High-pressure carbon dioxide pneumoperitoneum induces oxidative stress and mitochondria-associated apoptotic pathway in rabbit kidneys with severe hydronephrosis

Abstract

The primary aim of the present study was to investigate the potential effect of high-pressure carbon dioxide (CO2) pneumoperitoneum on kidneys with severe hydronephrosis and to investigate the possible underlying mechanism. A total of 18 rabbits underwent a surgical procedure inducing severe hydronephrosis. Rabbits were then divided at random into three groups (n=6 each) and subjected to intraabdominal pressure of 0, 8 or 18 mmHg, respectively. CO2 inflation lasted for 90 min in the pneumoperitoneum groups. Oxidative stress was assessed by measurements of reactive oxygen species (ROS). Activation of apoptosis was analyzed by western blot analysis of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated x protein (Bax), cytochrome c (Cyt c), caspase-3 and caspase-9 levels. In addition, TUNEL assay, hematoxylin and eosin (H&E) staining, measurement of mitochondrial membrane potential (MMP) and detection of changes to kidney ultramicrostructure were performed. In the 0 and 8 mmHg groups, all results were normal and similar. However, in the 18 mmHg group, the kidneys suffered oxidative damage and mitochondrial injuries, and increased ROS levels, lower MMP and mitochondrial vacuolization were observed. Furthermore, the mitochondrial/caspase-dependent pathway of apoptosis was activated, as indicated by the apoptotic index, and the expression levels and translocation of Bax, Bcl-2, Cyt c, caspase-3 and caspase-9. Therefore, it is concluded that high-pressure CO2 pneumoperitoneum induces oxidative damage and apoptosis in rabbit kidneys with severe hydronephrosis, which is associated with the mitochondrial apoptotic pathway.

Hydromorphone Protects against CO2 Pneumoperitoneum‐Induced Lung Injury via Heme Oxygenase‐1‐Regulated Mitochondrial Dynamics

“…Mitochondrial morphological and functional impairment during pneumoperitoneum has been shown to be involved in multiple organ injury [ 13 , 49 ]. Herein, we found that during pneumoperitoneum, excessive pressure and prolonged exposure led to mitochondrial swelling, vacuolization, and fragment, which was consistent with previous studies [ 13 , 50 ]. Multiple studies have demonstrated that mitochondrial DNA (mtDNA) is released after mitochondria are impaired [ 51 ], and mtDNA content has been shown to reflect the degree of mitochondrial dysfunction in response to cellular stress injury [ 51 ].…”

TUNEL Assay: A Powerful Tool for Kidney Injury Evaluation

“…Mixed acute-and-chronic injury, where toxic exposures, disease states, and/or assessments were both acute (i.e., hours, days) and chronic (i.e., weeks, months, years) in the same study, were observed in human transplant rejection (biopsy 1 week to 3 years post-transplant) [ 88 ], swine hepatitis E virus infection (7 and 14 days post-inoculation) [ 52 ], CO 2 pneumoperitoneum-induced stress in hydronephrotic kidneys (2 weeks hydronephrosis, 2 days post-pneumoperitoneum) [ 53 ], mesangial proliferative glomerulonephritis induced by snake venom (1 to 14 days post-injection) [ 99 ], unilateral ureteral obstruction (1 to 14 days of obstruction) [ 100 ], uranyl acetate exposure (1 to 28 days of exposure) [ 101 ], aristocholic acid nephropathy (5 and 30-day daily exposure) [ 102 ], and neonatal hyperoxia (tested 1 to 60 postnatal days, exposed to hyperoxia first 14 days) [ 103 ]. Fourteen days after impact is a very common assessment time point for these kinds of kidney injuries [ 52 , 53 , 99 , 101 , 103 ]. In the majority of cases, TUNEL-positive cells were localized in tubules and collective ducts.…”

“…TUNEL is applicable to all cell types, organs, and species that have DNA and DNases, which includes just about all species. TUNEL has been used to identify and quantify kidney injury in a wide array of animals, including zebrafish [ 50 ], Japanese rice fish [ 51 ], chickens [ 48 ], gerbils [ 52 ], mice [ 41 ], rats [ 45 ], rabbits [ 53 ], mini-pigs [ 54 ], and humans [ 55 ]. An important advantage of TUNEL is that it can be used in fixed cultured cells as well as fixed tissues.…”

“…Both TUNEL and cleaved caspase-3 assays showed exclusively tubular, not glomerular, staining in rat ischemia-reperfusion [ 64 ]. Other caspase studies used caspase-1 [ 27 ], -9 [ 53 ], -11 [ 28 ], and -12 [ 75 ]. Other apoptotic markers included the B-cell lymphoma 2 (Bcl2) family of proteins Bcl-2 [ 27 ], Bcl2L1 [ 43 ], Bcl-xl [ 71 ], Bax [ 99 ], Bak [ 49 ], and Bad [ 43 ]; apoptotic receptor Fas [ 52 ] and apoptotic receptor ligand FasL [ 66 ]; forkhead box (FOX) protein transcription factors Fox01 [ 133 ] and Fox03 [ 27 ]; and cytochrome c [ 53 ].…”

“…Other caspase studies used caspase-1 [ 27 ], -9 [ 53 ], -11 [ 28 ], and -12 [ 75 ]. Other apoptotic markers included the B-cell lymphoma 2 (Bcl2) family of proteins Bcl-2 [ 27 ], Bcl2L1 [ 43 ], Bcl-xl [ 71 ], Bax [ 99 ], Bak [ 49 ], and Bad [ 43 ]; apoptotic receptor Fas [ 52 ] and apoptotic receptor ligand FasL [ 66 ]; forkhead box (FOX) protein transcription factors Fox01 [ 133 ] and Fox03 [ 27 ]; and cytochrome c [ 53 ]. Inflammation was identified by IHC of tumor necrosis factor alpha (TNFα) [ 134 ]; tumor growth factors TGFβ [ 66 ] and TGF-β1 [ 135 ]; serine/threonine kinase 1 (SGK1) [ 135 ]; electron-transfer flavoprotein, beta-subunit (ETFβ) [ 136 ]; Janus kinase-2 (JAK2) [ 137 ]; signal transducer and activator of transcription 3 (STAT3) [ 137 ]; nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) [ 137 ] and its inhibitor protein IκB [ 137 ].…”