic postconditioning; Statins of HMG-CoA;

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kidney.

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INTRODUCTION

Reperfusion is a fundamental step in the treatment of ischemia. However, clinical and experimental evidences shows that the main events leading to cell and

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tissue dysfunction are related to reperfusion1.

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The ischemia and reperfusion (IR) injury constitutes a pathophysiological event common to several diseases of daily clinical practice. The kidney can be the

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target of the IR lesion directly, as in transplantation or renal surgery, or be reached at

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a distance, as in cases of shock or reperfusion injury in many organs, as occurs in

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the aortic clamping, used in aneurysm surgeries2.

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Some techniques for protection against reperfusion injury have already been

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tried and tested, and among them, ischemic postconditioning (IPC), which consists of

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one or more short cycles of reperfusion, followed by one or more short cycles of

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ischemia, immediately after the ischemic phase and before permanent reperfusion

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occurs. The IPC has already demonstrated its efficacy even in the kidney, and there

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is even a recent meta-analysis demonstrating its effectiveness in renal IR situations3,

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however, its efficacy in the prevention of remote renal injury is still poorly

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understood4.

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Much has been studied about the pathophysiology of reperfusion injury and

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some mechanisms have already been well evidenced such as the role of free

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radicals, vascular endothelial dysfunction, and neutrophil-mediated injury1. Recently,

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there has been an increase in interest in statins, drugs known for their

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antidislipidemic effect, this time due to its pleiotropic effect, which is characterized by

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anti-inflammatory properties, immunomodulating, antithrombogenic actions and

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improvement of endothelial function5. Recent experimental studies6 have shown 3

ACCEPTED MANUSCRIPT promising results with the use of statins demonstrating their role in the protection

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against IR injury, a fact that led us to inquire about their benefits facing reperfusion

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injury, the objective of this study being to evaluate the capacity of IPC associated

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with the use of statins in reducing the tissue injury of the renal parenchyma.

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The aim of the present study was to evaluate the effect of ischemic

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postconditioning and atorvastatin, alone and in combination, on the prevention of

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reperfusion injury in the kidney of rats subjected to ischemia and reperfusion by

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aortic clamping.

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MATERIAL AND METHODS

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The

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Experimentation of the University Anhanguera-Uniderp. A total of 41 Wistar norvergic

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male rats weighing 250g to 300g were collected from the Anhanguera-Uniderp

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University Animal Hospital. The animals were kept in cages at ambient temperature

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of approximately 23°C with 12h light cycles and rec eived water and feed ad libitum.

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The animals were distributed in the following groups:

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- Ischemia and Reperfusion Group (I/R): Nine rats were submitted to ischemia for 70

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minutes by aortic clamping (infra-renal aorta), followed by reperfusion of 70 minutes.

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- Ischemic Postconditioning Group (IPC): Nine rats were submitted to the ischemia

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procedure for 70 minutes by aortic clamping and reperfusion for 70 minutes. Between

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ischemia and reperfusion, four cycles of reperfusion (30 seconds each) were

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performed, interspersed by four cycles of ischemia (30 seconds each).

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ACCEPTED MANUSCRIPT - Ischemic Postconditioning + Statin Group (IPC+S): Nine rats received 3.4mg/day of

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atorvastatin, one dose per day through the gavage method, for seven days and then

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were submitted to the ischemia procedure for 70 minutes by aortic clamping and

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reperfusion for 70 minutes. Between ischemia and reperfusion, four cycles of

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reperfusion (30 seconds each) were performed, interspersed by four cycles of

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ischemia (30 seconds each).

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- Statin Group (S): Nine rats received 3.4mg/day of atorvastatin, one dose per day

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through the gavage method, for seven days, and then were subjected to the ischemia

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procedure for 70 minutes by aortic clamping and reperfusion for 70 minutes.

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- SHAM Group - Five rats submitted to laparotomy, dissection and isolation of infra-

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renal aorta.

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The animals were anesthetized by intraperitoneal injection of a 2:1 solution of

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Cetamine Hydrochloride (Cetamin®), 50mg/mL, and Xylasine Hydrochloride

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(Xilazin®), 20mg/mL, respectively, at a dose of 0.1mL/100g.

