The dysfunction of both motile and immotile cilia causes a spectrum of human disorders commonly known as “ciliopathies” (Benmerah, Durand et al. 2015, Reiter and Leroux 2017, Kempeneers and Chilvers 2018). This term was firstly used in 1984 and became popular in 21st century.The ciliopathies are symptomatically heterogeneous and mostly inherited autosomal recessive disease (Fig16). The ciliopathies can be classified into two major categories: motor ciliopathies (associated with motile cilia and mutations that
35 lead to motility loss) and sensory ciliopathies (associated mostly with primary cilia and defects in sensory/signalling functions). Additionally, first order ciliopathies (caused by disruption of ciliary proteins) are distinguished from the secondary order ciliopathies (associated with non-ciliary proteins which are required for cilia function) 104-105.
Figure 16. Different organ systems affected in ciliopathies and corresponding phenotypic manifestations of the disease are shown. Ciliopathies that are caused by defects in motile cilia are shown in orange, those that result from defects in primary cilia are shown in blue, those that are caused by defects in both cilia types are shown in green (Reiter and Leroux 2017).
Ciliopathies are distinguished from “extraciliary disorders”, a situation generated by a mutation in a protein which has both ciliary and non-ciliary functions; this causes a phenotype unrelated to the cilium. For example, IFT20 plays also a role in collagen trafficking, the ift20 mouse phenotype characterized by craniofacial skeletal abnormalities (Noda, Kitami et al. 2016).
36 The abnormalities found in ciliopathies includes dysfunction in the kidney, liver, respiratory organs, brain, eye, ear, skeleton and reproductive system (Fig.17).
Specifically, sensory ciliopathies are often characterized by kidney and liver diseases, organ laterality defects, polydactyly, retinal degeneration, skeletal and central nervous system malformations, obesity and diabetes (Fliegauf, Benzing et al. 2007, Kempeneers and Chilvers 2018).
1.16 Cystic kidney disorders
Primary cilia defects are often characterized by renal and liver cysts. The kidney disease spectrum in this case varies between the polycystic kidney disease (PKD) and syndromes where the renal cyst formation is accompanied by a broad range of abnormalities (Fig.8B, Fig.17). The mechanisms associated with renal cyst formation include increased cell proliferation, epithelial fluid and chloride secretion, extracellular matrix abnormalities, somatic and/or germ line mutations, planar cell polarity defects, apoptosis and inflammation (Paul and Vanden Heuvel 2014).
37 Figure 17. Major categories of ciliopathies (blue boxes) that are associated with the named proteins (right side) and a scheme of ciliary regions where these proteins localise are shown. The asterisks indicate proteins that are localized to other ciliary regions during ciliogenesis or participate in ciliary trafficking (Reiter and Leroux 2017).
Autosomal dominant (AD) PKD and autosomal recessive (AR) PKD are common potentially lethal hereditary disease in humans. ADPKD causes renal failure in approximately 50% of cases and its prevalence 1/400-1/1000 people. ADPKD is caused by mutation in PKD1 (85% affected individuals) on chromosome 16 and in PKD2 (15% of cases) on chromosome 4 (Ghata and Cowley 2017). The symptoms of the disease generally appear after 40 years and are hypertension, haematuria, abdominal pain, urinary tract infections, intestinal diverticulosis and cerebral aneurisms. PKD2 encodes
38 polycystin 2, a calcium channel of the TRP family that associates at the level of the primary cilium with polycytin 1, the PKD1 product. Cysts can originate from glomeruli or from any part of the kidney tubule including the collecting duct. Cysts begin as diverticula and slowly grow leading to compression of neighbouring tubules and vessels as well as interstitial inflammation and fibrosis. Cyst growth is driven by a combination of cell proliferation and fluid secretion. Suppression of autophagy may also play a pathogenical role in cyst formation and growth in ADPKD (Huber, Walz et al. 2011, Ravichandran and Edelstein 2014, Wang, Livingston et al. 2015). ARPKD is caused by mutation in PKDH1 gene encoding polyductin, a regulator of renal collecting duct and bile duct differentiation. The disease prevalence is 1/6000 live births and up to 75% of these patients die within a few days (Bergmann 2015). Nephronostisis is the most common cause of ESRD in children (end-stage renal disorders), it can be caused by mutations in the genes coding nephrocystins. Renal cysts are accompanied by congenital hepatic fibrosis, laterality defects, cerebellar vermis, hypoplasia and retinal degeneration. Mutations in HNF1ß or in other ciliopathy associated genes cause nephronophthisis, Bardet-Biedl or Joubert syndrome; these diseases are known to be PKD-mimetics (Kempeneers and Chilvers 2018).
