Inhibiting Effect Of Rosiglitazone And Rapamycin Combination On Cystic Epithelial Cells In ADPKD And Its Mechanism

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human hereditary kidney diseases with a prevalence of 1/400-1/1000, affecting more than 1.5 million people and accounting for about 5% of end-stage renal disease patients who require renal replacement therapy in China. The disease is characterized by progressive formation of multiple renal cysts affecting all segments of renal tubules. About 50% ADPKD patients eventually develop renal insufficiency in the fifth or sixth decades of life. Renal involvement is often accompanied by extra-renal manifestations, including hepatic and pancreatic cysts, cardiac valvular defects, colonic diverticulosis and intracranial aneurysm. So it is a fatal systematic disease. Great efforts have been attempted for years to find a cure for PKD. Although gene therapy seems to be a candidate, many problems need to be resolved before it could be used clinically. Seeking new drugs remains to be the focus of research at present. Dai Bing etc al from our lab reported that PPARy agonist pioglitazone prolonged survival of spontaneous mutational strain Han:SPRD rats and reduced renal cystogenesis, but the mechanism of TZDs on PKD is unknown. A report suggested mTOR pathway as a converging mechanism leading to renal cyst formation. Knowing that TZDs decrease phosphorylation and activity of p70S6 kinase (a downstream target of mTOR pathway), we hypothesized that TZDs might suppress cyst growth via inhibiting mTOR pathway. Multidrug therapy is usually required for optimal treatment of cancer. It is very likely that the treatment of ADPKD requires the same approach. Thus, we also examined the effect of rosiglitazone plus rapamycin on cystic cell growth. Rosiglitazone is an anti-glycemic agent and used clinically for patients with type 2 diabetes. We showed here for the first time that rosiglitazone decreased the proliferation of cystic epithelial cells. Addition of rosiglitazone to WT9-12 cell, an immortalized cystic epithelial cell line, inhibited cell growth in a dose-and time-dependent manner with an IC50 of 100μM. It was found in our study that 50 and 100μM rosiglitazone did not induce apoptosis in WT9-12 cells, but 200μM rosiglitazone induced an 8.18% increase in apoptotic cell death. As IC50 of growth inhibition by rosiglitazone occurred at a concentration of 100μM, the suppressive effect of rosiglitazone on cystic cell growth seemed to be largely mediated by its inhibition of cell proliferation. This was supported by the result of cell cycle analysis in which we found an increase in the number of cells in G0/G1 phase and a decrease in the number of cells in S phase after 50-100μM rosiglitazone treatment. mTOR pathway plays a critical role in regulating protein synthesis, cell growth and proliferation. The activation of mTOR results in increased p70S6K activity and translation machinery. mTOR pathway is shown to be activated and plays a role in the progression of ADPKD in both patients and mice. We therefore determined whether rosiglitazone would also affect mTOR/p70S6K in ADPKD cells. The result showed that rosiglitazone induced a dose-dependent decrease in p70S6K phosphorylation. The inhibition occurred 1 h after the treatment and was most obvious after 24 h. However, mTOR phosphorylation and 4E-BP1 phosphorylation, another downstream target of mTOR, remained unchanged after rosiglitazone treatment, indicating that the effect of rosiglitazone on p70S6K in ADPKD cells was not likely to be mediated by mTOR. The mechanisms responsible for the effects of rosiglitazone seem to involve both PPARγ-dependent and PPARγ-independent signals. Our study showed that the activation of PPARγmay play an important role in rosiglitazone-induced inhibition of p70S6K phosphorylation in WT9-12 cells, since blocking of PPARy activity by GW9662 or by knocking-down PPARγexpression largely prevented the effect of rosiglitazone.There is angiogenesis in ADPKD. Vascular endothelial growth factor (VEGF) is also known as a potent agent in angiogenesis. VEGF is strongly induced in hypoxic conditions via hypoxia inducible factor (HIF) regulated elements of the VEGF gene. We observed the expression of VEGF, HIFαand TNFαin urine and kidney tissue of Han.SPRD in different stages (4,8,12,16 and 24 weeks) by real-time PCR. The result showed that the expression of VEGF and TNFαmRNA was upregulated in Han:SPRD in the early stage of 4 weeks, but the expression of HIF1αwas normal. The expression of HIF1αbegan increasing from 8 weeks, indicating that VEGF upregulation might be related to TNFa in the early stage. To examine whether TNFαcontributed to the regulation of VEGF, WT9-12 cells were treated with TNFα, and the expression was detected by real-time PCR. TNFα(20ng/ml) led to a 15-fold increase of VEGF mRNA, but TNFαdid not induce increase of HIF1α. To determine whether other cytokines could also upregulate VEGF expression, WT9-12 cells were treated by LPS (20ng/ml), IGF-1(20ng/ml) and TGFβ(20ng/ml). The result showed that VEGF was increased by LPS, but not by IGF-1 and TGFβ, indicating that inflammatory cytokines, but not mitogens, could induce VEGF expression.In addition, we investigated whether rosiglitazone could inhibit angiogenesis in ADPKD. WT9-12 cells were treated with different concentrations of rosiglitazone, and the expression of VEGF was detected by real-time PCR. The result showed that rosiglitazone downregulated the level of VEGF, and 50μM rosiglitazone decreased VEGF level by 30%. However, rosiglitazone was able to block TNFa-induced VEGF expression in WT9-12 cells completely. Rosiglitazone has been used to treat diabetes for many years with few reported adverse effects. We supposed that rosiglitazone would also be suitable for ADPKD therapy, especially in the early stages of the disease.Since both rosiglitazone and rapamycin are clinical drugs that may have potential for ADPKD therapy, we tested the effects of combined use of the two drugs on cystic cell growth.50ng/ml rapamycin plus 50μM rosiglitazone significantly increased the inhibitory effect on cell growth as compared with either of the two drugs alone (p<0.05). R Value was 1.01, indicating an additive effect. This additive effect was still present when the dosage of rosiglitazone was increased to 100 and 200μM, R value being 1.14 and 1.08 respectively. Knowing that sequence-specific synergism is optimal cancer chemotherapy, we supposed that the same strategy may also be suitable for ADPKD treatment. We therefore further assessed the effect of sequential treatment with rapamycin and rosiglitazone on cystic cell proliferation. Interestingly, combination use in the sequence of rapamycin plus rosiglitazone, the R value was greater than 1 (mean 1.62), indicating that the interaction was synergistic. However, no such a synergistic effect was observed when rapamycin was used after discontinuation of rosiglitazone. Further, we investigated the effect of concomitant use of the two drugs on VEGF. Combination of 50ng/ml rapamycin and 50μM rosiglitazone decreased the expression of VEGF by 54%, as compared with either of the two drugs alone (rosiglitazone 18% and rapamycin 30%).In conclusion, rosiglitazone (a thiazolidinedione derivative) suppressed cystic cell growth. This effect was mediated partially via an mTOR-independent inhibition of p70S6K phosphorylation and PPARγ-independent manner. Rosiglitzone also inhibited angiogenesis by downregulating VEGF expression. Combination of rosiglitazone and rapamycin increased the efficiency of cell growth and angiogenesis inhibition, as compared with either of the two drugs alone. As rosiglitazone is an anti-diabetic drug clinically used for long-term treatment, it may have a potential for ADPKD therapy. Combination therapy may be a potential and a trend for ADPKD therapy.