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After anesthesia, the rats were submitted to median longitudinal laparotomy

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of approximately four centimeters, exteriorization of the small intestine, identification

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and dissection of infra-renal abdominal aorta.

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In all groups, except SHAM, the abdominal aorta was occluded by atraumatic

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vascular clamp that remained for 70 minutes (ischemia phase). After clamp

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placement, the small intestine was repositioned into the abdominal cavity and the

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surgical wound was closed with continuous suture of the skin with 4-0 monofilament

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nylon. After the ischemia phase, the abdominal wall was reopened by removal of the

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suture and in the I/R and S groups the vascular clamp was removed, initiating the

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reperfusion phase, lasting 70 minutes. In the IPC and IPC+S groups, preceding the 5

ACCEPTED MANUSCRIPT reperfusion phase, the ischemic postconditioning was performed by four cycles of

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reperfusion (removal of the atraumatic vascular clamping of the abdominal aorta)

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with duration of 30 seconds each, interspersed by four cycles of ischemia (occlusion

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of the abdominal aorta by atraumatic vascular clamp), also with duration of 30

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seconds each.

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In all groups after the beginning of the reperfusion phase, the abdomen was

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closed again by continuous suturing of the skin with 4-0 monofilament nylon thread

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until the end of the experiment.

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In the SHAM group, only a median longitudinal laparotomy of approximately

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four centimeters was performed, exteriorization of the small intestine, identification

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and dissection of infra-renal abdominal aorta.

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After the reperfusion phase, all animals were submitted to left kidney

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resection, and these specimens were washed with saline solution and placed in 10%

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formaldehyde solution for histological analysis.

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Euthanasia was performed by intraperitoneal administration of a lethal dose of cetamine and xylazine hydrochloride (0.4mL/100g).

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Slides were prepared with the harvested material, which were stained with

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hematoxylin-eosin and analyzed by optical microscopy by a single observer, without

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prior knowledge of which group the rat belonged to.

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The specimens were classified according to tissue injury degree stablished

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by Shih et al7. This is a classification of kidney injury initially described for nephritis,

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however, it has been used for IR injury analysis and cited by several authors for this

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purpose.

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- Grade 0 (normal): normal kidney. 6

ACCEPTED MANUSCRIPT - Grade 0.5: small focal areas of cellular infiltration and tubular damage.

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- Grade 1: involvement of less than 10% of the renal cortex.

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- Grade 2: involvement of 10%-25% of the renal cortex.

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- Grade 3: involvement of 25%-75% of the renal cortex.

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- Grade 4: involvement of more than 75% of the renal cortex.

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After the analysis of the data, the results were submitted to statistical

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treatment, using the Kruskal-Wallis non-parametric test, considering p<0.05.

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RESULTS

The averages of degrees of tissue injury were 4 in the I/R group,

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2.44 in the IPC group, 1.22 in the IPC+S group, 1.11 in the S group, and 1 in the

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SHAM group (Table 1 and Figure 1).

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Table 1. Degree of histopathological lesion in the renal parenchyma in rats per group.

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Figure 1: Comparison of the medians of the degrees of renal injury among the

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different groups analyzed (Kruskal-Wallis; p=0.0001; "a" p<0.001 in

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relation to I/R group, "b" p<0.05 in relation to IPC group).

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As can be observed, the method used for ischemia and reperfusion

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with infra-renal aorta clamping was effective in causing the distal injury in the kidney,

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since the histological analysis showed lesion degree 2.44, with statistical difference

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(p<0.001) when compared to the group SHAM, in which we would not expect injury. 7

ACCEPTED MANUSCRIPT Once the lesion was found in the control group, it was possible to

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verify the efficacy of the groups in which treatment was used. All treatment groups

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had a lower degree of lesion than control, demonstrating the efficacy of the IPC and

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statin.

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The IPC group had a lower lesion than the control group, confirming

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the method's effectiveness, according to other studies already published. The most

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relevant information is undoubtedly that of the statin group, presenting a lower

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degree of injury when compared to the control group (p<0.001)) and the IPC group

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(p<0.05), ie, if we had a treatment capable of minimizing the distance injury in the

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kidney such as the IPC, we now have a more effective and easier to apply method,

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since it can be administered orally.

The results also show that there was no advantage in the use of

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statins associated with IPC.