1.17 Caenorhabditis elegans as a model organism for kidney research.
The Caenorhabditis elegans reference strain N2 is a laboratory animal, it has been modified by domestication due to mutation appearance and stabilization. It was isolated in Bristol, (United Kingdom) and cultured on agar plates seeded with E.coli as a food source. The natural milieu for C. elegans is a rich soil or compost where they are often found in a non-feeding “dauer” stage, it is found worldwide, mostly in areas with humid warm climate (Frézal and Félix 2015). Two alternative life cycles have been described for C. elegans. If well fed, they pass through four larval stages and reach the adult stage after 3 days. Under stress conditions they may shift during L1 stage to predauer stage (L2d) followed by non-feeding diapause called dauer. This stage is very resistant to a variety of stress conditions and able to survive several months without food. C. elegans
39 have boom-and-bust lifestyle. A cycle of colonization starts when dauer larvae find a food source; they exit a dauer stage and seed a population of up to 104 feeding nematodes. As they run out of the food source, dauers may start explore the neighbouring environment for new resources. Dauer larva display a specific type of behaviour called nictation, it thought to be help in finding invertebrate host. A nematode stay on the tale and wave one`s body in the air. C. elegans can form a column and synchronize the group nictation. The population size of nematode varies: the densest population is detected in autumn followed by its decrease to the bottle necks in winter. The reproduction mode of these nematodes is called androdioecy. It happened ether through self-fertilizing (selfing) hermaphrodites or by breeding of XX (hermaphrodites) with X0 (males occur by non-disjunction of X chromosomes during meiosis at 0.1% frequency).
The introduction of the nematode to molecular biology and genetics led to tree Nobel prize awards: S. Brenner, R. Horvitz, J. Sulston in 2002 for apoptotic cell death description; A. Fire and C. Mello in 2006 for the discovery of gene silencing by dsRNA;
M. Chalfie in 2008 for the discovery and development of the green fluorescent protein, GFP. Starting in 1999, kidney researchers took advantage of this simple and time-saving fashion model organism which allows using modern molecular biology and genetics techniques combined with phenotype screening (Barr and Sternberg 1999). It is known that most human kidney disease genes associated with cyst formation, proteinuria or renal carcinoma development are highly conserved and C. elegans displays homologous genes with similar functions (Muller, Zank et al. 2011, Hsu 2012). Ciliary abortion leads to several defined phenotypes characterised by dysregulated mechanosensation, synaptogenesis, motility and life span (wormbook.org). Several landmark research on LOV-1, PKD-2 (homologues of human polycitin-1 and polycistin-2) describing functions and role in ciliogenesis and cyst formation were done in C. elegans (Barr and Sternberg 1999, Muller, Zank et al. 2011, Hsu 2012, Frézal and Félix 2015, Chen, Bharill et al. 2016, Bezares-Calderon, Berger et al. 2018). Many genes involved in juvenile cystic kidney disease including juvenile nephronophthisis (NHPH), Bardet-Biedl syndrome (BBS),
