P53 related apoptosis in kidneys in CO2 pneumoperitoneum ratmodel: an immunohistochemical study

Murat Tosun • Mehmet Yucel • Aysegul Kucuk •

Saban Sezen

Received: 7 October 2013 / Accepted: 19 June 2014 / Published online: 19 July 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Laparoscopic surgery techniques have been

increasingly preferred to classic laparotomy by surgeons

since 1987. However, this method may have some side

effects on different intraabdominal organs including kid-

neys. The aim of this study is to evaluate the effects of

different pressures of CO2 on p53 related apoptosis in

kidneys. Totally 24 male rats were divided into four equal

groups. CO2 is insufflated into rats’ intraabdominal cavity

in two different pressures of 10 and 20 mmHg during 1 h.

However, in sham group, only cannula was inserted, but no

gas was insufflated. After 1 h, 30 min reperfusion was

applied. At last, the kidneys were excised and p53

expression and apoptosis were evaluated immunohisto-

chemically. All the data revealed that the number of

apoptotic cell in kidney’ tubular cells significantly increa-

ses in proportion to CO2 pressure level. On the other hand,

p53 expression was detected only in the highest pressure.

Because the low CO2 pressured group’ rats had no p53

expression in kidneys, we suggest that this method can be

safely used for abdominal surgery. At the same time,

increasing in the number of apoptotic cells parallel to

pressure also suggest that CO2 pressure level and appli-

cation time are very important parameters during CO2

pneumoperitoneum.

Keywords Apoptosis � p53 � Kidney � CO2

pneumoperitoneum � Laparoscopy � Cell death

Introduction

Mouret in Paris first described laparoscopic surgery in 1987

[1]. This technique has many advantages such as faster

recovery, less postoperative pain, reduced hospital stays,

and better esthetic results [2]. Additionally, because the

used gases during surgical process such as CO2, Helium or

air easily eliminated from blood stream [3]. Today, this

technique is increasingly preferred to classic laparotomy.

However, in recent some studies have been revealed that

this technique may be harmful on intra-abdominal organs.

These side effects such as organ hypoxia/ischemia, changes

in physiological parameters are generally transient and

related to the type, temperature, and pressure level of the

gas and with the duration of the application [2–4]. In

kidneys, mechanical and hormonal factors have been

implicated in pneumoperitoneum-induced renal alterations.

The decline in urinary debt and in creatinine clearance may

observe during pneumoperitoneum [5]. On the other hand,

in a study, it was determined that the functions and mor-

phology of unilateral nephrectomy subjected rats’ kidneys

were not significantly influenced by prolonged and suc-

cessive pneumoperitoneum [6]. Experimental studies on

kidneys related to laparoscopic surgery have mainly

focused on physiopathology [3, 7, 8]. However, gene

M. Tosun (&)

Department of Histology Embryology, Afyon Kocatepe

University, Campus of Ali Cetinkaya, 03200 Afyonkarahisar,

Turkey

e-mail: drmtosun@yahoo.com

M. Yucel

Department of Urology, Dumlupinar University, Kutahya,

Turkey

A. Kucuk

Department of Physiology, Dumlupinar University, Kutahya,

Turkey

S. Sezen

Department of Histology Embryology, Kırıkkale University,

Kırıkkale, Turkey

123

Mol Biol Rep (2014) 41:6391–6395

DOI 10.1007/s11033-014-3519-5

expression and cell death mechanisms in kidneys and intra-

abdominal organs under intra-abdominal pressure were not

sufficiently evaluated. Among these genes, p53 a guardian

of genome, is very important, because it stimulates apop-

totic cascades to prevent malign cell formation and protect

genome stability in DNA damage [9]. p53 upregulation

generally means increasing destructive stress in tissue. This

activation is ended with either repairing DNA or physio-

logical cell death also known as apoptosis.

In this study, we aimed to evaluate the effects of dif-

ferent pressures of CO2 on p53 related apoptosis in rat’s

kidney during CO2 pneumoperitoneum.

Materials and methods

The study protocol was approved by Dumlupinar Univer-

sity Animal Care and Ethics Committee (605213).

Animals and surgery

In this study, 24 adult Sprague–Dawley male rats weighing

280–340 g were used. The rats were divided into four equal

groups. The first Group was Control (n = 6). In Sham

group (n = 6) only the cannule was inserted into intra-

abdominal cavity but no CO2 was insufflated (Group:2).

CO2 was insufflated into rats’ intra-abdominal cavity of a

pressure of 10 mmHg and 20 mmHg in Group 3(n = 6),

Group 4 (n = 6) respectively.

