Oxidative Damage and Mitochondrial Injuries Are Induced by Various Irrigation Pressures in Rabbit Models of Mild and Severe Hydronephrosis

<div><p>Objective</p><p>We aimed to study whether tolerance to irrigation pressure could be modified by evaluating the oxidative damage of obstructed kidneys based on rabbit models experiencing different degrees of hydronephrosis.</p><p>Methods</p><p>A total of 66 rabbits were randomly divided into two experimental groups and a control group. In the experimental groups, the rabbits underwent a surgical procedure inducing mild (group M, n=24) or severe (group S, n=24) hydronephrosis. In each experimental group, the rabbits were then randomly divided into 4 subgroups (M0-M3 and S0-S3) consisting of 6 rabbits each. Group 0 received no perfusion. Groups 1 through 3 were perfused with 20, 60 and 100 mmHg fluid, respectively. For the control group, after a sham operation was performed, the rabbits were divided into 4 subgroups and were perfused with fluid at 0, 20, 60 or 100 mmHg of pressure. Kidney injuries was evaluated by neutrophil gelatinase associated lipocalin (NGAL). Oxidative damage was assessed by analyzing superoxide dismutase (Mn-SOD) activity, malondialdehyde (MDA) levels, glutathione reductase (GR), catalase (CAT) and peroxide (H₂O₂) levels, mitochondrial injuries was assessed by mitochondrial membrane potential (MMP), the mitochondrial ultrastructure and tubular cell apoptosis.</p><p>Results</p><p>In the experimental groups, all results were similar for groups 0 and 1. In group 2, abnormalities were observed in the S group only, and the kidneys of rabbits in group 3 suffered oxidative damage and mitochondrial injuries with increased NGAL, decreased Mn-SOD, GR and CAT,increased MDA and H₂O₂, lower levels of MMP, mitochondrial vacuolization and an increased apoptotic index.</p><p>Conclusion</p><p>In rabbits, severely obstructed kidneys were more susceptible to oxidative damage and mitochondrial injury than mildly obstructed kidneys when subjected to higher degrees of kidney perfusion pressure.</p></div

Ultrastructural changes of the mitochondria.

<p>A: Swollen and vacuolar mitochondria in rabbits with mild and severe hydronephrosis under different perfusion pressures (×10000), the arrows showed the swollen and vacuolar mitochondria. B: Percentage of swollen and vacuolar mitochondria in rabbits with mild hydronephrosis. C: Percentage of swollen and vacuolar mitochondria in rabbits with severe hydronephrosis. ▽p<0.05 compared with the M0 group. #p<0.05 compared with the S0 group. Ctrl: normal kidneys perfused with fluid at 20 mmHg, 60 mmHg and 100 mmHg. M0, M1, M2, and M3 represent rabbits with mild hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively. S0, S1, S2, and S3 represent rabbits with severe hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively.</p

Expression of NGAL in the kidneys perfused at different pressures in rabbits with mild and severe hydronephrosis.

<p>A: NGAL expression (×400), the brown fields in the cytoplasm represented NGAL expression. B: Immunostaining scores of NGAL with different perfusion pressure in mildly kidneys (M group). C: Immunostaining scores of NGAL with different perfusion pressure in severely obstructed kidneys (S group). The bars represent means±SE; ▽p<0.05 compared with the M0 group. #p<0.05 compared with the S0 group. Ctrl: normal kidneys perfused with 20 mmHg, 60 mmHg or 100 mmHg. M0, M1, M2, and M3 represent rabbits with mild hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively. S0, S1, S2, S3 represent rabbits with severe hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively.</p

Levels of Mn-SOD, GR, and CAT, MDA and H₂O₂ in the kidneys perfused at different pressures in rabbits with mild and severe hydronephrosis.

<p>A: Activity of Mn-SOD, GR, CAT, MDA, H₂O₂ in rabbits with mild hydronephrosis. B: Activity of Mn-SOD, GR, CAT, MDA, H₂O₂ in rabbits with severe hydronephrosis.▽p<0.05 compared with the M0 group. #p<0.05 compared with the S0 group. Ctrl: normal kidneys perfused with 20 mmHg, 60 mmHg or 100 mmHg. M0, M1, M2, and M3 represent rabbits with mild hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively. S0, S1, S2, S3 represent rabbits with severe hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively.</p

Apoptosis in rabbits with mild and severe hydronephrosis subjected to different perfusion pressures.

<p>A: Apoptosis of renal cells in rabbits with mild and severe hydronephrosis after being perfused with fluid at different pressures (×400). B: Percentage of apoptotic cells in rabbits with mild hydronephrosis. C: Percentage of apoptotic cells in rabbits with severe hydronephrosis. ▽p<0.05 compared with the M0 group. #p<0.05 compared with the S0 group. Ctrl: normal kidneys perfused with 20 mmHg, 60 mmHg and 100 mmHg. M0, M1, M2, and M3 represent rabbits with mild hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively. S0, S1, S2, and S3 represent rabbits with severe hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively.</p

Mitochondrial membrane potential (MMP) of renal cells subjected to different amounts of pressure in mild and severe hydronephrosis.

<p>PE-A Represented red fluorescence, FITC-A represented green fluorescence. MMP values were expressed as the ratio of red fluorescence intensity (Q2) to the green fluorescence intensity (Q4). A: MMP analysis by flow cytometry in rabbits with mild and severe hydronephrosis. B: MMP of renal cells in rabbits with mild hydronephrosis. C: MMP of renal cells in rabbits with severe hydronephrosis. ▽p<0.05 compared with the M0 group. #p<0.05 compared with the S0 group. Ctrl: normal kidneys perfused with 20 mmHg, 60 mmHg and 100 mmHg. M0, M1, M2, and M3 represent rabbits with mild hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively. S0, S1, S2, and S3 represent rabbits with severe hydronephrosis subjected to perfusion pressure of 0 mmHg, 20 mmHg, 60 mmHg and 100 mmHg, respectively.</p