| Background and Objective:Polycystic kidney disease is a disorder disease characterized by the formation of multiplerenal tubular epithelial cells, liquid filled vesicles. The cysts mainly generated in the tubularand collecting duct. Excessive proliferation of epithelial cells is the mainly pathogenic andthe causes of developmental defects. Autosomal dominant polycystic kidney disease is oneof the most common type and the most common single-gene hereditary kidney disease. Itsincidence rate is about1/400-1/1000, and there are about13million patients in the world.According to the results of the2010Annual Survey of USRDS, ADPKD ranked fourth inthe cause of the original incidence of ESRD, second only to diabetic nephropathy,hypertensive nephropathy and primary glomerulonephritis. A half of the patients in the ageof60will progress to ESRD. Besides kidney cysts, ADPKD can also cause liver cysts,pancreatic cysts, splenic cysts, intracranial aneurysms, valvular heart disease and colondiverticula and other extra-renal lesions. The patients with this disease can transmit to futuregenerations. The offspring probability of illness was50%. There is5%-20%of spontaneousmutations in ADPKD.The treatment of polycystic kidney disease has plagued the majority of renal doctors.Gene therapy is undoubtedly the best choice for genetic diseases, however, the two genespkd1and pkd2’s cDNA has difficulties to introduce in. In the other hand, PKD1and PKD2distribute a wide range in vivo and it is difficult to overcome the downstream events such asinflammation, epithelial cell proliferation or fibrosis. At present, PKD gene therapy is failureand drug treatments get more attention.Some drugs such as rapamycin, tyrosine kinaseinhibitors, vascular vasopressin V2receptor antagonists, growth hormone analogues, metalprotease inhibitors are used to treat polycystic kidney disease, but there are no specific drugsfor the treatment of polycystic kidney disease yet. We found that the peroxisome proliferator-activated receptor (PPARγ) pioglitazone cantherapy polycystic kidney disease mice, but with cardiac enlargement. Taking into accountthe polycystic kidney disease is a chronic disease,we should pay attention to its long-termsafety of using medications.Our department and Shen Jianhua Group from the ShanghaiMateria Medica Institute cooperate to develop a variety of non-TZD class of PPARγagonists, namely carboxylic acids peroxisome proliferator-activated receptor agonist.Through the transformation of its chemical structure, we hope to be able to retain itstreatment of polycystic kidney disease effects while reducing the toxic side effects. Dr.Xiong Xishan in our Department screening of these compounds in tumor cells and nudemice with tumor found that DJ5can inhibit tumor cell and mice growth with very slightactivity of PPARγ. Based on the role of these compounds in tumor cells, we choose DJ5in-depth study. Study the compounds on inhibition of proliferation cyst substrate in humanepithelial cells WT9-12and the therapeutic effect of the zebrafish kidney cyst model, andinvestigate the protective effect mechanisms.Methods:1. Reproduce and identificate the zebrafish renal cysts model hi459/scorpion,hi4166/pkd2. Build pkd2morphants. Intervent the model with DJ5in50%epiboly.Observed trunk flexion, left-right asymmetry, cystic changes.2. Observed the three days zebrafish embryos’ glomerular and renal tube morphologicalchanges with the intervention of DJ5using HE stain, immunofluorescence of whole embryo.3. Using Western blot technique to detect the expression of p-Akt in zebrafish tissuse.4. Using the MTT assay, detect different concentrations (0,12.5,25,50,100,200μmol/L)of DJ5inhibit the proliferation of human renal cyst-lining epithelial cells WT9-12.5. Using the LDH method, detect different concentrations (0,12.5,25,50,100,200μmol/L) of DJ5affects human renal cyst-lining epithelial cells WT9-12cytotoxicity.6. Using Western blot technique to detect human renal cyst-lining epithelial cellsWT9-12Akt, its activated form p-Akt and its downstream pathway FoxO3a and its inhibitorp-FoxO3a expression after intervention of differernt concentrations (0,25,50,100μmol/L)of DJ5.7. Using flow cytometry, detect human renal cyst-lining epithelial cells WT9-12cellcycle and apoptosis changes after intervention at different concentrations (0,25,50,100μmol/L) of DJ5. 8. Using Western blot technique to detect renal cyst-lining epithelial cells WT9-12cellcycle and apoptotic proteins P21CIP/WAF1, cyclin A and PARP expression after intervention ofdifferernt concentrations (0,25,50,100μmol/L)of DJ5.Results:1. In the hi459model, the ratio of cysts was23.39percent without DJ5, verous15.41percent with DJ5; DJ5can affect the torso bending, left-right asymmetry in hi4166in somedegree; In the pkd2morphants, the ratio of cysts was33.28percent without DJ5, verous10.23percent with DJ5, and after DJ5treatment, pkd2morphants’s glomerular and renaltubular structure are close to normal, and the express of p-Akt was decreased after usingDJ5.2. DJ5can significantly inhibit the WT9-12cell proliferation, and this effects enhanceswith the time and concentration; The LDH release have a sudden increase in the200mmol/Lof DJ5, but at100mmol/L there is no significant difference with the control group.3p-Akt, p-FoxO3a is down-regulation, and FoxO3a is up-regulation in DJ5groups bywestern bolt.4DJ5can make WT9-12cells arrested in S phase, and P21CIP/WAF1is up-regulation,cyclin A is down-regulation.100mmol/L DJ5can make WT9-12apoptosis, and theexpression of PARP with89kd is enhanced.Conclusion:In this study, we use the zebrafish polycystic kidney disease model and found DJ5have asignificant therapeutic effect on renal cysts of the zebrafish; DJ5can obviously inhibitWT9-12cell proliferation by down-regulation p-Akt and p-FoxO3a and up-regulationFoxO3a.DJ5can make WT9-12cells arrested in S phase and promote apoptosis. This studyprovides an experimental basis and theoretical background, and has clinical value andpractical significance. |
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