Biochemical and Biophysical Research Communications
Deficiency of primary cilia in kidney epithelial cells induces epithelial to mesenchymal transition
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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2023, NeuronCitation Excerpt :IFT20 is a well-known IFTB protein, underlining the important role of primary cilia during corticogenesis. Interestingly, IFT20 was previously involved in epithelial-mesenchymal transition (Han et al., 2018), a process mechanistically linked to delamination of RGCs and their conversion into basal progenitors (Itoh et al., 2013), and was also found to be required for normal ciliogenesis and proliferation in mouse adult neural stem cells (Amador-Arjona et al., 2011). While our data provide a direct link between CROCCP2 and ciliogenesis, it is interesting to note that the overall length of the primary cilium in cortical progenitors appears to be similar in mouse and human species, indicating that additional mechanisms control the final length of the cilium in each species.
Overexpression of smad7 inhibits the TGF-β/Smad signaling pathway and EMT in NPHP1-defective MDCK cells
2021, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Knockdown (KD) of ciliary proteins Arl13b and Ift20 decreased cilia elongation and shortened primary cilia in MDCK cells, and increased α-SMA expression was observed. KD of Arl13b or Ift20 greatly promotes TGF-β1-induced EMT in MDCK cells [26]. Impaired cilia assembly and cilia function and short primary cilia were also observed in NPHP1-defective MDCK cells [27,28].
Oxidative stress following acute kidney injury causes disruption of lung cell cilia and their release into the bronchoaveolar lavage fluid and lung injury, which are exacerbated by Idh2 deletion
2021, Redox BiologyCitation Excerpt :Furthermore, we found that hepatic IR injury induces the deciliation of primary cilia of tubular cells of distant kidneys owing to oxidative stress in kidney tubule cells and that this deciliation is prevented by antioxidant treatment [20]. Moreover, primary cilia deficiency activates the epithelial to mesenchymal transition, which is critical for the progression of fibrosis [53], indicating that cilia are associated with the progression of post-injury responses. In the present study, we found that kidney IR causes the disruption of lung cell cilia, and these disrupted ciliary fragments are released into BALF.
Myocardin-related transcription factor and serum response factor regulate cilium turnover by both transcriptional and local mechanisms
2021, iScienceCitation Excerpt :In this regard, MRTF and SRF are central mediators in the pathogenesis of organ fibrosis ((Bialik et al., 2019; Fan et al., 2007; Luchsinger et al., 2011; Small et al., 2010; Tian et al., 2015) and reviewed in (Gasparics and Sebe, 2018; Small, 2012)) and dysregulated PC homeostasis has been associated with fibrotic conditions (Arrighi et al., 2017; Egorova et al., 2011; Han et al., 2018; Lee et al., 2018a; Li et al., 2020; Rozycki et al., 2014; Villalobos et al., 2019). While our understanding is rudimentary as yet, the PC alterations might play a biphasic role (Rozycki et al., 2014; Teves et al., 2019), wherein an initial PC growth or enhanced ciliation might contribute to repair or early fibrogenic signaling (Arrighi et al., 2017; Lee et al., 2018a; Rozycki et al., 2014), whereas subsequent loss of the cilium is associated with EMT, terminal myofibroblast differentiation and advanced fibrosis (Han et al., 2018; Li et al., 2020; Rozycki et al., 2014). Future research should address if functional or genetic alterations in SRF or MRTF, or their interactions with PC components, contribute to the pathogenesis of acquired ciliary disorders (e.g. in fibrosis and cancer) or inherited ciliopathies.
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2021, Kidney International ReportsCitation Excerpt :Interestingly, through the study of primary cilia in cultured epithelial cells, primary cilia undergo a dynamic biphasic change during epithelial-myofibroblast transition as well as fibroblast-to-myofibroblast transition induced by transforming growth factor-β.24 Furthermore, the study of inhibition of ciliogenesis demonstrated that deficiency of primary cilia induces epithelial-to-mesenchymal transition.25 Therefore, because it is well-known that products of genes causing NPH are localized at segments associated with primary cilia, it could be possible that abnormal primary cilia in NPH patients result in increased α-SMA–positive myofibroblasts and fibrosis, resulting in thick TBM duplication.