Cilia‐localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney
2018; Springer Nature; Volume: 37; Issue: 15 Linguagem: Inglês
10.15252/embj.201798615
ISSN1460-2075
AutoresAmandine Viau, Frank Bienaimé, Kamile Lukas, Abhijeet Todkar, Manuel Knoll, Toma A. Yakulov, Alexis Hofherr, Oliver Kretz, Martin Helmstädter, Wilfried Reichardt, Simone Braeg, Tom Aschman, Annette Merkle, Dietmar Pfeifer, Verónica I. Dumit, Marie‐Claire Gubler, Roland Nitschke, Tobias B. Huber, Fabiola Terzi, Jörn Dengjel, Florian Grahammer, Michael Köttgen, Hauke Busch, Melanie Boerries, Gerd Walz, Antigoni Triantafyllopoulou, E. Wolfgang Kuehn,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoArticle19 June 2018Open Access Source DataTransparent process Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney Amandine Viau Amandine Viau Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Frank Bienaimé Frank Bienaimé Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Service d'Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Kamile Lukas Kamile Lukas Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Abhijeet P Todkar Abhijeet P Todkar Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Manuel Knoll Manuel Knoll Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Search for more papers by this author Toma A Yakulov Toma A Yakulov Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Alexis Hofherr Alexis Hofherr orcid.org/0000-0003-2842-6161 Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Oliver Kretz Oliver Kretz Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Neuroanatomy, Albert-Ludwigs-University Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Martin Helmstädter Martin Helmstädter Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Wilfried Reichardt Wilfried Reichardt Faculty of Medicine, University of Freiburg, Freiburg, Germany Medical Physics, Department of Radiology, and Comprehensive Cancer Center, University Medical Center, Freiburg, Germany German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Simone Braeg Simone Braeg Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Tom Aschman Tom Aschman Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Search for more papers by this author Annette Merkle Annette Merkle Medical Physics, Department of Radiology, and Comprehensive Cancer Center, University Medical Center, Freiburg, Germany Search for more papers by this author Dietmar Pfeifer Dietmar Pfeifer Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center, Freiburg, Germany Search for more papers by this author Verónica I Dumit Verónica I Dumit Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Marie-Claire Gubler Marie-Claire Gubler INSERM UMR1163, Laboratory of Inherited Kidney Diseases, Necker-Enfants Malades Hospital, Paris, France Imagine Institute, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Roland Nitschke Roland Nitschke Center for Biological Systems Analysis (ZBSA), Life Imaging Center, Albert-Ludwigs-University Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Tobias B Huber Tobias B Huber Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Fabiola Terzi Fabiola Terzi INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Jörn Dengjel Jörn Dengjel orcid.org/0000-0002-9453-4614 Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, Albert-Ludwigs-University Freiburg, Freiburg, Germany Department of Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Florian Grahammer Florian Grahammer Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Michael Köttgen Michael Köttgen Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Hauke Busch Hauke Busch orcid.org/0000-0003-4763-4521 German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany Search for more papers by this author Melanie Boerries Melanie Boerries German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Systems Biology of the Cellular Microenvironment Group, Institute of Molecular Medicine and Cell Research (IMMZ), Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Gerd Walz Gerd Walz Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Antigoni Triantafyllopoulou Antigoni Triantafyllopoulou Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Department of Rheumatology and Clinical Immunology, Charité - University Medical Centre Berlin, Berlin, Germany Search for more papers by this author E Wolfgang Kuehn Corresponding Author E Wolfgang Kuehn [email protected] orcid.org/0000-0002-7753-9616 Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Amandine Viau Amandine