40 Joubert syndrome (JBTS) and Meckel-Gruber syndrome (MKS) are also conserved in C.elegans.
1.18 Salt sensing and sensory transduction in Caenorhabditis elegans
The roundworm C. elegans is a well-known model for sensory guided behaviours which rely on the ability of the animal to detect chemical, mechanical or thermal stimuli (Martinac 2008). Chemosensation is used to avoid noxious conditions, find food and mate. The nematodes display two kinds of sensory-dependent behaviour: taxes (movement towards the stimuli) and avoidances (movement away from the stimuli). The nematode chemosensory system comprises 70 sensory neurons, 60 of them have ciliated endings. Some of these neurons are organized into sensory organs – amphids, phasmid and inner labial organs. The neurons can be divided into several groups: ASE, ASH, ASI, ASG, ASJ, ASK (amphid neurons with single ciliated endings); AWC, AWA, AWB (amphid wing cells neurons); ADF, ADL (amphid neuron), URX (ring interneuron), AQR (neuron basal body is not a part of a sensillum, projects into ring); PQR (Neuronal basal body is not a part of a sensillum, projects into preanal ganglion); PHA, PHB (phasmid chemosensory neurons). Water soluble attractants including Na+, Cl-, cAMP, biotin, lysine and serotonin are sensed by amphid single (ASE) gustatory neurons. The two ASE neurons function in a different manner: the right neuron (ASER) detects Cl- and K+ and the left neuron (ASEL) detects Na+ (wormbook). These neurons exhibit voltage-regulated potassium and calcium currents. ASE neurons which do not express voltage activated sodium channels may display calcium-based amplification of the signal but no sodium-based action potentials.
The sensory transduction goes via ion channels that belong to cyclic nucleotide-gated (CNG), transient receptor potential (TRP) and amiloride-sensitive degenerin/epithelial sodium (DEG/ENaC) protein superfamilies. These proteins are concentrated in a neuronal ciliary membrane.
41 Figure 18. Hypothetical CPRG-TRPV signal transduction pathway. The repellents are detected by CPGRs and by other ion channels. This leads to Gi-like proteins ODR-3 and GPA-3 activation which controls lipid metabolism and enables lipid mobilization. TRPV channels (OSM-9/OCR-2) open in response to lipid mobilization and cell membrane depolarization occurs. TRPV channels can be activated directly by chemical and mechanical stimuli (wormbook.org).
A CNG channel encoded by tax-2 and tax-4 genes is essential for a signal transduction downstream of G-protein signalling in many sensory neurons. TAX-1/TAX-2 channel is highly sensitive to cGMP and indifferent to cAMP (Hellman and Shen 2011, Li, Zhou et al. 2017). In response to external stimuli, G-protein signalling regulates either cGMP phosphodiesterase or guanylate cyclase function, thus it regulates open/closed state of the cGMP-gated channel. Mutants for tax-2 and tax-4 have defects in behaviour, activity-dependent gene expression and in the structure of sensory axons (Hellman and Shen 2011). The TRPV channels OSM9/OCR-2 are localised in amphid sensory neurons of C. elegans and serve for osmosensation signalling (Lee and Ashrafi 2008, Phua, Lin et al. 2015). In other species, they also function in phototransduction (insects) and pain sensation (vertebrates). In AWA and ASH neurons they provide signal transduction downstream of GPCRs and ODR-3 G-protein signalling (Tobin, Madsen et al. 2002, Liedtke, Tobin et al. 2003, Lee and Ashrafi 2008). The CPCR signal reception leads to the TRPV channel activation likely through the lipid mobilization where Gq and
42 phospholipase C-mediated Ca+ influx plays a role(Minke 2006, Putney and Tomita 2012, Gonçalves, Cordeiro et al. 2014, Hasan and Zhang 2018) (Fig. 17). Gentle touch sensing in Caenorhabdits elegans touch receptor neurons (TRNs) is driven though degenerin/epithelial sodium channel (DEG/ENaC) complex proteins such as MEC-4 and MEC-10 (MEC derives from mechanosensory). Nature, composition and functioning of the channel complexes are poorly understood. It is known that there are several membrane proteins involved in touch sensing: somatostatin-like MEC-2, paraoxonase-like MEC-6, channel proteins MEC-4 and MEC-10. These proteins, co-immunoprecipitated with each other in heterologous expression systems. DEG/ENaC proteins form a variety of complexes i.e. MEC-4 homotrimer and MEC-4:MEC-4:MEC-10 heterotrimer… MEC-2, MEC-4 and MEC-6 but not MEC-10 are important for production of the transduction current. Furthermore, MEC-2 and MEC-6 regulate surface expression of MEC-4 channels. (Chen, Bharill et al. 2016). Recently, it has been also reported that DEG/ENaC subunits del-2 and del-6 are involved in chemotaxis to NaCl (Cilia 2016, Poster - A-T1-P-16, S. van der Burght, G.Jansen).