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The main histological findings observed in the different groups can

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be observed in figure 2.

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Figure 2 - Photomicrographs of histological changes of the kidney considering the

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classification of Shih et al. Focal edema (fe); glomerular atrophy (ga); tubular

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congestion (tc); necrosis of tubular cells (white arrows); hydropic degeneration (black

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arrows); medular congestion (mc); HE, 10x and 40x.

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DISCUSSION

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Ischemia followed by reperfusion may induce apoptosis and an inflammatory

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response that affects tissue repair. As a result, many have evaluated the impact of 8

ACCEPTED MANUSCRIPT IPC on subsequent apoptotic and inflammatory responses. In experimental IR

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models in rats with 30 minutes of ischemia and three hours of reperfusion there was

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a significant decrease in tissue necrosis with IPC. There is also a decrease in

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reactive oxygen species (ROS) generation and protection of mitochondrial integrity,

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suggesting that the protective effect of IPC may be the result of a reduction in the

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inflammatory response. However, few studies have directly assessed the impact of

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IPC on inflammation. IPC may limit the expression of P-selectin, which is required for

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neutrophil bearing and its recruitment. In addition, it may reduce the accumulation of

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neutrophils in the affected region, decrease adhesion to ischemic vascular

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endothelium, and attenuate the endothelial dysfunction of the involved vessel, events

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that normally occur in IR8.

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In the present study, renal protection was observed with IPC, demonstrating the

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efficacy of the method against this IR model. Once produced, ROS spread

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throughout the body, causing local and remote injury, so that a method capable of

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controlling ROS production can protect the directly affected organ as well distant

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organs.

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The study of the remote reperfusion injury on the kidney is a relatively new

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subject, therefore, lacking publications that allow us to further compare with the

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present results. Jiang et al.4 also observed IPC efficacy in renal IR, but this was not

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an evaluation study of its remote effect as presented here, since those authors

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performed the IR directly on the renal hilum.

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Jonker et al.3 recently published a meta-analysis in which they researched

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articles on renal IPC. They found four publications about the remote effect of IPC in

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the kidneys and confirmed the efficacy of the method also in this situation, as

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observed in this publication. 9

ACCEPTED MANUSCRIPT Thus, it is clear the efficacy of IPC in renal local or remote IR, however, there is

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great interest in the research of drugs that can attenuate the reperfusion injury,

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especially well-known and safe drugs, exactly the case of statins and that at least

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their pleiotropic effect could be useful in such situations.

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In the present study renal protection was obtained with the use of atorvastatin,

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more than IPC alone. As there are no studies with the same design used here, i.e.

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aortic clamping and atorvastatin use, the comparison with the literature is also

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impaired. Moreover, since the use of statins for the prevention of reperfusion injury is

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relatively new, the best route of administration and ideal dose are items to be better

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clarified in future research. It has been chosen here administration by gavage with

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the intention of simulating what is practiced in humans, that is, the absorption by the

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gastrointestinal tract, aiming its clinical applicability.

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Statins have been successfully tested for this purpose in several situations.

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Wu et al.9 performed renal IR in rats and demonstrated that atorvastatin decreased

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the tissue injury in the control group. The same results were obtained by Cusomano

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et al.10 in renal IR of rats using atorvastatin, but in both studies, despite the good

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results, the statin was not investigated for its remote effect, since these authors

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performed IR in the renal hilum.

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The statins also protect other tissues in the presence of IR such as heart11-13,

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nervous system14 and liver15. In the lung, the efficiency of statins was also

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demonstrated, as published by Matsuo et al.16, however, with a method different from

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the one used here, since these authors performed IR directly in the pulmonary hilum,

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thus not being a study of remote protection. In addition, these authors used

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rosuvastatin as a protective medicine and not atorvastatin as in the present research.

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ACCEPTED MANUSCRIPT The mechanism of protection of statins to IR situations is due to its pleiotropic

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effect. By inhibiting a HMG-CoA conversion to L-mevalonate, such as statins prevent

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a synthesis of isoprenoids, which are precursors of cholesterol biosynthesis, which

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serve as important lipid ligands for post-translational modification of intracellular

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proteins such as small GTPases, Rho , Rac and Ras. This protein isoprenylation

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allows a suitable subcellular localization and an intracellular circuit of proteins, which

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control various cellular functions, and an inhibition and pathways are important

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components of the pleiotropic effects of statins. The Rho pathway is related to

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oxidative stress, atherosclerosis and elevated blood pressure. Signaling the pathway

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between the two crucial mechanisms, such as cytoskeletal remodeling and the ROS

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synthesis10.

