| Objective:As the application of the intervention techniques in diagnosis and therapy increasing, the effects of Contrast media on Renal function have been taken seriously more and more. In the study we establish reliable and repeatable experimental model of contrast induced acute kidney injury(CIAKI) in diabetic rats, to simulate the common background of CIAKI in clinic and investigate the effection of Oxidative stress and renal cell apoptosis on CIAKI in vivo. At the same time, we discuss the molecular mechanism of renal cell apoptosis induced by contrast media, especially their effect on Akt/mTOR/P70S6K and MAPK(ERK、JNK) signal pathways. In addition, we also Probe further into the renoprotective effect of Probucol on CIAKI and apoptotic signal pathways in diabetic rats.Methods:(1) First, we established early diabetic nephropathy rats model, further induced CIAKI by intravenous injection meglumine diatrizoate(DTZ).Moreover,we examed blood sugar and renal function (mainly blood/urine creatinine, calculate creatinine clearance rate), evaluate the reliability and repeatablility of the model.In the second part of the study,we investigate the effect of Probucol intragastric administration on CIAKI (DP and DCP group).(2) The expression level of MDA、SOD and GSH-PX in Renal tissue homogenate have been studied, to discuss the effect of Probucol on Oxidative stress in CIAKI.(3) Observe histopathological changes by HE staining of the kidney; TUNEL staining was using to detect renal cell apoptosis and calculate apoptotic index. In addition, we observe the expression of apoptosis related protein caspase-3and9in the kidney By immuno-histochemistry; quantitative analysis of the Bcl-2and Bax protein expression in renal tissue by western blotting, in order to study the protective effect of probucol to renal cell apoptosis in the CIAKI.(4) Application of Western Blotting for quantitative analysis of the key proteins expression level of Akt/mTOR/P70S6K and MAPK (p-ERK, p-JNK) apoptosis signal pathways in renal tissue was used. Explore signal transduction pathways in renal cell apoptosis in CIAKI, and further analyze the association between these two signaling pathways. Research renoprotective effects of Probucol on CIAKI in diabetic rats, reveal its effection on signaling transduction pathways in renal cell apoptosis.Results:(1) The changes of renal function before and after contrast induce acute kidney injury(CIAKI):compared with D group, the serum creatinine was significantly increased in DC group after operation (108.34±10.37vs75.67±6.29, p<0.01), The rate of increase in serum creatinine after operation was greater too(39.11±8.51vs4.67±0.87, p<0.01). The rate of increase in serum creatinine is56.7%after injection of DTZ48hours in DC group, so the diabetic CIAKI model was founded successfully. Compared with D group, the creatinine clearance rate (Ccr) was significantly decreased in DC group after operation(2.93±0.57vs3.83±0.69, p<0.05), the rate of decrease in Ccr was significantly greater in DC group(-2.64±0.57vs-1.16±0.27, p<0.01).The experimental result with probucol intervention display: Compared with DC group, the serum creatinine was significantlyly decreased in DCP group after operation(87.23±9.15vs108.34±10.37, p<0.01). The rate of increase in serum creatinine in DCP group was lower than DC group(14.17±3.16vs39.11±8.51, p<0.01). The rate of increase in serum creatinine reaches56.7%after injection of DTZ48hours in DC group, but the DCP group only reaches19.4%. The rate of decrease in Ccr after operation in DCP group was lower than DC group(-1.73±0.57vs-2.64±0.57, p<0.01).(2) The effect of CIAKI on oxidative stress:After injection of DTZ, MDA content increased in kidney, DC group was higher than D group(1.41±0.19vs1.05±0.07, p<0.05); while the activity of antioxidase GSH-PX decreased, DC group was lower than D group(87.91±4.31vs99.35±7.41, p<0.05).The experimental result with probucol intervention display: The probucol could depress the level of MDA in kidney of diabetic CIAKI rat, DCP group was lower than DC group(0.90±0.10vs1.41±0.19, p<0.05); At the same time, it could reverse the activity of antioxidase GSH-PX, DCP group was higher than DC group(102.08±10.32vs87.91±4.31, p<0.01).(3) The apoptotic index of renal cells was significantly increased in diabetic rat after injection of DTZ, the expression of apoptosis GAP-associated protein caspase-3and caspase-9are significantly increased in kidney. The apoptotic index of renal cells was significantly decreased after intervention of probucol, the protein expression of caspase-3and caspase-9were also decreased.Compared with D group, the expression of anti-apoptosis Bcl-2protein was significantly decreased in DC group after the induction of STZ, while expression of promoting apoptosis protein Bax was significantly increased. Both express tendency were reversed by probucol:the protein expression of Bcl-2was increased and Bax was decreased.(4) The activity of apoptosis upstream signal molecule p-Akt(S473)、p-mTOR、 p-P70S6K were decreased in diabetic CIAKI rats, the expression of p-ERK in MAPK pathway was decreased, while the expression of p-JNK was significantly increased.The effect of high osmolality CM on upstream signal molecule could be reversed by probucol:the expression of p-ERK、p-Akt(S473)、p-mTOR and p-P70S6K were increased, the expression of p-JNK was decreased.Conclusion:(1) The diabetic CIAKI rat model was founded successfully in our experiment, the result is reliable and repeatable.(2) It appears significantly oxidative stress in diabetic rats after kidney damage induced by CM:MDA was increased, while the activity of endogenous antioxidase GSH-PX was decreased.(3) The ionic high osmolality CM induce severe apoptosis in renal cells of diabetic rats. The expression of caspase-3and caspase-9were significantly increased in kidney, at the same time the expression of promoting apoptosis protein Bax was increased and anti-apoptosis protein Bcl-2was decreased.(4) The effects of ionic high osmolality CM on renal cells through mitochondrial caspase pathway developing apoptosis may be mediated through upstream Akt/mTOR/P70S6K and MAPK pathways.(The anti-apoptosis signal of Akt/mTOR/P70S6Kand ERK are inhibited, but the activity of the promoting apoptosis protein JNK is increased.)(5) The probucol can protect kidney of CIAKI, the mechanisms may involve that it can extenuate oxidative stress and apoptosis of renal cells. The inhibition of renal cells apoptosis may be mediated through upstream Akt/mTOR/P70S6K and MAPK (ERK and JNK) pathways. |
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