Study On The Effect And Mechanism Of Combination Use Of Rapamycin And Rosiglitazone In ADPKD

Background and objectiveAutosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited human renal diseases with an incidence of approximately1:500-1:1,000. ADPKD is the4th most common cause of end-stage renal disease (ESRD) after diabetes, hypertension and glomerulonephritis and constitutes2.3%of patients on chronic dialysis. It is characterized by the progressive enlargement of numerous fluid-filled cysts in both kidneys and includes extra renal manifestations, such as hepatic cysts, aneurysm, pancreatic duct dilatation, colonic diverticulitis, and heart valve deformed. ADPKD is a serious system disease and contributes a significant burden to the society and families. Mutations in the pkdl and pkd2genes encoding the polycystin-1(PC1) and polycystin-2(PC2) are responsible for the85%and15%of ADPKD cases, but the fundamental pathogenesis of the disease is not clear."two-hit hypothesis" and "primary cilium theory’ are the two main hypotheses for disease initiation. A third-hit theory has also been proposed recently. Cyst development and enlargement are associated with dysfunctions in epithelial cell proliferation, fibrosis, inflammation, fluid secretion into the cyst and extracellular matrix accumulation.Cyst epithelial cells over-proliferation is one of the most important pathogeneses, so ADPKD is also thought to be a tumor like disease. Multiple signaling pathways involve in cyst formation and progression. The most dominating pathways mediated are EGFR, cAMP and mTOR pathways. The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase which coordinates cell growth, proliferation, protein synthesis and transcription. According to the core part, mTOR can be divided into mTORC1and mTORC2complex. The mTORC1complex mainly regulates cell growth and metabolism whereas the mTORC2complex regulates cell actin synthesis and cell apoptosis. Regulatory mechanisms of mTOR pathway in complex, mutual regulated relations exist in mTORC1, mTORC2and mTORC1negative feedback pathways. Activation of the mTORC1pathway can not only inhibit mTORC2-Akt pathway through its downstream of ribosomal S6kinase (S6K) but also activate TSC1/2complex through the PI3K-Akt pathway feedback loop. Under normal circumstances, polycystin1has depressant effect on mTORCl and mTORC2pathway. With the dysfunction of polycystic protein, mTORC1pathway and mTORC2pathway active and cause abnormal proliferation and dedifferentiation of kidney cyst lining epithelial cell. Rapamycin can specifically inhibit mTORC1. In recent years, mTOR inhibitors have been studied in PKD animal models and PKD cell culture showing effectively suppressing effect to the growth of cysts. At present, researchers have focused on the application of mTOR inhibitors in the treatment of ADPKD patients with randomized controlled trials. But the results are not satisfactory.Peroxisome proliferator-activated receptor-γ(PPAR-γ), a member of the ligand dependent nuclear receptor superfamily, is expressed in a variety of tissues. It plays important roles in cell proliferation, fibrosis, and inflammation and has anti-tumor, anti-fibrosis, anti-inflammation effects. In recent years, the study found PPAR-γagonists also have protective effect on polycystic kidney, polycystic liver and cardiac defects. These protective effects may mediate through β-catenin, c-Myc, CFTR, MCP-1, S6, ERK and TGF-β signaling pathways.Combined inhibition of mTOR and ERK or other pathways shows synergistic effects and better effect than single drug therapy in cancer models. As a kind of tumor like disease, ADPKD patients may also benefit from the multitargeted drug therapies. The purpose of this study is to observe the expression changes of mTOR pathway in renal tissue in ADPKD and effect on polycystic kidney in Han:SPRD rats model using rapamycin and rosiglitazone, to explore the influence of combined application of rapamycin and rosiglitazone on renal cyst lining epithelial cell, to establish the possible therapeutic value of multitargeted drug therapy using mTOR inhibitor and PPAR-γ agonists in the treatment of defects in progressive ADPKD.Methods1、We analyzed blood specimens and renal tissue specimens from male heterozygous Han:SPRD rat of different week age, to observe the changes of renal function and renal morphological in heterozygous Han:SPRD rat of different week age.2、We used western blotting method to study the variance of mTOR, P-mTOR, p70S6K and P-p70S6K between kidney tissues of Han:SPRD rats Cy/+and Han:SPRD rats+/+.3、We used western blotting method to study the variance of mTOR, P-mTOR, p70S6K and P-p70S6K between kidney tissue of ADPKD patients and normal human control. 