Viau Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Frank Bienaimé Frank Bienaimé Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Service d'Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France Search for more papers by this author Kamile Lukas Kamile Lukas Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Abhijeet P Todkar Abhijeet P Todkar Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Manuel Knoll Manuel Knoll Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Search for more papers by this author Toma A Yakulov Toma A Yakulov Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Alexis Hofherr Alexis Hofherr orcid.org/0000-0003-2842-6161 Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Oliver Kretz Oliver Kretz Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Neuroanatomy, Albert-Ludwigs-University Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Martin Helmstädter Martin Helmstädter Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Wilfried Reichardt Wilfried Reichardt Faculty of Medicine, University of Freiburg, Freiburg, Germany Medical Physics, Department of Radiology, and Comprehensive Cancer Center, University Medical Center, Freiburg, Germany German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Simone Braeg Simone Braeg Renal Department, University Medical Center, Freiburg, Germany Search for more papers by this author Tom Aschman Tom Aschman Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Search for more papers by this author Annette Merkle Annette Merkle Medical Physics, Department of Radiology, and Comprehensive Cancer Center, University Medical Center, Freiburg, Germany Search for more papers by this author Dietmar Pfeifer Dietmar Pfeifer Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center, Freiburg, Germany Search for more papers by this author Verónica I Dumit Verónica I Dumit Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Marie-Claire Gubler Marie-Claire Gubler INSERM UMR1163, Laboratory of Inherited Kidney Diseases, Necker-Enfants Malades Hospital, Paris, France Imagine Institute, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Roland Nitschke Roland Nitschke Center for Biological Systems Analysis (ZBSA), Life Imaging Center, Albert-Ludwigs-University Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Tobias B Huber Tobias B Huber Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Fabiola Terzi Fabiola Terzi INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France Search for more papers by this author Jörn Dengjel Jörn Dengjel orcid.org/0000-0002-9453-4614 Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, Albert-Ludwigs-University Freiburg, Freiburg, Germany Department of Biology, University of Fribourg, Fribourg, Switzerland Search for more papers by this author Florian Grahammer Florian Grahammer Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Search for more papers by this author Michael Köttgen Michael Köttgen Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Search for more papers by this author Hauke Busch Hauke Busch orcid.org/0000-0003-4763-4521 German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany Search for more papers by this author Melanie Boerries Melanie Boerries German Cancer Consortium (DKTK), Freiburg, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Systems Biology of the Cellular Microenvironment Group, Institute of Molecular Medicine and Cell Research (IMMZ), Albert-Ludwigs-University, Freiburg, Germany Search for more papers by this author Gerd Walz Gerd Walz Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Antigoni Triantafyllopoulou Antigoni Triantafyllopoulou Faculty of Medicine, University of Freiburg, Freiburg, Germany Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany Department of Rheumatology and Clinical Immunology, Charité - University Medical Centre Berlin, Berlin, Germany Search for more papers by this author E Wolfgang Kuehn Corresponding Author E Wolfgang Kuehn [email protected] orcid.org/0000-0002-7753-9616 Renal Department, University Medical Center, Freiburg, Germany Faculty of Medicine, University of Freiburg, Freiburg, Germany Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany Search for more papers by this author Author Information Amandine Viau1,2,3,‡, Frank Bienaimé1,2,3,4,‡, Kamile Lukas1, Abhijeet P Todkar1, Manuel Knoll5, Toma A Yakulov1,2, Alexis Hofherr1,2, Oliver Kretz1,2,6,7, Martin Helmstädter1,2, Wilfried Reichardt2,8,9,10, Simone Braeg1, Tom Aschman5, Annette Merkle8, Dietmar Pfeifer2,11, Verónica