Deficiency of primary cilia in kidney epithelial cells induces epithelial to mesenchymal transition

https://doi.org/10.1016/j.bbrc.2018.01.079Get rights and content

Highlights

  • Length of primary cilia decreased in TGF-β-induced EMT.

  • Gene silencing of Arl13b and IFT20 inhibited primary cilia elongation in MDCK cells.

  • Lack of cilia by knock down of Arl13b and Ift20 triggers EMT in resting condition.

  • Lack of cilia by knock down of Arl13b and Ift20 exacerbated TGF-β-induced EMT.

Abstract

Primary cilium is a microtubule-based non-motile organelle that plays critical roles in kidney pathophysiology. Our previous studies revealed that the lengths of primary cilia decreased upon renal ischemia/reperfusion injury and oxidative stress, and restored with recovery. Here, we tested the hypothesis that lack of primary cilium causes epithelial to mesenchymal transition (EMT) of kidney tubule cells. We investigated the alteration of length of primary cilia in TGF-β-induced EMT via visualization of primary cilia by fluorescence staining against acetylated α-tubulin. EMT was determined by measuring mesenchymal protein expression using quantitative PCR and indirect fluorescence staining. As a result, TGF-β treatment decreased ciliary length along with EMT. To test whether defect of primary cilia trigger onset of EMT, cilia formation was disturbed by knock down of ciliary protein using siRNA along with/without TGF-β treatment. Knock down of Arl13b and Ift20 reduced cilia elongation and increased expression of EMT markers such as fibronectin, α-SMA, and collagen III. TGF-β-induced EMT was greater as well in Arl13b and Ift20-knock down cells compared to control cells. Taken together, deficiency of primary cilia trigger EMT and exacerbates it under pro-fibrotic signals.

Introduction

The primary cilium is evolutionarily conserved organelle that projects from surface of the most mammalian cells [1] performing diverse biological roles including mechano-, chemo-, and photosensation [2]. In the kidney, primary cilia are observed in most part including parietal layer of Bowman's capsule, proximal tubule, distal tubule, and the collecting duct except for intercalated cells [3].

The lengths of primary cilia decrease under physiological condition such as mitosis during cell cycle [4], pathological condition during kidney damage such as kidney transplantation-induced tubular necrosis in human [5] and kidney ischemia/reperfusion (I/R) injury in mice [6]. Primary cilia were restored accompanied with functional recovery following injury [5,6]. Deciliation in the Madin Darby Canine Kidney (MDCK) cells occurred by oxidative stress and primary cilia were restored by ERK activation [6]. Additionally, defects in renal tubular primary cilia cause polycystic kidney disease in which epithelial cell proliferation is out of control [7]. This suggests that defect of primary cilia occurs under pathological microenvironment such as oxidative stress, inflammation, pro-fibrotic signals, and failure to restore primary cilia undergoes irreversible kidney damage such as fibrosis via epithelial to mesenchymal transition (EMT) of kidney tubule cells.

Successful ciliogenesis is critical to achieve functional cilia [8]. Intraflagellar transport protein (Ift) machinery is known to be critical in building cilia of many different organs [9]. Ift complex plays as a vehicle for transporting cargos to regulate cilia assembly, maintenance, and function [10]. Tissue specific inactivation of Ift20 in the mouse kidney collecting duct cells promoted cystic kidneys lacking cilia [11]. ADP ribosylation factor-like GTPase 13b (Arl13b) is also highly enriched within the cilium and essential for the ciliogenesis in zebrafish and mouse [12,13]. Inactivation of Arl13b at the perinatal stage in the distal nephron led to cyst formation and renal failure in mice [14].

This research was designed to study whether lack of primary cilia observed in pathological condition is just a result from kidney tubule cell damage or can be a trigger of further damage such as kidney EMT and fibrosis. Therefore, we induced deficiency of primary cilia by inhibition of ciliogenesis via knock down of Arl13b and Ift20 to test the effect of defective cilia on EMT and pro-fibrotic change of kidney tubule cells.

Section snippets

Cell culture

MDCK cells were purchased from ATCC (Manassas, VA, USA) and cultured in Eagle's Minimum Essential Medium (EMEM) containing 5% fetal bovine serum (Thermo Scientific, Waltham, MA, USA) and 100 U/mL streptomycin/penicillin (S/P) (WelGENE Inc., Daegu, Korea) at 37 °C in a humidified atmosphere containing 5% CO2. For Fig. 1, 50% confluent cells were treated with TGF-β (R&D Systems, Minneapolis, MN, USA) in final concentration of 5 ng/mL or vehicle for 48 h. The pictures were taken under bright field

TGF-β induced EMT in kidney epithelial cells accompanied with defect of primary cilia

In order to test whether deficiency of primary cilia is associated with pro-fibrotic alteration, we treated MDCK cells with TGF-β, and determined TGF-β-induced EMT by immunofluorescence staining and qRTPCR. TGF-β-treated cells became longer and expressed α-SMA and collagen III while vehicle-treated cells did not show fluorescence signal (Fig. 1A). mRNA level of α-SMA increased 1.4 ± 0.2 folds (p = .046) (Fig. 1B) and Collagen III increased 1.6 ± 0.2 folds (p = .009) (Fig. 1C) in the

Discussion

The most critical finding of the present study is that deficiency of primary cilia triggers EMT under resting condition and exacerbates it upon pro-fibrotic signal such as TGF-β. This finding reveals the importance of keeping intact primary cilia upon acute kidney injury such as kidney I/R or kidney transplantation, to prevent proceeding to pro-fibrotic alteration of the kidney.

Sub-lethal acute kidney injury such as I/R involves hemodynamic alterations, inflammation, and endothelial and

Acknowledgement

This work was supported by the National Research Foundation of Korea (NRF) Grant (MSIP No. 2014R1A5A2010008, NRF-2014R1A1A2055041, and NRF-2017R1D1A1B03032729) funded by the Korean Government.

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