C. elegans behavioural responses to NaCl and other salt solutions occurs via a molecular pathway which include ion channels localized to the primary cilia of ASE neurons and their downstream signalling mechanisms. Being a member of this family, ENaC is the only mammalian homologue, which functions as a sodium channel and mediates apical sodium transport in the terminal part of the kidney tubule. We hypothesize that perception of NaCl occurs via degenerin/epithelial sodium channel (DEG/ENaC) functioning as a receptor. We used chemotaxis assay with different concentration of NaCl to investigate this issue.
Conclusion
The kidney is an organ responsible for elimination of soluble waste metabolites as well as excess of water and electrolytes in order to control body fluid homeostasis. At the same time, the kidney is a sensory organ. At the body level, it monitors the tissue oxygenation status by deep cortical peritubular fibroblast-like cells and blood pressure
43 via glomerular afferent arteries. At the nephron level, the myogenic response of the glomerular afferent artery controls renal blood flow and the tubulo-glomerular feedback by the macula densa modulates glomerular filtration rate. At the cell level, the single non-motile primary cilium may function as a local urinary flow/composition detector in renal tubule epithelium (Pluznick and Caplan 2015). The non-motile primary cilium acts as a transducer of extracellular stimuli into intracellular signalling. Its function might be modulated by changes of cilium length (Besschetnova, Kolpakova-Hart et al. 2010, Avasthi and Marshall 2012, Wang, Livingston et al. 2015, Han, Jang et al. 2016). The physiological processes and signalling pathways that are controlling and being under regulation of primary cilium are being intensively studied. Nonetheless, the interplay between primary cilium and transepithelial sodium transport has not been characterized yet.
V HYPOTHESIS AND PROJECT AIMS Hypothesis
Primary cilium is primarily involved in the pathogenesis ciliopathies characterized by the presence of renal cysts. It was proposed to be a regulator of directed cell migration and tissue repair by controlling Wnt signalling (Lienkamp, Ganner et al. 2012, Veland, Lindbæk et al. 2014). Therefore, we assessed whether the primary cilium of renal epithelia can play a role in regulation of the cyst formation process by controlling cell migration, proliferation and differentiation.
According to the literature, the primary cilium metabolism might be linked to the transepithelial ion transport. For instance, the primary cilia-associated signalling pathways participate in ion transport regulation and modulation of Na+ transport by ouabain, an inhibitor of the Na,K-ATPase, affects cilium length (Larre, Castillo et al. 2011, Vallon and Rieg 2011, Avasthi and Marshall 2012, Praetorius and Leipziger 2013, Wang, Livingston et al. 2015, Lang and Pearce 2016). In addition, primary cilia of renal epithelial cells display increase length in response to uninephrectomy characterized by hyperfiltration by the remnant kidney which suggest a potential
44 chemo/mechanosensory function (O'Connor, Malarkey et al. 2013, Han, Jang et al.
2016). Therefore, we hypothesized that in mature renal epithelia the primary cilium is a structurally dynamic organelle that modulates Na+ transport.
Project aim
The main aims of this study were to characterize a role of primary cilium in tubulogenesis and in renal reabsorption of Na+ using both cultured cells and animal models.
In addition, using C.elegans as a model organism, we aimed to analyse the role of ENAC/DEG family members in Na+ sensing.
VI RESULTS 2.1 Published results
My contribution to the scientific article:
Ernandez T*, Komarynets O*, Chassot A, Sougoumarin S, Soulié P, Wang Y, Montesano R, Feraille E. “Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium” Am J Physiol Cell Physiol. 2017 Jul 1;313(1):C94-C107. doi: 10.1152/ajpcell.00290.2016.
This paper examines a role of renal primary cilia in branching tubulogenesis and lumen development control; it shows that the organelles regulate tubular lumen maturation in 3D cell model of a renal collecting duct under no flow conditions. We found that this primary cilium function is not achieved via modulation of cell motility or proliferation but rather by regulating luminal fluid accumulation in cyst like structures made of collecting duct cells.
As a project participant I was responsible for planning and performing experiments with IFT88KD, a cell line line characterized by inducible IFT88 gene silencing. I performed work including molecular cloning and creation of shRNA containing plasmids; vector production; stable transfection and establishment of a mCCDcl1
derived cell line; 3d cell culture and wound healing experiments as well as data analysis. I participated in drafting and reviewing a paper manuscript; performing additional experiments after we received a paper revision.
