| Objective: The study was designed to establish experimental models of contrast induced acute kidney injury(CIAKI) in renal insufficiency rats and investigate the role of renal cell apoptosis in renal insufficiency rats of CIAKI. The study was also designed to explore the role of m TOR/P70S6 K signaling pathway on renal apoptosis through the use of m TOR inhibitor rapamycin and activator lysophosphatidic acid in renal insufficiency rats of CIAKI.Methods: 1. We established the rat model with chronic renal dysfunction by 5/6 nephrectomy, then induced CIAKI by injecting intravenously different schemes of iohexol to NE rats. At the same time, the serum creatinine, blood urea nitrogen(BUN) was examined to determine the reliable modeling scheme. 2. We gave lysophosphatidic acid and rapamycin intervention on CIAKI to observe their effects on contrast induced acute kidney injury. 40 healthy male clean grade SD rats were fed for 1 weeks before the experiment. One week later, they were randomly divided into sham operation group(C group, n=8) and 5/6 nephrectomy group(n=32). 5/6 nephrectomy group underwent one-step nephrectomy, two weeks later, rats in operation group were randomly divided into 5/6 ne group(NE group, n = 8), NE+ iohexol(CIAKI group, n = 8), CIAKI+ lysophosphatidic acid(LPA group, n = 8), CIAKI+ rapamycin(RAPA group, n = 8), then each rat of RAPA group was daily given rapamycin sodium carboxymethyl cellulose solution 1mg / kg / D by gavage, at the same time, rats from other groups were given the same amount of sodium carboxymethyl cellulose(a total of 2 weeks). Two weeks later, each rat of LPA group was injected intravenously with 1mg/kg lysophosphatidic acid phosphate buffer injection, other groups were intravenously injected with the same amount of phosphate buffer. And half an hour later each rat from CIAKI group, LPA group and RAPA group was induced by tail vein injection of 20ml/kg(water-depriving for 24 h), rats from other groups were injected with the same amount of normal saline. 3.1 ml of urine was collected and used for the determination of urinary KIM-1 at 24 hours after injecting Iohexol.We anesthetized rats and collected inferior vena cava blood 2ml at 24 hours after injectting Iohexol, then the rats were sacrificed and the kidney tissue were collected. The half kidney tissue were used for HE staining and TUNEL staining, the other half kidney tissue were used for western blotting determining the expression of p-P70S6 K, p-m TOR, m TOR, and P70S6 K.Results: 1. The Serum creatinine resluts showed that 24 hours after injection Serum creatinine from each rat that was injected 20ml/kg iohexol and then was banned from water for 24 hours surpassed baseline serum creatinine more than 25%. Renal insufficiency CIAKI rat model was successfully established. 2. There was no significant difference in baseline body weight between the surgical group and the sham operation group. Surgery group, the left kidney weight and left kidney weight index is higher than that of sham operation group(P < 0.05). In 24 h after the injectin of iohexol, compared with the NE group, the Scr values in CIAKI group was significantly higher(97.16 ± 9.15 vs 77.15±4.30, P < 0.05).The Scr values in PLA group was lower than that in CIAKI group(91.23 ± 10.13 vs 97.16 ± 9.15, P?0.05). The Scr values in PLA group was lower than that in RAPA group(91.23 ± 10.13 vs 106.1 ± 6.31, P<0.05). The Scr values in RAPA group was significantly higher than that in group CIAKI(106.1 ± 6.31 vs 97.16 ± 9.15, P < 0.05). Compared with the NE group, the values of urinary KIM-1 in the CIAKI group was significantly higher than that in NE group(11.72 ± 1.52 vs 2.19 ± 0.68, P < 0.01). The values of urinary KIM-1 in PLA group was significantly less than that in CIKIA group(8.01 ± 2.77 vs 11.72 ±1.52, P < 0.01). The values of urinary KIM-1 in PLA group was significantly less than that in RAPA group(8.01 ± 2.77 vs 14.49 ± 3, P < 0.01). The values of KIM-1 in RAPA group was significantly higher than that in CIAKI group(14.49 ± 3 vs 11.72 ± 1.52, P < 0.01). 3. Compared with NE group, the renal tubular injury scores from CIAKI group was significantly higher(17.73±4.2 vs 10.84±2.63, P < 0.05). Compared with the CIAKI group, the scores in LPA group was lower(15.96 ± 4.77 vs 17.73 ± 4.2, P < 0.05). The scores in LPA group was lower than that in RAPA group(15.96 ± 4.77 vs 19 ± 2.92, P < 0.05). The scores of renal tubular injury in RAPA group was significantly higher than that in CIAKI group(19 ± 2.92 vs 17.73 ± 4.2, P < 0.05). TUNEL staining result showed that the apoptosis index in the NE group was higher than that in the C group(5.35 ± 0.73 vs 3.90 ± 0.83, P < 0.05). Compared with group NE, the apoptosis index in CIAKI group was significantly increased(16.51 ± 2.43 vs 5.35 ± 0.73, P < 0.01). Compared with group CIAKI, the apoptosis index in LPA group was decreased(14.08 ± 3.90 vs 16.51 ± 2.43, P < 0.01). The apoptosis index in LPA group was lower than that in RAPA group(14.08 ± 3.90 vs 19.01 ± 3.61, P < 0.01). Compared with group CIAKI, the apoptosis index in RAPA group was significantly increased(19.01 ± 3.61 vs 16.51 ± 2.43, P < 0.01). 4. The phosphorylation level of m TOR and P70S6 K in CIAKI group was significantly lower than that in NE group, and the difference was significant(P < 0.05). LPA group showed a less decrease in the phosphorylation of m TOR and P70S6 K than CIAKI group(P < 0.05). The phosphorylation level of m TOR and P70S6 K in LPA group was significantly lower than that in RAPA group(P < 0.05). RAPA group showed a greater decrease in the phosphorylation of m TOR and P70S6 K than CIAKI group.Conclusion:1. The renal insufficiency CIAKI rat model was successful founded in our experiment, and the model is reproducible. 2. Renal dysfunction rats that underwent CIAKI induced by iohexol showed that renal tubular cells apoptosis and dephosphorylation of m TOR and P70S6 K was increased. 3. Lysophosphatidic acid activated the m TOR/P70S6 K signaling pathway in renal tissue and decreased the apoptosis of renal tubular cells and acute kidney injury in CIAKI model of rats with renal insufficiency. 4. Rapamycin inhibited the m TOR/P70S6 K signaling pathway in renal tissue and increased renal tubular cell apoptosis and acute kidney injury in CIAKI model of rats with renal insufficiency. |
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