I Dumit12, Marie-Claire Gubler13,14, Roland Nitschke15,16, Tobias B Huber1,2,7,16,17, Fabiola Terzi3, Jörn Dengjel12,18, Florian Grahammer1,2,7, Michael Köttgen1,2, Hauke Busch9,10,19, Melanie Boerries9,10,20, Gerd Walz1,2,16, Antigoni Triantafyllopoulou2,5,21 and E Wolfgang Kuehn *,1,2,16 1Renal Department, University Medical Center, Freiburg, Germany 2Faculty of Medicine, University of Freiburg, Freiburg, Germany 3INSERM U1151, Institut Necker Enfants Malades, Department of Growth and Signaling, Université Paris Descartes-Sorbonne Paris Cité, Paris, France 4Service d'Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France 5Department of Rheumatology and Clinical Immunology, University Medical Center, Freiburg, Germany 6Department of Neuroanatomy, Albert-Ludwigs-University Freiburg, Freiburg, Germany 7III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 8Medical Physics, Department of Radiology, and Comprehensive Cancer Center, University Medical Center, Freiburg, Germany 9German Cancer Consortium (DKTK), Freiburg, Germany 10German Cancer Research Center (DKFZ), Heidelberg, Germany 11Department of Hematology, Oncology and Stem Cell Transplantation, University Medical Center, Freiburg, Germany 12Center for Biological Systems Analysis (ZBSA), Core Facility Proteomics, Albert-Ludwigs-University Freiburg, Freiburg, Germany 13INSERM UMR1163, Laboratory of Inherited Kidney Diseases, Necker-Enfants Malades Hospital, Paris, France 14Imagine Institute, Université Paris Descartes-Sorbonne Paris Cité, Paris, France 15Center for Biological Systems Analysis (ZBSA), Life Imaging Center, Albert-Ludwigs-University Freiburg, Freiburg, Germany 16Center for Biological Signaling Studies (BIOSS), Albert-Ludwigs-University Freiburg, Freiburg, Germany 17Center for Biological Systems Analysis (ZBSA), Albert-Ludwigs-University Freiburg, Freiburg, Germany 18Department of Biology, University of Fribourg, Fribourg, Switzerland 19Institute of Experimental Dermatology, University of Lübeck, Lübeck, Germany 20Systems Biology of the Cellular Microenvironment Group, Institute of Molecular Medicine and Cell Research (IMMZ), Albert-Ludwigs-University, Freiburg, Germany 21Department of Rheumatology and Clinical Immunology, Charité - University Medical Centre Berlin, Berlin, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 761 270 32270; Fax: +49 761 270 32860; E-mail: [email protected] The EMBO Journal (2018)37:e98615https://doi.org/10.15252/embj.201798615 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis. Synopsis The cilium instructs immune cell behaviour and tissue homeostasis via a complex of ciliopathy-related proteins and the kinase LKB1 that together regulate chemokine signalling. Dysregulation of this system presents an unexpected disease mechanism for ciliopathies. The primary cilium transmits a signal that activates expression of chemokine CCL2. This signal is regulated through an intra-ciliary complex of LKB1, NPHP1, polycystin 1 (PC1), ANKS3 and NEK7. Loss of LKB1 or PC1 increases CCL2 expression and peritubular macrophage numbers and promotes ciliopathy phenotypes. The Polycystic Kidney Disase (PKD) phenotype seen upon PKD1 depletion is ameliorated in the absence of cilia or tubular CCL2. Introduction Polycystic renal diseases belong to a spectrum of inherited disorders termed ciliopathies. They include genetically distinct and morphologically heterogeneous disorders that manifest at any time from early childhood to late in life and lead to end-stage kidney disease in the majority of cases (Braun & Hildebrandt, 2016). In adults, they present as autosomal dominant polycystic kidney disease (ADPKD), one of the most common monogenetic disorders in man, with large numbers of cysts leading to massive enlargement of the kidneys (Ong et al, 2015). In children, the most prevalent form is nephronophthisis (NPHP). These kidneys are mostly small and fibrotic and contain only few cysts (Wolf, 2015; Konig et al, 2017). Important progress has been made in identifying the genetic causes of these entities: ADPKD is caused by mutations in either PKD1 or PKD2 (The International Polycystic Kidney Disease Consortium, 1995; Mochizuki et al, 1996). The number of NPHP genes has surpassed 20 and is growing annually (Hildebrandt et al, 1997; Saunier et al, 1997; Braun & Hildebrandt, 2016). Yet, very few insights exist into what links these diverse genes to the disease manifestations of cysts, inflammation, and fibrosis. A key to this question lies in cilia, cell organelles that