RESEARCH ARTICLE
Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium
Thomas Ernandez,1* Olga Komarynets,2* Alexandra Chassot,2Soushma Sougoumarin,2Priscilla Soulié,2 Yubao Wang,2Roberto Montesano,2and Eric Feraille1,2
1Service of Nephrology, University Hospital of Geneva, Geneva, Switzerland; and2Department of Cell Physiology and Metabolism, University Medical Center, Geneva, Switzerland
Submitted 3 October 2016; accepted in final form 28 April 2017
Ernandez T, Komarynets O, Chassot A, Sougoumarin S, Soulié P, Wang Y, Montesano R, Feraille E. Primary cilia control the maturation of tubular lumen in renal collecting duct epithelium.Am J Physiol Cell Physiol 313: C94 –C107, 2017. First published May 3, 2017; doi:10.1152/ajpcell.00290.2016.—The key role of the primary cilium in developmental processes is illustrated by ciliopathies result-ing from genetic defects of its components. Ciliopathies include a large variety of dysmorphic syndromes that share in common the presence of multiple kidney cysts. These observations suggest that primary cilia may control morphogenetic processes in the developing kidney. In this study, we assessed the role of primary cilium in branching tubulogenesis and/or lumen development using kidney collecting duct-derived mCCDN21cells that display spontaneous tu-bulogenic properties when grown in collagen-Matrigel matrix. Tubu-logenesis and branching were not altered when cilium body growth was inhibited by Kif3A or Ift88 silencing. In agreement with the absence of a morphogenetic effect, proliferation and wound-healing assay revealed that neither cell proliferation nor migration were altered by cilium body disruption. The absence of cilium following Kif3A or Ift88 silencing in mCCDN21cells did not alter the initial stages of tubular lumen generation while lumen maturation and enlargement were delayed. This delay in tubular lumen maturation was not observed after Pkd1 knockdown in mCCDN21 cells. The delayed lumen maturation was explained by neither defective secre-tion or increased reabsorpsecre-tion of luminal fluid. Our results indicate that primary cilia do not control early morphogenetic processes in renal epithelium. Rather, primary cilia modulate tubular lumen mat-uration and enlargement resulting from luminal fluid accumulation in tubular structures derived from collecting duct cells.
cilium; epithelium; ion transport
MOST EPITHELIAL CELL TYPESdisplay an apical nonmotile primary cilium formed by microtubules surrounded by a membrane in continuity with the apical plasma membrane. The importance of the primary cilium is highlighted by a group of human syndromes, currently called ciliopathies, covering a large va-riety of dysmorphic syndromes that share the presence of multiple renal cysts (10). Ciliopathies are caused by mutations of genes whose products are localized at the base or in the body of the primary cilium, leading to absence or dysfunction of primary cilia (8).
The primary cilium body is a microtubule-based structure, or axoneme, consisting of nine doublets of microtubules of␣ -tu-bulin that are stabilized by acetylation. It is a dynamic organ-elle normally absent in dividing cells. It grows from the apical membrane of epithelial cells after polarity is established. The primary cilium initially grows following the formation of basal bodies from centrioles and their migration to the apical pole of epithelial cells. Basal bodies first associate with intracellular vesicles that form the initial ciliary membrane domain, and then the cilium elongates following tubulin polymerization.
The primary cilium is subjected to a constant turnover since steady-state tubulin polymerization is balanced by its depoly-merization. Intraflagellar transport with anterograde kinesin motors and retrograde dynein motors is mandatory for building primary cilia as well as trafficking along the cilium (15). The primary cilium is believed to function as a mechanosensor since bending of primary cilia induces an increase in intracel-lular calcium concentration (27) that might be mediated by activation of TRP family channels found in abundance along this organelle (2, 7, 22). It also behaves as a signaling platform and plays a critical role in the Hedgehog (1, 32) and Wnt signaling pathways (5, 16), two key players in epithelia devel-opment.
Renal epithelial cell lines grown in a three-dimnensional (3D) matrix represent a useful in vitro model of tubulogenesis and generation of tubular lumen (6, 19, 21). The widely used
Renal epithelial cell lines grown in a three-dimnensional (3D) matrix represent a useful in vitro model of tubulogenesis and generation of tubular lumen (6, 19, 21). The widely used