For anesthesia, the rats were subjected to ether and just

after sedation 50 mg/kg ketamine hydrochloride (Ketalar,

Parke Davis, Morris Planes, NJ) was injected intramuscu-

larly. An angiocatheter cannule set that can synchronously

transport CO2 coming from insufflator to six rats’ intra-

abdominal cavity was constituted. The abdominal wall was

incised and these cannules were inserted into the rats’ intra-

abdominal cavities. The other end of the cannule was

connected to CO2 insufflator (Insufflator Duo Lab, Carl

Zeiss, Germany). All connections in the system were

carefully checked and insufflation was started for each

group as it was planned. After 60 min, CO2 insufflation

was stopped and desufflation was applied. Then, after

30 min for reperfusion of kidneys, the rats were sacrificed,

laparotomy was applied and kidneys were excised and

immediately put into 10 % neutral formalin for fixation for

histology.

Histology

All the specimens were fixed in 10 % neutral formalin,

dehydrated in increasing alcohol series, cleared in xylene

and embedded in paraffin. Several 5 l sections obtained

from these specimens were mounted on poly-L-lysine-

coated slides for immunohistochemistry.

Immunohistochemistry

Apoptosis in tissues was detected by terminal TDT-medi-

ated dUTP-biotin nick-end labeling (TUNEL) technique

using a commercial kit (TDT-Fragel DNA fragmentation

detection kit, Calbiochem, Darmstadt, Germany). The

solutions provided by manufacturer to staining were used

in order to manufacturer’ recommendations during

staining.

For detecting p53 expression, immunohistochemistry

was used. For antigen retrieval, microwave treatment was

used in 10 mM citrate buffer, pH 6.0 for 20 min. After the

retrieval process, 10 % H2O2 was used for inactivation of

endogen peroxidase in the specimens during 12 min.

Specimens were then reacted with mouse monoclonal

antibody against human p53 protein (Clone DO-7) during

2 h in order to manufacturer recommendation. Horse

Radish Peroxidase detection system was used as the sec-

ondary antibody for 2 9 20 min and DAB Substrate Sys-

tem for chromogen for 10 min in order to manufacturer

recommendation (all chemicals obtained from Labvision

Corporation, Fremont, CA). Mayers Hematoxylin (Sigma,

St. Louis, MO) was used for counterstaining for 1 min.

Then all slides were dehydrated, mounted with Entellan

and evaluated under light microscope (Nikon E600, Japan).

Image analysis

The immunopositive cells in ten different places at 209

objective magnification were counted in Nikon NIS Ele-

ments D4 Image Analysis Software. However, because p53

expression was detected only in Group 4, immunoreactivity

by HSCORE of p53 was not calculated.

Statistical analysis

Statistical analysis was performed with the Statistical

Package for the Social Sciences for Windows (SPSS ver-

sion 16.0, Chicago, IL, USA). For statistical analysis, only

apoptotic cell death data were used. Because p53 expres-

sion was detected only in Group 4 statistical analyses was

not performed. Statistical analyses of data were performed

using a Mann Whitney U test. A value of p \ 0.05 was

considered statistically significant.

Results

It was determined that the number of apoptotic cells

increased in parallel to the increasing intraabdominal

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pressure. Apoptotic cells were detected both Group: 4

(Fig. 1a) and Group: 3 (Fig. 1b) but not in Control

(Group:1) (Fig. 1c) and Sham (Group:2). Apoptotic cells

were detected only in tubular cells especially in cortico-

medullary junction but not in glomerulus. Increasing in the

number of apoptotic cells was statistically significant

between Group: 3 (n = 41.0 in ten different microscopic

area for each slides) and Group: 4 (n = 96.0 in ten dif-

ferent microscopic area for each slides) (p = 0.021). At the

same time, there were also significant differences between

Sham and Control and the other two groups (p = 0.000 for

all). On the other hand, p53 expression was detected only

high CO2 pressured applied Group:4 (n = 34.0 in ten dif-

ferent microscopic area for each slides) (Fig. 1d). There

were no p53 expression in Group:3 (Fig. 1e), Group:2 and

Group:1 (Fig. 1f). All the data were summarized in

Table 1.

Discussion

Some of experimental studies about CO2 pneumoperito-

neum revealed that this application have some important

risks. However, it was revealed that the side effects of CO2

Fig. 1 a Group:4. 20 mmHg insufflated group. There are many

brown in color apoptotic cells (Arrows) between green in color

nonapoptotic cells in tubules. 920. (Tdt-Fragel kit). b Group:3.