contain proteins mutated in the ciliopathies. Apart from kidney disease, ciliopathies also affect the liver, the central nervous system, and the skeleton (Hildebrandt et al, 2011). Motile cilia occur in bundles in the airways and brain, whereas primary cilia are non-motile filiform structures at the surface of most cell types, including renal tubules (Drummond, 2012). Cilia contain a microtubule-based cytoskeleton, an ultrastructural gate, and a complex macromolecular transport system (Nachury, 2014; Stepanek & Pigino, 2016; Garcia-Gonzalo & Reiter, 2017). They act as signal transducers receiving physical and chemical stimuli (Praetorius & Spring, 2001; Corbit et al, 2005; Lechtreck et al, 2009; Boehlke et al, 2010b) and affect a wide range of autonomous cellular functions such as cell size, polarity, cell cycle, and migration (Kim et al, 2004; Park et al, 2006; Robert et al, 2007; Jones et al, 2008; Vasilyev et al, 2009; Boehlke et al, 2010b; Schneider et al, 2010; Orhon et al, 2016). However, it is unclear how the dysfunction of cilia explains the phenotype of renal ciliopathies. When exploring this question, two observations prompted our attention. First, apart from cysts, ADPKD and NPHP share an inflammatory phenotype: In NPHP, inflammation and subsequent fibrosis are much more prominent than cysts (Hildebrandt et al, 1997; Saunier et al, 1997; Wolf, 2015; Braun & Hildebrandt, 2016; Slaats et al, 2016). Likewise, in ADPKD macrophage infiltration occurs early on and promotes cyst enlargement (Karihaloo et al, 2011). Similarly to NPHP, the late stages of ADPKD are characterized by a severe degree of fibrosis (Norman, 2011; Ong et al, 2015). The second aspect concerns the fact that metabolic signaling has been observed to contribute to ADPKD and NPHP (Chaki et al, 2012; Rowe et al, 2013; Menezes et al, 2016). We therefore asked the question how cilia, metabolic signal transducers, and inflammation are linked. An interesting candidate to explore this question is the kinase liver kinase B1 (LKB1). Liver kinase B1 is a metabolic sensor localized in cilia (Boehlke et al, 2010b; Mick et al, 2015). We have previously shown in vitro that bending of cilia under flow activates LKB1 to phosphorylate its target AMPK (5′-adenosine monophosphate-activated protein kinase) at the base of cilia and to regulate metabolic signaling linked to PKD (Boehlke et al, 2010b; Walz et al, 2010; Liu et al, 2014; Orhon et al, 2016). To explore the role of LKB1 in vivo, we inactivated LKB1 in the kidney. First, we asked whether renal deletion of LKB1 induces a renal ciliopathy phenotype. Second, we sought to uncover molecular mechanisms connecting LKB1, cilia, and PKD proteins to cysts, inflammation, and fibrosis. Results Deletion of LKB1 in the kidney disrupts renal morphology and function Mono-allelic mutations in the gene STK11/LKB1 in humans cause Peutz-Jeghers disease, an inherited cancer syndrome characterized by benign tumors of the skin and intestine, as well as the development of malignancies (Hemminki et al, 1998). Lkb1−/− mice display severe developmental defects leading to embryonic lethality. We therefore pursued a kidney-specific knockout strategy and crossed Lkb1flox/flox mice (Nakada et al, 2010) with a kidney-specific deleter strain to excise Lkb1 in the distal nephron. LKB1 was absent from the cilia of these tubule segments (Lkb1ΔTub; Figs 1A and B, and EV1A–C), but the number and morphology of cilia were unchanged (Fig EV1D–F). The first sign of a renal disturbance was impaired urine concentration, which occurred at 5 weeks (Figs 1C and EV1G–I); magnetic resonance imaging (MRI) studies confirmed that increased amounts of urine were present in the renal pelvis and occasionally demonstrated a few cysts (Fig 1C). Macroscopically, the kidneys diminished in size over time (Fig EV1J and K; Appendix Fig S1). The occurrence of cysts was noted at the cortico-medullary junction (Fig 1C and F; 14 and 23 weeks). Histology revealed dilated tubules, thickened tubular basement membranes, interstitial inflammation, and fibrosis (Fig 1G and H). These findings are similar to those of a previous study, which investigated renal metabolism using the same mouse genotype that was created independently (Han et al, 2016).1 Collagen synthesis, a marker for extracellular matrix deposition, was increased (Fig EV1L), and renal failure ensued (Fig 1D and E). Analogous to humans with Peutz-Jeghers syndrome, heterozygote animals had normal renal function and histology (Appendix Fig S1). These findings