Sixty SPF healthy male C57BL/6 mice (6-8 weeks) were randomly divided into
sham operation group (
Sham group),
sepsis model group (CLP group) and Klotho
protein injection group (CLP+KL group), with 20 in each group. The septic AKI mice model was established by cecal
ligation and
puncture (CLP);
Sham group had the same procedure except that the cecal was not ligated. The CLP+KL group was received Klotho
protein (0.02 mg/kg) by intraperitoneal consecutive injection for 4 days after operation;
Sham group and CLP group were injected with the same amount of saline. Blood samples were obtained at 24 hours after operation, the levels of serum
creatinine (SCr) and
urea nitrogen (BUN) were measured by
sarcosine oxidase and
urease method. The mice were sacrificed under
anesthesia at 5 days after operation to harvest renal tissues, and the pathological damage of the kidney was evaluated by
hematoxylin-
eosin (HE) staining. The ultrastructure of mitochondria in mouse renal tubular epithelial cells was observed under transmission electron microscope. The levels of
reduced glutathione hormone (GSH),
malondialdehyde (MDA) and
nitric oxide synthase (NOS) in mitochondrion were determined by micro-
enzyme method,
thiobarbituric acid method, colorimetry method, respectively. The
protein expressions of Klotho, Bcl-2 and
cytochrome C (Cyt C) were detected by Western Blot.
RESULTS: The pathological structure of the kidneys in the
Sham group was clear and intact. Compared with the
Sham group, the renal tissue
edema of the mice in the CLP group was significant, and the transparent tube type was observed in the small lumen, and the interstitial inflammatory cells infiltrated; the levels of SCr and BUN were significantly increased [SCr (μmol/L): 182.60±6.97 vs. 47.20±5.37, BUN (mmol/L): 53.70±5.12 vs. 18.70±2.62, both P < 0.01]; the mitochondria were swollen and deformed, the sputum structure was destroyed, the matrix density was decreased, the outer membrane was lost, and the levels of MDA, GSH and NOS were significantly increased [MDA (μmol/g): 1.172±0.046 vs. 0.746±0.094, GSH (μmol/g): 5.765±0.059 vs. 4.223±0.072, NOS (kU/g): 0.91±0.05 vs. 0.68±0.03, all P < 0.01]; the
protein expressions of Klotho and Bcl-2 in renal tissue were decreased, and the
protein expression of Cyt C was increased (Klotho/β-actin: 0.188±0.020 vs. 0.538±0.024, Bcl-2/β-actin: 0.311±0.010 vs. 0.391±0.015, Cyt C/β-actin: 0.226±0.010 vs. 0.135±0.006, all P < 0.01). Comparing with the CLP group, the glomerular and tubular tissue epithelial
edema and the small lumen in the CLP+KL group were reduced; the levels of SCr and BUN were significantly decreased [SCr (μmol/L): 85.70±7.23 vs. 182.60±6.97, BUN (mmol/
L): 35.30±3.50 vs. 53.70±5.12, both P < 0.01]; the mitochondrial structure was relatively intact; the levels of MDA, GSH and NOS were significantly decreased [MDA (μmol/g): 0.958±0.072 vs. 1.172±0.046, GSH (μmol/g): 4.756±0.107 vs. 5.765±0.059, NOS (kU/g): 0.79±0.02 vs. 0.91±0.05, all P < 0.01]; the
protein expressions of Klotho, Bcl-2 were significantly increased, but the
protein expression of Cyt C was significantly decreased (Klotho/β-actin: 0.336±0.011 vs. 0.188±0.020, Bcl-2/β-actin: 0.474±0.017 vs. 0.311±0.010, Cyt C/β-actin: 0.168±0.006 vs. 0.226±0.010, all P < 0.01).
CONCLUSIONS: