Author's Accepted Manuscript Pneumoperitoneum in the presence of acute and chronic kidney injury: an experimental model in rats Oleg Dolkart, Wisam Khoury, Eyal Amar, Avi A. Weinbroum
PII: DOI: Reference:
S0022-5347(14)03313-8 10.1016/j.juro.2014.03.114 JURO 11378
To appear in: The Journal of Urology Accepted Date: 31 March 2014 Please cite this article as: Dolkart O, Khoury W, Amar E, Weinbroum AA, Pneumoperitoneum in the presence of acute and chronic kidney injury: an experimental model in rats, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.03.114. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed 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. All press releases and the articles they feature are under strict embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date.
ACCEPTED MANUSCRIPT
Pneumoperitoneum in the presence of acute and chronic kidney injury: an experimental model in rats
Oleg Dolkart1*, Wisam Khoury2*, Eyal Amar1, Avi A Weinbroum1 Pre-Clincial Research Laboratory and Post Anesthesia Care Unit, Tel Aviv Sourasky
RI PT
1
Medical Center and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Department of General Surgery, Rambam Health Care Campus, and the Bruce and
M AN U
Ruth School of Medicine, Technion, Haifa, Israel
SC
2
*Drs Dolkart and Khoury equally contributed to the study and manuscript preparation.
All Correspondence to:
TE D
Dr. Wisam Khoury Department of General Surgery Rambam Health Care Campus
Haifa, Israel
EP
8, Ha'alia Street
AC C
Phone: 972-4-8541730 Fax: 972-4-8542231
Email: [email protected]
Short Running Title: Pneumoperitoneum and kidney injury
Key Words: Pneumoperitoneum, kidney injury, acute, chronic, dysfunction
ACCEPTED MANUSCRIPT
ABSTRACT Background: There is evidence that pneumoperitoneum (PNP) induces transient changes in renal function in healthy individuals. Its effect on malfunctioned kidney is not well known. We investigated the effects caused by PNP in animals with impaired
RI PT
renal function.
Methods: Male Wistar rats underwent subtotal (5/6) nephrectomy in two sequential
steps to induce renal failure. Two weeks after surgery rats were classified as suffering
SC
from acute kidney injury (AKI group), and 10 weeks after surgery – as chronic kidney
M AN U
injury (CKI group). All rats were exposed to PNP at the above time point, at 0, 5, and 8 mmHg for 60 min. Changes in creatinine (Cr), blood urea nitrogen (BUN) and creatinine clearance (CCT) levels were measured. Histopathological changes and apoptosis were also evaluated in the different subgroups.
Results: Thirty-six rats (18 with AKI, 18 with CKI) terminated the study. While Cr
TE D
and BUN levels did not change after applying PNP in the different pressure subgroups, CCT decreased significantly in the 5 and the 8 mmHg subgroups, in both the AKI and the CKI rats. Histopathological findings in the AKI pressurized (5, 8
EP
mmHg) subgroups revealed ischemic changes, while a compensatory hypertrophy was
AC C
noticed in the CKI pressurized subgroups. Apoptotic count was significantly higher in the CKI subgroups as compared to their AKI pressurized counterparts. Conclusions: PNP seems feasible in animals with impaired baseline renal function. In particular, chronic cases should not be considered as contraindication for PNP, while in acute cases PNP might be detrimental.
ACCEPTED MANUSCRIPT
INTRODUCTION Pneumoperitoneum (PNP) induces pathophysiological changes that may affect renal perfusion1-3. Augmented sympathetic activity, increased renal vascular resistance, endocrine response, triggering oxidants, or mechanical compression of the
RI PT
parenchyma and renal vasculature, are each and all possible detrimental factors1-5.
Such changes immediately translate into transitory functional damage and oliguria1, 3. Indeed, kidneys which were harvested laparoscopically to be transplanted,
SC
experienced greater delayed graft function rate as compared to those removed in the
M AN U
open technique6, 7. This latter impairment is transient without definite squeals in the healthy population; however, as the use of laparoscopic surgery expands, subtle kidney injury may become clinically evident, particularly if applied to patients already suffering from reduced (baseline) renal function.