show that LKB1 is required for normal morphology and function of the kidney. Loss of LKB1 in the kidney results in alterations of tubular architecture, cysts, and chronic inflammation with progressive scarring leading to renal failure, reminiscent of a renal ciliopathy phenotype. Figure 1. Loss of Lkb1 in the mouse kidney disrupts renal architecture and causes inflammation and fibrosis LKB1 immunostaining in kidneys from control and Lkb1ΔTub mice at 5 weeks. Representative images of n = 5 mice/group. Scale bars: 200 μm. See also Fig EV1A and B. Representative confocal microscopy images of collecting duct (CD, AQP2 expressing) from 5-week-old control (n = 4) and Lkb1ΔTub (n = 5) animals. LKB1 is not expressed in CD primary cilia (Ac-Tub) of Lkb1ΔTub mice. Scale bars: 2 μm. See also Fig EV1C. Magnetic resonance tomography images obtained at 10 and 16 weeks. Representative images of coronal kidney sections of n = 3 mice/group at 10 weeks and n = 5 (control) and n = 8 (Lkb1ΔTub) at 16 weeks. Scale bars: 2 mm. See also Appendix Fig S1A. Plasma blood urea nitrogen (BUN) at 5, 14, and 23 weeks (w). Kaplan–Meier survival curves of control (n = 13) and Lkb1ΔTub (n = 15) mice. Periodic acid–Schiff (PAS) staining from control (5 weeks) and Lkb1ΔTub mice at 5, 14, and 23 weeks. Arrowheads indicate cysts. Lkb1ΔTub mice: representative of n = 13 (5 weeks), n = 12 (14 weeks), and n = 10 (23 weeks). Scale bar: 1 mm. Higher magnification of PAS- and Picrosirius Red (lower right)-stained sections showing dilated tubules (upper left), thickened tubular basement membranes (arrowheads, upper right), interstitial inflammation (lower left), and interstitial fibrosis (lower right) in 23-week-old Lkb1ΔTub mice. Scale bar: 50 μm. See also Fig EV1L. Transmission electron microscopy of distal tubule from 14-week-old Lkb1ΔTub mice. Dotted lines indicate thickening of basement membrane (lower panel). Representative images of n = 3 mice. Scale bars: 2 μm. Data information: In (D, E), filled circles: control mice; and open circles: Lkb1ΔTub mice. Each circle represents one individual mouse. In (D), bars indicate mean. Mann–Whitney, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Characterization of kidneys from Lkb1ΔTub mice A, B. Western blot (A) and quantification (B) of LKB1 expression in kidney medulla lysates from control and Lkb1ΔTub mice at 5 weeks. The remaining LKB1 protein derives from proximal tubule segments and glomeruli that are not targeted by Ksp-Cre. C. Representative confocal microscopy images of descending thin limb of Henle (AQP1 expressing) and thick ascending limb of Henle (Tamm-Horsfall expressing, THP) from 5-week-old control (n = 4) and Lkb1ΔTub (n = 5) animals. LKB1 is present in cilia (Ac-Tub) from AQP1-expressing tubules (upper panel: control mice; lower panel Lkb1ΔTub mice) and THP-positive tubules of control animals, but not in cilia from THP-positive tubules of Lkb1ΔTub mice, where Ksp-Cre is active. Scale bars: 2 μm. D, E. Staining (D) and quantification (E) of primary cilia (Ac-Tub) in collecting duct (CD) (Dolichos Biflorus Agglutinin expressing, DBA) at 5 weeks. Representative images of 5 mice/group. Blinded quantification of ten fields of view per biological sample. Scale bar: 50 μm. F. Scanning electron micrographs of CD at 5 weeks. Representative images of 5 mice/group. Scale bar: 20 μm, high magnification (right): 1 μm. G. Spot urine from 5-week-old animals. H, I. Urinary flow rate (H) and urine osmolality (I) at 5, 14, and 23 weeks. J. Representative kidneys from control and Lkb1ΔTub mice at 23 weeks. Scales in cm. K. Kidney weight (KW)-to-body weight (BW) ratio at 5, 14, and 23 weeks. L. Renal collagen mRNA content evaluated by qRT–PCR at 5 and 23 weeks. Data information: In (B, E, H, I, K, L), filled circles: control mice; and open circles: Lkb1ΔTub mice. Each circle represents one individual mouse. Bars indicate mean. Mann–Whitney, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not statistically different. w: weeks. Source data are available online for this figure. Download figure Download PowerPoint LKB1 interacts with ciliopathy proteins, and a functional interaction exists between LKB1 and NPHP1 To obtain insights into what might explain the phenotype of LKB1 deletion in the kidney, we performed a proteomic screen in protein lysates from renal medulla, aiming to identify endogenous interactors of LKB1 that might illuminate its function. The LKB1 precipitates obtained from these ly
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