4、We used different doses of rapamycin and rosiglitazone on male Han:SPRD heterozygous rats separately or jointly for24weeks, analyzed blood specimens and renal tissue specimens to observe the renal morphological change and protective effect of combined medication on renal function.5、We detected the expression of proliferating cell nuclear antigen PCNA and mTOR downstream molecule p70S6K using immunohistochemical method to observe the effect of combined therapy on cell proliferation and regulation of the mTOR pathway.6、We used western blotting method to study the variance of mTOR, P-mTOR, p70S6K, P-p70S6K, ERK, P-ERK, Akt and P-Akt in Han:SPRD rat kidney tissue to observe the regulation of the mTOR pathway in rat models.7、We assessed the combination effect of rapamycin and rosiglitazone to cystic lining epithelium cell line WT9-12viability by using the MTT assay.8、We assessed the combination effect of rapamycin and rosiglitazone on cell cycle and apoptosis in WT9-12cell by using flow cytometry assay.9、We used western blotting method to study the variance of mTOR, P-mTOR, p70S6K, P-p70S6K, ERK, P-ERK, Akt and P-Akt in WT9-12cell to observe the their regulation effect to mTOR pathway.10、After blocking mTORCl negative feedback loop with PI3K specific inhibitor LY294002, we used western blotting method to observe the combination effect to mTOR pathway.Results1、The renal function in male heterozygous rats and wild-type rats renal function at4week are almost the same level, then renal function of male heterozygous rats deteriorated rapidly. The speed of renal dysfunction reached relative platform at20weeks. The cyst variation index and the ratio of kidney weight to body weight had a similar change as renal function. Renal cyst occurred mainly in the renal medulla, increased in the renal cortex.2、The P-mTOR and P-p70S6K are upregulated in kidney tissue of Han:SPRD rats Cy/+, but we did not find a significant trend in rat of different week-old.3、The P-mTOR and P-p70S6K are upregulated in kidney tissue of ADPKD patients.4、Compared to using rosiglitazone or rapamycin alone, the combined application can significantly inhibit the development of cyst, decreased the ratio of kidney weight to body weight, improved renal function, significantly reduced the dose of rapamycin, obtained better effect. Rosiglitazone can ameliorate the hyperlipidemia induced by rapamycin. Combination application can also significantly improve cyst index and improve the renal interstitial inflammatory and fibrosis.5、Combined use of the two drugs can inhibit cell proliferation, showing a better effect to using rosiglitazone or rapamycin alone. Rapamycin and rosiglitazone reduced the number of p70S6K stained cells. Combined use of the two drugs can reduce the number more significant.6、The P-Akt and P-ERK are downregulated in kidney tissue of Han:SPRD rats Cy/+after rosiglitazone treatment while rapamycin upregulated the P-Akt and P-ERK level.7、Either rosiglitazone or rapamycin could decrease proliferation rate of WT9-12cell, and combined use of the two drugs had a better effect.8、Rapamycin can induce cell apoptosis. Rosiglitazone can’t induce cell apoptosis, but enhance the effect of rapamycin. Either rosiglitazone or rapamycin can inhibit the cell cycle in G0/G1phase, and the combination application improved this effect significantly.9、Rosiglitazone can decrease the expression of phosphorylated Akt, ERK while rapamycin upregulate the phosphorylation of Akt, ERK in WT9-12cell. It suggest that rosiglitazone may play have a suppression effect to PI3K.10、After blocking mTORCl negative feedback loop, rosiglitazone can further reduce the expression of phosphorylated Akt. Although the expression of phosphorylated ERK was also decreased, but having no significant difference comparing to the control group.ConclusionThe result suggests that the mTOR signaling pathway is aberrantly activated in cystic epithelia of ADPKD patients and Han:SPRD rat model. The abnormal activation of mTOR signal pathway and its downstream molecules S6K lead to excessive cell proliferation, cell apoptosis and cytoskeleton structure. This the one of the most important virulence factors of ADPKD. The in vivo experiments confirmed that rosiglitazone combined with rapamycin can obtain better effect than treat with rapamycin alone. In vitro experiments showed that synergistic effect of combined medication may contribute to the inhibition effect of rosiglitazone on the molecule Akt which plays an important role in mTORC1negative feedback pathway and mTORC2pathway.