Inactivation of max-interacting protein 1 induces renal cilia disassembly through reduction in levels of intraflagellar transport 20 in polycystic kidney.

Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.

Blockade of interleukin-8 receptor signalling inhibits cyst development in vitro, via suppression of cell proliferation in autosomal polycystic kidney disease.

AIM: Autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent inherited disorder and results in the progressive development of cysts in both kidneys. In recent studies, several cytokines and growth factors secreted by the cyst-lining epithelia were identified to be upregulated and promote cyst growth. According to our previous study, chemokines with a similar amino acid sequence as human interleukin-8 (IL-8) are highly expressed in a rodent model with renal cysts. Therefore, in this study, we focused on whether IL-8 signalling is associated with renal cyst formation, and tested the possibility of IL-8 as a new therapeutic target for ADPKD. METHODS: Expression of IL-8 and its receptor were screened either by enzyme linked immunosorbent assay (ELISA) or Western blot. Inhibited IL-8 signalling by antagonist for IL-8 receptor or gene silencing was tested in molecular levels, mainly through Western blot. And cell proliferation was measured by XTT assays. Finally, a three-dimensional culture was performed to understand how IL-8 affected cyst formation, in vitro. RESULTS: Interleukin-8 secretion and expression of its receptor highly increased in two different human ADPKD cell lines (WT9-7 and WT9-12), compared to normal human renal cortical epithelial cell line. Cell proliferation, which is mediated by IL-8 signal, was inhibited either by an antagonist or siRNA targeting for IL-8 receptor. Finally, a three-dimensional culture showed an alleviation of cystogenesis in vitro, after blocking the IL-8 receptor signals. CONCLUSION: These results suggest that IL-8 and its signalling molecules could be new biomarkers and a therapeutic target of ADPKD.

Over-expression of Mxi1 represses renal epithelial tubulogenesis through the reduction of matrix metalloproteinase 9.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease. ADPKD is characterized by cyst development that leads to abnormal kidney structure. Renal tubules are a fundamental unit of architecture, so controls of tubular growth and formation are important for proper kidney function. The molecular mechanisms of tubulogenesis are being actively studied as the basis of diagnosis and treatment of ADPKD. Mxi1 is a member of the MAD family of proteins that functions in terminal differentiation, inhibition of cell cycle progression and tumor suppression, while the Myc protein, which is antagonized by Mxi1, causes renal cystogenesis. Based on these molecular relationships, the present study implicated Mxi1 with ADPKD be demonstrating that curtailed Mxi1 gene expression caused cyst formation in Mxi1-deficient mice. To ascertain whether Mxi1 affects renal epithelial cell tubulogenesis, three-dimensional cultures (3D culture) of mIMCD-3 cells and stably Mxi1 over-expressed mIMCD-3 cells were established. The results indicated that over-expression of the Mxi1 gene plays a role in the regulation of tubulogenesis by regulating some genes participating in renal epithelial branching tubulogenesis such as matrix metalloproteinase 9 (MMP9), integrins, fibronectin, and E-cadherin. The results support the suggestion that over-expression of Mxi1 can suppress renal epithelial tubulogenesis. In particular, MMP9 is greatly affected by the expression level of Mxi1. It can be concluded that mIMCD-3 cells that stably over-express Mxi1 fail to form renal epithelial tubules because of abnormally reduced expression of MMP9.

Mxi1 influences cyst formation in three-dimensional cell culture.

Cyst formation is a major characteristic of ADPKD and is caused by the abnormal proliferation of epithelial cells. Renal cyst formation disrupts renal function and induces diverse complications. The mechanism of cyst formation is unclear. mIMCD-3 cells were established to develop simple epithelial cell cysts in 3-D culture. We confirmed previously that Mxi1 plays a role in cyst formation in Mxi1-deficient mice. Cysts in Mxi1 transfectanted cells were showed by collagen or mebiol gels in 3-D cell culture system. Causative genes of ADPKD were measured by q RT-PCR. Herein, Mxi1 transfectants rarely formed a simple epithelial cyst and induced cell death. Overexpression of Mxi1 resulted in a decrease in the PKD1, PKD2 and c-myc mRNA relating to the pathway of cyst formation. These data indicate that Mxi1 influences cyst formation of mIMCD-3 cells in 3-D culture and that Mxi1 may control the mechanism of renal cyst formation.