| Objectives IgA nephropathy(IgAN) was described for the first time by Berger and Hinglais in 1968, so it was also named Berger's disease whose clinical characteristic is hematuria. Now, it is considered that IgA nephropathy is one of the most common glomerular diseases in the world. The constituent ratio of IgA nephropathy in glomerular diseases in China is about 26% to 43%. It is found that there are 9% to 50% patients with IgAN got end-stage renal disease(ESRD) irreversibly in five to twenty-five years when they were diagnosed as Ian. The pathological characteristics of IgAN are mesangial matrix deposition, crescent formation and interstitial damage in adult, while it is mesangial cell proliferation in children, which means that the clinical features are hematuria and albuminuria in children while they are hematuria, albuminuria and renal dysfunction in adult. But the pathogenesis of IgAN is unknown. Many literature data are about deposit of immunity complex in the mesangium while there are a few of reports about mesangial cell proliferation. Extracellular signal regulated MAP kinase(ERK), which is also named p42P44 MAPK, is a focus in research field. Ras-raf-MEK-ERK1/2 is one of the most classical signal transduction pathways in mitogen–activated protein kinase(MAPK). When extracellular signal regulated MAP kinase is stimulated by some factors, the immediate early genes(IEG) is stimulated too, which makes cells change. It is proved that the change of expression of MAPK especially ERK1/2 affects mesangial cell proliferation heavily, but it is unknown whether ERK1/2 involve in pathogenesis of IgAN. The objectives of this experiment is to observe the expression of ERK1/2 in IgAN rats by IgAN model established and rats with rapamycin, to find out the onset characteristics of IgAN and to find pathogenesis of it. IgAN treat with a large dosage of glucocorticoid, fish oil, ACEI, ARB and immunosuppressant like CsA, cyclophosphamide and so on, but until recently there is no treatment standard. Rapamycin(RAP) is one of macrolides antibiotics which are got from actinomycetes culture medium by Ayerst research institute in 1970s, and then Martal found immunosuppressive effect of RAP. It is proved that RAP can inhibit activation of signal transduction pathways, inhibit mesangial cell proliferation and interfere with cyclin and cell dependent kinase by different cytokine. Rapamycin is high effect and low toxicity immunosuppressive agents, and it is used to treat tumor, organ transplantation and so on. But there are a few of reports about rapamycin used to treat IgAN. This experiment make we investigate the effect of RAP on IgAN by different experimental methods and provide evidence for pathogenesis of IgAN. Methods1 Animal and agentsExperimental animal were male Wistar rats about 80 to 100g from Experimental Animals Center of Hebei.2 Set up IgAN model and divide groups2.1 Set up rat model of IgAN: the rats of IgAN group had been administered with bovine serum albumin by gavage at a dose of 400mg·kg-1·d-1 every two days for 6 weeks and injected 0.4ml castor oil plus 0.1ml CCl4 once a week for 9 weeks. Lipoplysaccharides were injected at 6th and 8th weeks. These rats were observed until 10th week. Renal tissue was cut into 3μm frozen section. Deposition of IgA in mesangium by the fluorescence microscope is positivity, mesangial cell proliferation and deposition of matrix by PASM and Masson staining. These results indicated IgAN model is successful and achievement rate is 90%(Fig.1). Unsuccessful rats got same agents until they are IgAN.2.2 Divide groups: male Wistar rats were divided into three groups: Control group(15), IgAN group(15) and rapamycin treated group(RAP group, 15). The method to make IgAN group and RAP group is as same as that of part 2.1. After the IgAN model was affirmed to be successful, the rats of rapamycin treated group were administered daily with rapamycin by gavage for 4 weeks. These rats were observed until 14th week. And rats of IgAN group had water and meals daily. The rats of Control group were administered daily with normal saline at same time.3 Detection of biochemistry targetsCollect 24 hours urine to detect 24 hours urine protein and bloodletting to get BUN and Scr before they were sacrificed.4 Detection of ERK1/2 in renal tissue of rats4.1 Detection of ERK1/2 in rats by immunohistochemistryPartial renal tissue was fixed in 4% formaldehydum and embedded with paraffin after model established. The microscope slides were washed by polylysine and sections were about 3μm. The primary antibody was rabbit anti-ERK1/2 polyclone antibody, and secondary antibody was PV9000 multiplex. Tissue with PBS was used as negative control. Detect the expression of ERK1/2 by PV9000 Kit. Positive site was colored brown-yellow.4.2 Detection of ERK1/2 protein in rats by Western blotPartial renal tissues were sheared by scissor, and then lap them. Put them in ice bath 1h, then centrifuge them at 14000r/min for 25 minutes in 4℃and extract total protein. We analyze specific protein band after electrophoresis and development.4.3 Detection of ERK1/2 cDNA in rats by RT-PCRPartial renal tissues about 3×3mm were grinded with Trizol and extracted mRNA. ERK1/2 mRNA was reverse transcribed to cDNA. Detect ERK1/2 cDNA in rats by RT-PCR and get the data about cDNA. Results1 The results of UPO, BUN and Scr showed that content of 24hUPO, BUN and Scr of IgAN rats are more higher than those of RAP group(P<0.001) and those of Control group(P<0.05). 24hUPO, BUN and Scr of RAP group are higher than those of Control group(P<0.05, Table 1).2 Comparing with the Control group, expression of ERK1/2 protein in renal tissue of IgAN rats was increased which detected by immunohistochemistry method(P<0.05). Comparing with the RAP group, expression of ERK1/2 protein in renal tissue of IgAN rats was increased obviously(P<0.001). Expression of ERK1/2 protein in RAP group is higher than that in Control group(P<0.05).3 Expression of ERK1/2 protein in IgAN group was increased comparing with Control(P<0.05) and RAP group(P<0.001) by Western blot. Expression of ERK1/2 protein in RAP group is higher than that in Control group(P<0.05).4 Expression of cDNA in Control group(P<0.05) and RAP group(P<0.001) is weaker than that in IgAN group by RT-PCR. Expression of cDNA in Control group is weaker than that in RAP group(P<0.05).Conclusions1 Expression of ERK1/2 in IgAN rats rises comparing with the others, at the same time, expression of ERK1/2 in RAP group was increased comparing with that in Control group, which indicates that many factors could stimulate ERK1/2 signal transduction parthway which will probably lead to IgAN.2 Rapamycin could inhibit ERK1/2 signal transduction pathway on glomerular mesangial cell of IgAN rats, which indicates rapamysin could treat IgAN rats but it is unknown the dose of it to cure IgAN.3 Inhibitor of ERK1/2 signal transduction pathway may be a new routine of treating IgAN.
|
PDF Full Text Request