Medical literature is scarce with regard to data pertaining to the effects of PNP on
TE D
renal function in individuals with kidney impaired function. We therefore investigated changes induced by PNP in animals that had undergone removal of large part of renal mass, an established chronic kidney injury mode 8. We tested kidney's
EP
response to various PNP pressures both in an acute (immediately after) and chronic
AC C
(long after) phases after kidney mass reduction.
MATERIALS AND METHODS The experimental protocol was approved by the IRB Committee for Animal Experimentation at the Tel Aviv Medical Center. The animals were acclimatized in the pre-clinical research laboratory for 7 days before starting experimentation, and were always maintained on a 12-h light/12-h dark cycle starting at 06:00 AM and at temperature of 21–220C.
ACCEPTED MANUSCRIPT Male Wistar rats (body weight 250-300 g) were used in all experiments; they were always anaesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) each time they underwent an interventional procedure (Fig. 1).
RI PT
Rat model of kidney injury
The establishment of kidney injury followed the technique described by Windt et al,8 consisting of subtotal (5/6) nephrectomy in two sequential steps: two-thirds of the left
SC
kidney were removed initially, followed by the removal of the entire right kidney two weeks later. PNP experimentation started 14 days after the second intervention, the
M AN U
phase being labelled acute kidney injury state (AKI), while experimentation after 10 weeks was termed chronic kidney injury state (CKI).
Renal function assessment
TE D
Renal function was established by measuring serum creatinine (Cr), blood urea nitrogen (BUN), and creatinine clearance test (CCT) levels. All blood samples were obtained from the retro-orbital cavity of the right eye. All values represent conditions
EP
at 24h before and 24h after exposing the rats to PNP. Urine samples were then
AC C
collected along the following period of 24h, during which the animals were kept in a metabolic cage (Labotal Scientific Equipment Ltd., Abu Gosh, Israel) each, where they were allowed to eat and drink ad libitum. The collected blood was centrifuged at 4,000 rpm for 5 min, and the sera was removed and stored. Urine samples were also centrifuged, in order to remove any trace of food or feces. Urine Cr, as well as serum Cr, and BUN, were later determined, using laboratory techniques used at the Tel-Aviv Medical Center’s biochemical laboratory. CCT was later calculated, using the following formula:(urine
ACCEPTED MANUSCRIPT creatinine x urine volume)/(1440 x serum creatinine) where serum and urine creatinine are measured in mg/dl, urine volume is measured in ml, and 1440 stands for the total number of min in 24h9. The blood and urine samples were assessed both prior to and after each PNP
RI PT
application in an experimented animal.
Determination of acute and chronic kidney injury
SC
Two weeks after the second intervention, when the total of 5/6 of the entire renal mass had been removed and the animal recovered, AKI was defined8 and characterized
M AN U
based on the new blood and urine values10. CKI was established 10 weeks after the second subtotal nephrectomy8; the blood and urine values were considered as the baseline for either condition.
TE D
Application of pneumoperitoneum in acute or chronic kidney injury conditions Eighteen animals per condition were subjected to PNP, while anesthetized and mechanically ventilated with room air, using a piston-type rodent ventilator set to
EP
deliver 10 ml/kg body weight tidal volume, at 45 breaths/min. This resulted in ETCO2 ranging between 26-30 mmHg. Positive end-expiratory pressure was maintained at 4
AC C
mmHg, as previously reported1. Rats in each condition, i.e., AKI or CKI, were randomly divided into 3 subgroups (n=6/subgroup): each of these was subjected to intra-abdominal pressure of 0, 5 or 8 mmHg; PNP was always maintained for 60 min. These pressures and the experimental time frame were previously reported by this group1, showing 1) that 8 mmHg in the rat is the equivalent to 12 mmHg in humans11; 2) that the 60-min duration of PNP is sufficiently long to generate kidney metabolic damage1.