10 mmHg insufflated group. There are a little brown in color

apoptotic cells (Arrows) between green in color nonapoptotic cells in

tubules. 920. (Tdt-Fragel kit). c Control Group. No apoptotic cells

are seen. 920. (Tdt-Fragel kit). d Group 4. p53 positive cells (Arrows)

are seen in different localizations. 920. (Anti-p53 Clone DO-7).

e Group 3. No p53 positive cells are seen. 920. (Anti-p53 Clone DO-

7). f Control Group. No p53 positive cells are seen. 920. (Anti-p53

Clone DO-7). (Color figure online)

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pneumoperitoneum such as organ ischemia, cell death,

physiopathological changes were minimal and reversible

without any apparent sequel when the shorter time and

lower pressure was used. Because we have studied the

effects of CO2 pneumoperitoneum in different intraab-

dominal organs in our different studies before [10–14], we

have standardized CO2 pressure for experimental studies.

In these studies, we found that if CO2 pressure is higher

than 20 mmHg, p53 related apoptosis was clearly activated

in tissues. Nevertheless, it was considered that this pressure

is very high when compared to abdominal region capacity

of rat. On the other hand, under 10 mmHg pressure we had

not detected any side effects on all intraabdominal organs

we studied. So, we designed pressure limits between

10–20 mmHg for this study. On the other hand, many

studies support our model. For example, it was reported

that the increasing of intra-abdominal pressure to

20–25 mmHg caused a decrease of 63 % in mucosal blood

flow in rats with a normal mean arterial pressure of

102–123 mmHg [15]. On the other hand, in a study, it was

revealed that prolonged intraabdominal pressure of

15 mmHg might predispose to multi organ dysfunction in

pigs [8]. At the same time, in another study, it was dem-

onstrated that the production of Tumor Necrosis Factor-a(TNF-a), a very important cytokine in immune response

and tissue scarring, of Interleukin-1b (IL-1b), a major

proinflammatory cytokine, and of superoxide radicals and

several-hour transient impairment on mitochondrial meta-

bolic functions occur in a higher pressure in pneumoperi-

toneum models5. Furthermore, many studies indicated that

if the pressure level is well-controlled, there are no nega-

tive effects on hemodynamic parameters [16, 17]. Also, it

was revealed that abdominal gas in 8 mmHg insufflation

does not have any adverse effect on the renal function of

the kidney donor 1 week after laparoscopic donor

nephrectomy. It means that pressure level is very important

concept to protect from tissue damage [18]. The renal

effects of pneumoperitoneum generally related with renal

vascular insufficiency from central venous compression.

However, some studies maintain that renal dysfunction

related to CO2 pneumoperitoneum is reversible as in other

abdominal organs [19–22].

In our study, we determined that the higher intraab-

dominal pressure causes evident increasing in the number

of apoptotic cell in kidney tubular cells. It means that CO2

pneumoperitoneum in higher pressures have lethal effects

on kidney tubular cells. Generally, apoptotic pathways

were activated by p53 expression in our before studies [10–

14].However, in this study, we detected that p53 expression

was detected only 20 mmHg applied groups. This protec-

tion might be related with mechanical resistance of renal

capsule and also retroperitoneal location of kidneys.

Because p53 expression indicates DNA damage in that

cells, we can say that CO2 pneumoperitoneum does not

cause any genome damage in kidney’ cells in lower pres-

sures. This data is very important for safety of CO2

pneumoperitoneum in molecular level. On the other hand,

apoptosis in tissues can also be activated by different fac-

tors such as bcl-2, Bax, caspases etc. However, different

agents such as hyperthermia, cytokines, and different

inflammatory agents activate these pathways without DNA

damage. In our studies, we suggested that apoptotic cell

death after CO2 pneumoperitoneum might be related to low

rate and transient hypoxic injury. Just as, the corticome-

dullary location of apoptotic cells supports our hypothesis.

The decreasing blood stream may cause hypoxic mito-

chondrial respiration destruction and caspases cascade

activated apoptosis occurred. However, it was considered

that the rate of the number of apoptotic cells to all the

number of total renal tubular cells, apoptotic index, was

very low, we can hypothesized that the all the renal tubular

functions might be compensated by other tubular cells.

Conclusion

We suggest that because higher pressures cause increasing

in the number of kidney tubular cells death, regulation of

CO2 pressure level, and application time is very important

during CO2 pneumoperitoneum.

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Table 1 The number of immunopositive cells in all groups

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Apoptotic cells

The number of p53

positive cells

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* p \ 0.05

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