| Objectives:to observe the effect of insulin sensitizer pioglitazone (PIO) on kidney of Diabetic rats with insulin resisitance,and its effect on c-Jun N-terminal kinase(JNK)expression.To probe the protective mechanism of PIO on renal function of diabetic rats and to provide a new thought for the prevention and cure of Diabetic Nephropathy.Methods:①After animal model of diabetic rats with insulin resistance (IR)was established,rats were randomly divided into three groups: normal control group(NC),diabetes mellitus group(DM),and pioglitazone group(PIO)in which rats were treated with PIO at dose of 10mg/kg.d by intragastric administration.②Rats were executed through the method of cervical spine dislocation after 6-week's intervention. Kidney specimens were harvested were stored in liquid nitrogen for use.③Pathological changes was observed by means of HE, PAS, Masson's, and PASM.④JNK, p-JNK was analyzed semiquantitatively by using the method of immunol histochemistry.⑤JNK mRNA expression of kidney tissue was examined by reverse transcription-polymerase chain raction(RT-PCR).⑥Protein expression of JNK and p-JNK was examined by using Western Blot.Results: 1.Pathological changes of kidney structure:We observed the pathological changes of rat kidney tissue in the diabetes mellitus group (DM), and found that,compared with normal control group(NC), Bowman's space of the former group became small,the size of glomeruli increased, the amount of collagen and extracellular matrix increased, and basement membrane was irregularly thickened.We also found that extra glomerularmesangial region expanded, and the amount of mesangial cells increased and part of those cells dissolved. In addition, renal tubules deformed with narrowing,and nterstitium distributed unevenly with scattered fibration.Compared with DM group,glomeruli of the PIO group were uniform,and basement membrane distributed evenly with slight hyperplasia. Besides, dyeing of extra glomerularmesangial region was uneven,and slight paramorphia was found in renal tubules, moreover there was no fibration in nterstitium. 2.Immunol histochemistry(IHC):①There was little JNK and P-JNK expression in the glomeruli , renal tubules, and nterstitium of NC group.②JNK and P-JNK expression in these three parts of DM group were variant , and compared with that of NC group the expression increased.The difference of expression levels between these two groups was statistically significant(P<0.05).③There was no statistical significance between the JNK and P-JNK expression levels of the PIO group and that of NC group(P>0.05),but compared with DM group the change level of JNK and P-JNK expression in PIO group was statistically significant(P<0.05).3.RT-PCR:①There was almost no JNK mRNA expression in NC group.②The JNK mRNA expression in DM group increased dramatically when compared with that in NC group.③JNK mRNA expression level of PIO group decreased remarkably in contrast to that of DM group.④There was no evidently difference between the JNK mRNA expression of the PIO group and that of NC group.4.Western Blot:①Compared with that of DM group ,P-JNK and JNK protein expression of PIO group decreased with statistical significance (P<0.05).②P-JNK and JNK protein expression of DM group increased dramatically when compared to that of NC group,and the difference was statistically significant(P<0.05).③The ratio of JNK protein expression level to P-JNK protein expression level in PIO group increased with statistical significance(P<0.05),compared with that in DM group.④In PIO group,JNK and P-JNK protein expression level and their ratio was similar to what we observed in NC group and there was no statistical significance(P>0.05).Conclusions:①PIO can improve the Pathological changes of kidney structure of diabetic rats.②JNK and P-JNK are less expressed in kidney tissue of SD rat under normal condition, but their expression level are increased dramatically in kidney tissue of DM group with Insulin resistance(IR).Furthermore, the ratio of P-JNK to JNK are also increased.③JNK and P-JNK expression can be down regulated by the treatment of pioglitazone (PIO),along with the reduced ratio of P-JNK to JNK.④Suppressed expression level of phosphorylated JNK may play roles in the mechanism of the protective effect of pioglitazone(PIO)on rat kidney in DM group with insulin resistance. |
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