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.

Figure 1

Levels of ROS in severely hydronephrotic rabbit kidneys perfused at different pneumoperitoneum pressures. ^#P<0.05 vs. the 0 mmHg group. ROS, reactive oxygen species.

Figure 2

MMP of renal cells subjected to different pneumoperitoneum pressures in rabbit kidneys with severe hydronephrosis. (A) Flow cytometric analysis of MMP changes. PE-A represents the red fluorescence, while FITC-A represents the green fluorescence. MMP levels are expressed as the ratio of red to green fluorescence intensity, indicated in Q2 and Q4. (B) Ratio of red to green fluorescence intensity of renal cells in rabbits with severe hydronephrosis under different intraabdominal pressures. ^#P<0.05 vs. the 0 and 8 mmHg groups. MMP, mitochondrial membrane potential.

Figure 3

Histological evaluation of rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) Hematoxylin and eosin staining images of kidney sections (magnification, ×200). Asterisks indicate examples of expansive kidney tubules caused by severe hydronephrosis, while the arrows display examples of necrotic tubular cells. (B) Tubular necrosis scores in each group. ^#P<0.05 vs. the 0 and 8 mmHg groups.

Figure 4

TUNEL detection of rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) TUNEL analysis images of kidney sections (magnification, ×200). The arrows in the figure indicate examples of TUNEL-positive cells. (B) Apoptosis index of renal cells in each group. ^#P<0.05 vs. the 0 and 8 mmHg groups.

Figure 5

Ultramicrostructural changes of rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) Transmission electron microscopy images of ultramicrostructural changes in different groups (magnification, ×3,000). The arrows indicate examples of swollen and vacuolar mitochondria. (B) Percentage of swollen mitochondria in each group, indicating apoptotic changes. ^#P<0.05 vs. the 0 and 8 mmHg groups.

Figure 6

Expression levels of Bax and Bcl-2 in rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) Representative western blots and (B) relative protein expression levels of Bax, Bcl-2 and β-actin in rabbit kidneys from different groups. All data are expressed as the mean ± standard deviation of three experiments, and each experiment included triplicate repeats. ^#P<0.05 vs. the 0 and 8 mmHg groups. ^*P<0.05 vs. the 0 and 8 mmHg groups. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated x protein.

Figure 7

Expression of Cyt c in mitochondria and cytosol in rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) Representative western blots showing the protein expression levels of Cyt c, COX-IV and β-tubulin in mitochondria and cytosol in rabbit kidneys from different groups. (B) Distribution of Cyt c in mitochondria and cytosol in each group. All data are representative of three independent experiments. ^#P<0.05 vs. the 0 and 8 mmHg groups. ^*P<0.05 vs. the 0 and 8 mmHg group. Cyt c, cytochrome C; COX-IV, Cyt c oxidase-IV.

Figure 8

Expression levels of caspase-3 and caspase-9 in rabbit kidneys with severe hydronephrosis subjected to different pneumoperitoneum pressures. (A) Representative western blots demonstrating the protein expression levels of caspase-3, caspase-9 and β-actin in rabbit kidneys from different groups. (B) Relative expression of pro-caspase-3 and cleaved-caspase-3 in each group compared with β-actin. (C) Ratio of cleaved-caspase-3/pro-caspase-3 expression in each group. (D) Relative expression of pro-caspase-9 and cleaved-caspase-9 in each group compared with β-actin. (E) Ratio of cleaved-caspase-9/pro-caspase-9 expression in each group. All data are expressed as the mean ± standard deviation, and each experiment was conducted in triplicate. ^#P<0.05 vs. cleaved-caspase in 0 and 8 mmHg groups; ^*P<0.05 vs. cleaved-caspase/pro-caspase levels in 0 and 8 mmHg groups.

Figure 9

Proposed mechanisms of cell apoptosis induced by high CO₂ pneumoperitoneum pressure in rabbit kidneys with severe hydronephrosis. High CO₂ pneumoperitoneum pressure induced renal blood flow changes and increased the accumulation of ROS, resulting in upregulated Bax and downregulated Bcl-2 levels in kidneys. Consequently, the MMP was reduced, which then accelerated the release of Cyt c into the cytoplasm, leading to apoptosis via the caspase-3 and caspase-9-dependent pathway. CO₂, carbon dioxide; MMP, mitochondrial membrane potential; ROS, reactive oxygen species; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated x protein; Cyt c, cytochrome C; AIF, apoptosis inducing factor.