ACCEPTED MANUSCRIPT Histopathology The experimented kidneys in either condition were removed from the rats 24h after the experiments, and were fixed with 4% paraformaldehyde, sliced into transverse sections and paraffinized. The slices were sectioned (5 µm) and stained with
RI PT
hematoxylin and eosin (H&E). The severity of kidney injury was assessed by
histologically identifying tubular cell swelling, brush-border loss and nuclear loss.
All sections were examined by a pathologist who was completely blinded as to the
SC
study design and to the slice's origin.
M AN U
Apoptosis
Renal cell rate of apoptosis was assessed by TUNEL staining, using the ApopTag® Plus Peroxidase in Situ Apoptosis Detection Kit (S7101, Chemicon International, Inc. Temecula, CA, US), following the manufacturer’s instructions. The rates of renal
TE D
apoptosis found within the renal cells' nuclei were quantified by counting the TUNEL-positive cells, as reported previously12, and examined by an experienced
EP
technician who was blinded as to the stage or group of the slice analyzed.
AC C
Statistical analyses
Data are presented as mean ± standard deviation (SD) of the mean or median [interquartile range] according to the data distribution. Analysis of variance (ANOVA), and the Tukey's post hoc test that followed ANOVA, were used. A P value ≤ 0.05 was considered significant.
RESULTS
ACCEPTED MANUSCRIPT All rats survived both the two-phase nephrectomy and the acute and the chronic experimental phases. Following renal mass reduction procedures, the preexperimental (baseline) blood and urine renal parameters exhibited a mild, though
provided by non-nephrectomized rats13 (data not shown).
Pneumoperitoneum and biochemical measurements
SC
A. Acute kidney injury (AKI)
RI PT
insignificant, elevation, including creatinine and BUN levels, as compared to data
24h after the exposure of the rats to a 60min-pressure it appeared PN did not
M AN U
detriment renal function, in terms of Cr and BUN levels, independently of the grade of pressure being applied during the experiments. However, CCT values did decrease significantly (P
PII: DOI: Reference:
S0022-5347(14)03313-8 10.1016/j.juro.2014.03.114 JURO 11378
To appear in: The Journal of Urology Accepted Date: 31 March 2014 Please cite this article as: Dolkart O, Khoury W, Amar E, Weinbroum AA, Pneumoperitoneum in the presence of acute and chronic kidney injury: an experimental model in rats, The Journal of Urology® (2014), doi: 10.1016/j.juro.2014.03.114. DISCLAIMER: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our subscribers we are providing this early version of the article. The paper will be copy edited and typeset, and proof will be reviewed 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. All press releases and the articles they feature are under strict embargo until uncorrected proof of the article becomes available online. We will provide journalists and editors with full-text copies of the articles in question prior to the embargo date so that stories can be adequately researched and written. The standard embargo time is 12:01 AM ET on that date.
ACCEPTED MANUSCRIPT
Pneumoperitoneum in the presence of acute and chronic kidney injury: an experimental model in rats
Oleg Dolkart1*, Wisam Khoury2*, Eyal Amar1, Avi A Weinbroum1 Pre-Clincial Research Laboratory and Post Anesthesia Care Unit, Tel Aviv Sourasky
RI PT
1
Medical Center and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Department of General Surgery, Rambam Health Care Campus, and the Bruce and
M AN U
Ruth School of Medicine, Technion, Haifa, Israel
SC
2
*Drs Dolkart and Khoury equally contributed to the study and manuscript preparation.
All Correspondence to:
TE D
Dr. Wisam Khoury Department of General Surgery Rambam Health Care Campus
Haifa, Israel
EP
8, Ha'alia Street
AC C
Phone: 972-4-8541730 Fax: 972-4-8542231
Email: [email protected]
Short Running Title: Pneumoperitoneum and kidney injury
Key Words: Pneumoperitoneum, kidney injury, acute, chronic, dysfunction
ACCEPTED MANUSCRIPT
ABSTRACT Background: There is evidence that pneumoperitoneum (PNP) induces transient changes in renal function in healthy individuals. Its effect on malfunctioned kidney is not well known. We investigated the effects caused by PNP in animals with impaired
RI PT
renal function.
