The Therapeutic Effect And Mechanism Research Of Rapamycin On IgA Nephropathy Rat

IgA nephropathy (IgAN) is the most frequent glomerulonephritis worldwide. The primary form of the disease used to be considered quite harmless. Now, IgAN is recognized as a major health problem because20to40%of patients progress to end-stage renal disease (ESRD) within20years of disease onset and an additional20%exhibit progressive kidney damage. The present study was designed to explore the renoprotective potential of low-dose mTOR inhibitor rapamycin in an IgA nephropathy rat model and the possible mechanism of action, at the same time explore the mechanism of rapamycin inhibition mesangial cell proliferation and effect on signal transduction pathway PI3K/AKT/mTOR and MAPK/ERK1/2.Part one The curative effect observation and mechanism research of Rapamycin treatment on IgA nephropathy ratObjective IgA nephropathy (IgAN) is the most frequent glomerulonephritis worldwide. Different therapeutic approaches have been tested against IgAN. The present study was designed to explore the renoprotective potential of low-dose mTOR inhibitor rapamycin in an IgA nephropathy rat model and the possible mechanism of action.Methods After establishing an IgA nephropathy model, the rats were randomly divided into four groups:control, control with rapamycin treatment, IgA nephropathy model and IgA nephropathy model with rapamycin treatment. Rapamycin (1.0mg/kg per day) was dosed intragastrically. The body weight of the rats was measured weekly. At the end of4,8,12,16weeks, a24-h urine collection was obtained using metabolic cages. Ponceau’s stain was utilized to measure24-h urinary protein levels. The rats were deeply anesthetized, and tissue samples were processed and stored at16weekend. Hepatic and renal function was determined with an DxC800autoanalyzer. Direct immunofluorescence technique was used to test the intensities of fluorescence of mice kidney frozen sections. The cell morphologic changes of renal tissue were observed by HE、PAS、Masson staining.Proliferation was assayed via BrdU incorporation. Real-time PCR and immunohistochemistry were utilized to detect the expression of α-SMA, Collagen I, Collagen III, TGF-β1and PDGF. Western blotting and immunohistochemistry were performed to determine p-S6protein levels.Results Low-dose mTOR inhibitor rapamycin prevented an additional increase in proteinuria and protected kidney function in a model of IgA nephropathy. Rapamycin directly or indirectly interfered with multiple key pathways in the progression of IgAN to end-stage renal disease (ESRD):1) reduced the deposition of IgA and inhibited cell proliferation;2) decreased the expression of fibrosis markers:a-SMA、 type III collagen; and3) down-regulated the expression of the profibrotic growth factors PDGF and TGF-β1. The expression of p-S6was significantly elevated in IgAN rats.Conclusions The mTOR pathway was activated in IgAN rats, early application of small dose rapamycin can block mTOR pathway. Rapamycin reduce proteinuria and IgA deposits, inhibit glomerular mesangial cell (MC) proliferation, cytokine and extracellular matrix (ECM) secretion and the interaction between of them, slow the renal injury of IgAN in rats. Part two The mechanism research of rapamycin inhibition on rat mesangial cells proliferationObjective Mesangial cell proliferation is the main pathological characteristics of IgA nephropathy, so we cultured rat mesangial cells. The present study was designed to explore the mechanism of rapamycin inhibition mesangial cell proliferation and effect on signal transduction pathway PI3K/Akt/mTOR and MAPK/ERK1/2.Methods Culture HBZY-1cell Conventionlly, divided into the control group, PDGF groups, RAPA lnmol/L、RAPA10nmol/L、RAPA100nmol/L、RAPA1000nmol/L group. MTT method to detected24h,48h cell proliferation, the cell cycle and cell apoptosis were detect by flow cytometry; Protein extraction and Western blotting analysised the expression of renal cortical cell cycle protein Cyclin D1, Cyclin E, CDK2and CDK4; Real-time PCR and Western blotting were used to determine p27mRNA and protein levels; Western blotting detected the expression quantity of signaling pathways protein p-P70S6K/P70S6K, p-AKT/AKT, p-ERKl/2/ERKl/2.Results1) Rapamycin dose dependent inhibited the mesangial cell proliferation was caused by PDGF, block the cell cycle in GO-G1phase, but in100nmol/L and1000nmol/L group did not see obvious difference; Rapamycin didn’t affect mesangial cell apoptosis;2) rapamycin did’t affect the expression of increased Cyclin D1, E and CDK2,4were stimulated by PDGF; But it could regulate the transcription of mRNA, increase the expression of p27protein;3) rapamycin dose dependent inhibited phosphorylation of P70S6K, the strongest inhibitory effect appeared in1000nmol/L group; rapamycin were no significant impact on phosphorylation of AKT and ERK1/2in10nmol/L、100nmol/L groups, p-AKT、p-ERK protein expression quantity slightly higher in1000nmol/L group.Conclusions Rapamycin through mTORCl/P70S6K passway regulated mRNA transcription, increased the expression of p27protein, thereby inhibited the activity of cyclin E-CDK2, block the cell cycle by GO-G1phase to S phase, inhibited cell proliferation. In rat mesangial cells, large dose of rapamycin (1000nmol/L) may be feedback activate the passway of AKT and Ras/Raf/ERK, thus weakened the inhibition effect of rapamycin on cell proliferation.