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In the development of this project we did not know that the therapeutic

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methods applied would present the results showed here, so that an association

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group (IPC+S) was created aiming at enhancing tissue protection. The use of statin

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offered greater remote renal protection than the IPC, and there was also no

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advantage in the association of the two therapeutic methods, since the results were

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similar in the IPC+S and S groups, thus demonstrating in this model of renal distant

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injury that atorvastatin presents promising role as pharmacological protection against

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reperfusion injury.

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We do not have publications that have used atorvastatin in preventing

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reperfusion injury in humans, but this study may be a starting point for such research.

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It may be asked whether individuals who are on continuous use of statins for other

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reasons would be better protected from remote reperfusion injury, which needs to be

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scientifically confirmed. Another question that arises is whether, in acute ischemia 11

ACCEPTED MANUSCRIPT situation, when reperfusion occurs, the use of statins could minimize local and distant

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lesions. Our research has shown its efficacy in such situation, but with previous use

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of the drug, and not in the act of reperfusion. These are still obscure situations, but

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we believe that, based on the good results of atorvastine shown here, further

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research can be initiated to clarify such questions.

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CONCLUSION

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Atorvastatin and postconditioning were able to protect against kidney reperfusion

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injury in rats submitted to ischemia and reperfusion by aortic clamping, and

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atorvastatin presented more pronounced protection than postconditioning.

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REFERENCES

1. Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: new

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strategies for cardioprotection. Diabetes Obes Metab 2008;10(6):451-9. 2. Sotoudeh A, Takhtfooladi MA, Jahanshahi A, et al. Effect of N-acetylcysteine on

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histopathological study in rat model. Acta Cir Bras 2012;27(2):168-171.

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skeletal

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ischemia-reperfusion:

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3. Jonker SJ, Menting TP, Warlé MC, Ritskes-Hoitinga M, Wever KE. Preclinical

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evidence for the efficacy of ischemic postconditioning against renal ischemia-

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reperfusion injury, a systematic review and meta-analysis. PLoS One 2016;11(3):e0150863.

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4. Jiang H, Chen R, Xue S, et al. Protective effects of three remote ischemic

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conditioning procedures against renal ischemic/reperfusion injury in rat kidneys:

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a comparative study. Ir J Med Sci 2015;184(3):647-53.

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5. Tuttolomondo A, Di Raimondo D, Pecoraro R, et al. Early High-dosage

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10. Cusumano G, Romagnoli J, Liuzzo G, et al. N-Acetylcysteine and high-dose

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kidney. Transplant Proc 2015;47(9):2757-62.

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12. Han QF, Wu L, Zhou YH, et al. Simvastatin protects the heart against ischemia

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13. Kelle I, Akkoç H, Uyar E, et al. The combined effect of rosuvastatin and

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Thorac Cardiovasc Surg 2015;149(3):902-9.

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ACCEPTED MANUSCRIPT Table 1. Degree of histopathological lesion in the renal parenchyma in rats per group. Groups Rats IPC

IPC+S

S

4

2

1

1

1

2

4

2

1

1

1

3

4

2

1

1

1

4

4

4

2

1

1

5

4

2

1

1

1

6

4

2

1

1

-

7

4

4

2

2

-

8

4

2

1

1

-

9

4

2

1

1

-

Average

4

2.44

1.22

1.11

1

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SHAM

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Figure 1: Comparison of the medians of the degrees of renal injury among the different groups analyzed (Kruskal-Wallis; p=0.0001; "a" p<0.001 in relation to I/R group, "b" p<0.05 in relation to IPC group).

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Figure 2 - Photomicrographs of histological changes of the kidney considering the classification of Shih et al.7 (Histological classification depends on the percentage of alterations found): Focal edema (fe); glomerular atrophya (ga); tubular congestion (tc); necrosis of tubular cells (white arrows); hydropic degeneration (black arrows); medular congestion (mc); HE, 10x and 40x.