Methods: Male Wistar rats underwent subtotal (5/6) nephrectomy in two sequential
steps to induce renal failure. Two weeks after surgery rats were classified as suffering
SC
from acute kidney injury (AKI group), and 10 weeks after surgery – as chronic kidney
M AN U
injury (CKI group). All rats were exposed to PNP at the above time point, at 0, 5, and 8 mmHg for 60 min. Changes in creatinine (Cr), blood urea nitrogen (BUN) and creatinine clearance (CCT) levels were measured. Histopathological changes and apoptosis were also evaluated in the different subgroups.
Results: Thirty-six rats (18 with AKI, 18 with CKI) terminated the study. While Cr
TE D
and BUN levels did not change after applying PNP in the different pressure subgroups, CCT decreased significantly in the 5 and the 8 mmHg subgroups, in both the AKI and the CKI rats. Histopathological findings in the AKI pressurized (5, 8
EP
mmHg) subgroups revealed ischemic changes, while a compensatory hypertrophy was
AC C
noticed in the CKI pressurized subgroups. Apoptotic count was significantly higher in the CKI subgroups as compared to their AKI pressurized counterparts. Conclusions: PNP seems feasible in animals with impaired baseline renal function. In particular, chronic cases should not be considered as contraindication for PNP, while in acute cases PNP might be detrimental.
ACCEPTED MANUSCRIPT
INTRODUCTION Pneumoperitoneum (PNP) induces pathophysiological changes that may affect renal perfusion1-3. Augmented sympathetic activity, increased renal vascular resistance, endocrine response, triggering oxidants, or mechanical compression of the
RI PT
parenchyma and renal vasculature, are each and all possible detrimental factors1-5.
Such changes immediately translate into transitory functional damage and oliguria1, 3. Indeed, kidneys which were harvested laparoscopically to be transplanted,
SC
experienced greater delayed graft function rate as compared to those removed in the
M AN U
open technique6, 7. This latter impairment is transient without definite squeals in the healthy population; however, as the use of laparoscopic surgery expands, subtle kidney injury may become clinically evident, particularly if applied to patients already suffering from reduced (baseline) renal function.
Medical literature is scarce with regard to data pertaining to the effects of PNP on
TE D
renal function in individuals with kidney impaired function. We therefore investigated changes induced by PNP in animals that had undergone removal of large part of renal mass, an established chronic kidney injury mode 8. We tested kidney's
EP
response to various PNP pressures both in an acute (immediately after) and chronic
AC C
(long after) phases after kidney mass reduction.
MATERIALS AND METHODS The experimental protocol was approved by the IRB Committee for Animal Experimentation at the Tel Aviv Medical Center. The animals were acclimatized in the pre-clinical research laboratory for 7 days before starting experimentation, and were always maintained on a 12-h light/12-h dark cycle starting at 06:00 AM and at temperature of 21–220C.
ACCEPTED MANUSCRIPT Male Wistar rats (body weight 250-300 g) were used in all experiments; they were always anaesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) each time they underwent an interventional procedure (Fig. 1).
RI PT
Rat model of kidney injury
The establishment of kidney injury followed the technique described by Windt et al,8 consisting of subtotal (5/6) nephrectomy in two sequential steps: two-thirds of the left
SC
kidney were removed initially, followed by the removal of the entire right kidney two weeks later. PNP experimentation started 14 days after the second intervention, the
M AN U
phase being labelled acute kidney injury state (AKI), while experimentation after 10 weeks was termed chronic kidney injury state (CKI).
