From Vascular Endothelial Growth Factor And Other Molecular Expression To Explore Yiqi Heat Paste On The Influence Of Glomerular Filtration Barrier

Objective:To observe the influence of Yiqi Qingre Extract on the glomerular filtration barrier of puromycin aminonucleoside (PAN) treated rats, and discuss the renoprotective mechanism of Yiqi Qingre Extract.Method:48male Wistar rats were randomly divided into4groups:sham group, PAN model group, Yiqi Qingre Extract group and the fosinopril group; Rats of the experimental groups were performed with injection of PAN at a dose of40mg/kg via the left jugular vein, while rats of the sham group were given an injection of0.9%normal saline at the same volume. Twenty-four hour urinary protein levels were measured at last day before the injection and the3rd,5th,10th day after the injection.6rats form each group were sacrificed at the3rd and10th day after the injection and get blood form abdominal aorta.The levels of serum total protein, albumin, creatinine, urea nitrogen, cholesterol and triglycerin levels were examined. Use Renal tissue and observe the alterations of each rat with light microscope and electron microscope. mRNA level of nephrin,podocin,CD2AP and VEGFA were examined with RT-PCR. Protein expression of podocin, CD2AP and VEGF were tested with Western blot. The level of Protein of nephrin was examined with immunohistochemistry.Result:1. Yiqi Qingre Extract can decrease24hour urinary protein of rats:Each group rats did not have urinary protein at the3rd day after injection(P>0.05). At the5th day after the injection, except form the sham group, the rest of the group have a great deal of urinary protein(P<0.01).But there was no great discrepancy with each group; At the10th day after the injection, the Yiqi Qingre Extract group and fosinopril group can decrease24hour urinary protein of rats greatly(P<0.05).2. The influence of Yiqi Qingre Extract to serum level:There was no discrepancy of serum level with each group at the3rd day after injection. At the10th day, both Yiqi Qingre Extract and the fosinopril can improve disorder of blood-lipid and increase the volume of total protein and albumin in serum.3. Result of light microscope and electron microscope:At the 3rd day after the injection, compare with the sham group, the model group, the YiQi QingRe group and the fosinopril group didn’t have evident renal tissue pathologic damage under light microscope. But under the electron microscope, the model group, the YiQi QingRe Extract group and the fosinopril group had extensive damage of glomerular filtration barrier structure. There was no great discrepancy among the three groups. At the10th day after the injection, observed under both light microscope and electron microscope, Yiqi Qingre Extract group showed less structural damage of glomerular filtration barrier compared with model group.4. Result of molecular biology:The result of Western blot and immunohistochemistry showed:At the third day after injection, compared with the sham group, nephrin、podocin and CD2AP protein expression of the model group decreased, the level of VEGF protein expression increased, YiQi QingRe extract can raise the level of nephri、podocin and CD2AP protein expression (P<0.05), both YiQi QingRe Extract and fosinopril can inhibit VEGF expression significantly (P<0.05). At the10th day after the injection, compared with the sham group, nephrin、 poducin and CD2AP protein expression decreased, VEGF-A mRNA expression increased, YiQi QingRe extract can raise the level of nephrin、podocin and CD2AP protein expression (P<0.05), both YiQi QingRe extract and fosinopril can inhibit VEGF expression (P<0.05). PT-PCR result showed: At the3rd day after the injection, compared with the sham group, the level of mRNA of nephrin、podocin and CD2AP protein did not have evident alteration,The mRNA expression of VEGF-A increased, especially the VEGF161had a significant growth (P<0.05), both YiQi QingRe extract and fosinopril had a evident inhibition to mRNA expression of VEGF-A. At the10th day after injection, compared with the sham group,The mRNA expression of nephrin、poducin and CD2AP decreased, the level of mRNA expression of nephrin、podocin and CD2AP increased at the model group, both YiQi QingRe extract and fosinopril can inhibit the growth (P<0.05), compared with the result at the3rd day after injection, The mRNA expression of VEGF-A increased in all groups, especially at the model group,YiQi QingRe extract and fosinopril can inhibit it’s growth Conclusion:1. YiQi QingRe extract can reduce urinary protein of PAN model rats, improve dyslipidemia of rats, enhance the volume of albumin and total protein, improve renal pathological damage effectively. The mechanism maybe increase the protein expression of nephrin, podocin and CD2AP, inhibit protein expression of GEGF, keep the integrity of podocyte slit diaphragm and endothelial fenestrae to relieve the damage of glomerular filtration barrier.2. There was no proteinuria at the3rd day after injection, the renal filtration barrier structure was significantly damaged, foot process inosculated and endothelial fenestrae was disorganized. The mRNA expression of VEGF increased earlier than the mRNA alteration of nephrin、podocin and CD2AP at the model group.