Renal function assessment
TE D
Renal function was established by measuring serum creatinine (Cr), blood urea nitrogen (BUN), and creatinine clearance test (CCT) levels. All blood samples were obtained from the retro-orbital cavity of the right eye. All values represent conditions
EP
at 24h before and 24h after exposing the rats to PNP. Urine samples were then
AC C
collected along the following period of 24h, during which the animals were kept in a metabolic cage (Labotal Scientific Equipment Ltd., Abu Gosh, Israel) each, where they were allowed to eat and drink ad libitum. The collected blood was centrifuged at 4,000 rpm for 5 min, and the sera was removed and stored. Urine samples were also centrifuged, in order to remove any trace of food or feces. Urine Cr, as well as serum Cr, and BUN, were later determined, using laboratory techniques used at the Tel-Aviv Medical Center’s biochemical laboratory. CCT was later calculated, using the following formula:(urine
ACCEPTED MANUSCRIPT creatinine x urine volume)/(1440 x serum creatinine) where serum and urine creatinine are measured in mg/dl, urine volume is measured in ml, and 1440 stands for the total number of min in 24h9. The blood and urine samples were assessed both prior to and after each PNP
RI PT
application in an experimented animal.
Determination of acute and chronic kidney injury
SC
Two weeks after the second intervention, when the total of 5/6 of the entire renal mass had been removed and the animal recovered, AKI was defined8 and characterized
M AN U
based on the new blood and urine values10. CKI was established 10 weeks after the second subtotal nephrectomy8; the blood and urine values were considered as the baseline for either condition.
TE D
Application of pneumoperitoneum in acute or chronic kidney injury conditions Eighteen animals per condition were subjected to PNP, while anesthetized and mechanically ventilated with room air, using a piston-type rodent ventilator set to
EP
deliver 10 ml/kg body weight tidal volume, at 45 breaths/min. This resulted in ETCO2 ranging between 26-30 mmHg. Positive end-expiratory pressure was maintained at 4
AC C
mmHg, as previously reported1. Rats in each condition, i.e., AKI or CKI, were randomly divided into 3 subgroups (n=6/subgroup): each of these was subjected to intra-abdominal pressure of 0, 5 or 8 mmHg; PNP was always maintained for 60 min. These pressures and the experimental time frame were previously reported by this group1, showing 1) that 8 mmHg in the rat is the equivalent to 12 mmHg in humans11; 2) that the 60-min duration of PNP is sufficiently long to generate kidney metabolic damage1.
ACCEPTED MANUSCRIPT Histopathology The experimented kidneys in either condition were removed from the rats 24h after the experiments, and were fixed with 4% paraformaldehyde, sliced into transverse sections and paraffinized. The slices were sectioned (5 µm) and stained with
RI PT
hematoxylin and eosin (H&E). The severity of kidney injury was assessed by
histologically identifying tubular cell swelling, brush-border loss and nuclear loss.
All sections were examined by a pathologist who was completely blinded as to the
SC
study design and to the slice's origin.
M AN U
Apoptosis
Renal cell rate of apoptosis was assessed by TUNEL staining, using the ApopTag® Plus Peroxidase in Situ Apoptosis Detection Kit (S7101, Chemicon International, Inc. Temecula, CA, US), following the manufacturer’s instructions. The rates of renal
TE D
apoptosis found within the renal cells' nuclei were quantified by counting the TUNEL-positive cells, as reported previously12, and examined by an experienced
EP
technician who was blinded as to the stage or group of the slice analyzed.
AC C
Statistical analyses
Data are presented as mean ± standard deviation (SD) of the mean or median [interquartile range] according to the data distribution. Analysis of variance (ANOVA), and the Tukey's post hoc test that followed ANOVA, were used. A P value ≤ 0.05 was considered significant.
RESULTS
ACCEPTED MANUSCRIPT All rats survived both the two-phase nephrectomy and the acute and the chronic experimental phases. Following renal mass reduction procedures, the preexperimental (baseline) blood and urine renal parameters exhibited a mild, though
provided by non-nephrectomized rats13 (data not shown).
Pneumoperitoneum and biochemical measurements
SC
A. Acute kidney injury (AKI)
RI PT
insignificant, elevation, including creatinine and BUN levels, as compared to data
24h after the exposure of the rats to a 60min-pressure it appeared PN did not
M AN U
detriment renal function, in terms of Cr and BUN levels, independently of the grade of pressure being applied during the experiments. However, CCT values did decrease significantly (P
