Investigating nephronophthisis using a novel murine and cell model. Ann Marie Hynes A thesis submitted for the Degree of Doctor of Philosophy Institute of Genetic Medicine, Newcastle University, September 2013 i Abstract Nephronophthisis (NPHP) is a major cause of pediatric renal failure. Currently there is little understanding of the aetiology of the disease. In order to identify the molecular events leading to NPHP, we have created a novel mutant mouse strain containing a truncating mutation in the Cep290 gene. Patients with mutations in CEP290 present with a ciliopathy phenotype that includes retinal dystrophy, cerebellum defects and NPHP. Characterisation of Cep290LacZ/LacZ mice confirms that they display all the features of the human condition. Microarray analysis of newborn kidney tissue was used to explore initiating events leading to NPHP. Ciliopathies have recently been associated with either disrupted Wingless integrated (Wnt) or sonic hedgehog (Shh) signaling. We show that mutant kidneys display abnormal Shh signaling in the absence of Wnt signaling abnormalities. Primary cell cultures of collecting duct (CDT) cells (isolated from Cep290LacZ/LacZ mice and wild-type litter mates crossed with the “immorto” mouse) were established and characterised. CDT cells expressed the mineral corticoid receptor (MR) and the epithelial sodium channel (ENaC) alpha subunit. The CDT cell lines formed epithelial layers and formed tubules when maintained in 3D culture media. Cep290LacZ/LacZ CDT cells displayed ciliogenesis abnormalities as well as abnormal spheroids with loss of lumen when grown in 3D culture. Pharmacological activation of Shh signaling (purmorphamine) partially rescues the spheroid and ciliogenesis defects in Cep290LacZ/LacZ CDT cells. This implicates abnormal Shh signaling in the onset of NPHP and suggests that targeted treatment of Shh antagonists have therapeutic potential. ii Dedication In loving memory of my Mam. I’ve missed you every day iii Acknowledgements Firstly and most importantly, I would like to thank my two supervisors Dr. Colin Miles and Dr. John Sayer for their expert guidance, support, help and supervision during my PhD. I would also like to thank Professor Nicholas Simmons for his many interesting ideas and my internal assessors Professor Judith Goodship and Dr. Simon Bamforth for your helpful advice. I would also like to thank my collaborators; Dr. Rachel, H. Giles, who assisted with analysis on 3D cultures, Dr. Matthew Bashton, who carried out the data analysis on the microarray data, Dr. Kath White, Ms Tracey Davey and Caroline Miller who assisted with the electron microscopy images and Dr. Pete Thelwall who assisted with analysis on brain MRI scanning. A special thank you to, Dr. Lorraine Eley, for all the technical advice, written advice and finally for the relentless encouragement to get this thesis finished. A huge thank you to the willing volunteers who proof read various chapters of my thesis, including Dr. Roslyn Jane Simms, Dr. Georgina Carr and Dr. Kevin Gillinder. Thank you to all the members of the kidney/muscle/endocrine lab for all your support, friendship and enabling me to borrow labs supplies/equipment when I had run out. Also a big thank you to Holly Anderson who kindly let me film her so I would know how to transfer a western blot on my own. Special thanks to Dr. Adya Misra and Amy-Leigh Johnson for all your moral support and encouragement. A big thanks to my life time friends Claire Feeney and Claire Burns back at home in Ireland who have supported me through everything life has thrown at me, I’m grateful to have such wonderful friends. I would like to thank all my family who have marked their calendars and will be lighting their candles for me on the day of my viva. Special thanks to my dad for your continuous support. I would also especially like to thank my big brother Cathal who I know is always there for me, listening to my problems and giving me support and encouragement when I needed it most. Finally I would like to thank my partner Dean Hallam for your constant support, wonderful meals, looking after me when I was unwell and for your invaluable expertise in the final formatting of my thesis. iv Table of Contents Abstract ........................................................................................................ ii Chapter 1 Introduction ................................................................................... 1 1.1 Polycystic Kidney Disease (PKD) .......................................................... 1 1.2 Autosomal Dominant Polycystic Kidney Disease (ADPKD) ................... 1 1.3 Autosomal Recessive Polycystic Kidney Disease (ARPKD) .................. 2 1.4 Pleiotropic PKD disorders. ..................................................................... 2 1.5 NPHP ..................................................................................................... 3 1.6 Clinical effect of NPHP genetic mutations.............................................. 5 1.7 NPHP6 mutations .................................................................................. 6 1.8 Current Nphp mouse models ................................................................. 6 1.9 JBTS ...................................................................................................... 9 1.10 MKS .................................................................................................. 11 1.11 BBS .................................................................................................. 11 1.12 Sub cellular location of NPHP and JBTS protein products ............... 12 1.13 The primary cilium and NPHP/JBTS protein products ...................... 15 1.14 Linking Shh and Wnt signalling with NPHP and JBTS ...................... 17 1.15 Defective Shh/Wnt signalling identified in Nphp mouse models ....... 20 1.16 Defective Shh signalling in JBTS/MKS mouse models ..................... 21 1.17 CEP290 mutations and Cep290 animal models ............................... 22 1.18 CEP290 and its sub cellular location in primary cilia ........................ 23 1.19 CEP290’s interacting partners. ......................................................... 24 1.20 Expression patterns of CEP290 in human embryonic and fetal tissue . ......................................................................................................... 25 v 1.21 The normal physiology of collecting duct tubule (CDT) cells are required to aid in understanding disease pathology of NPHP. ...................... 26 1.22 ARPKD CDT cell lines previously studied ........................................ 27 1.23 Using animal models to test potential target ADPKD drugs. ............. 27 1.24 Treating PKD patients ...................................................................... 29 1.25 Aims of this study ............................................................................. 31 Chapter 2 Materials and Methods ................................................................ 32 2.1 DNA extraction. .................................................................................... 32 2.2 Mouse genomic DNA extraction. ......................................................... 32 2.3 Genotyping, Polymerase Chain Reaction (PCR) ................................. 32 2.4 PCR protocol ....................................................................................... 33 2.5 Agarose Gel Electrophoresis ............................................................... 33 2.6 Ribosomal Nucleic Acid (RNA) extraction from Tissue. ....................... 33 2.7 Illumina Microarray .............................................................................. 34 2.7.1 Illumina Microarray sample preparation. ....................................... 34 2.7.2 Normalisation of Illumina Microarray Data. .................................... 35 2.7.3 Data analysis ................................................................................. 35 2.8 Reverse Transcription (RT) .................................................................. 35 2.9 Quantitative PCR (qPCR) .................................................................... 36 2.10 Mouse Husbandry. ........................................................................... 37 2.11 Tissue preparation for histology ........................................................ 37 2.11.1 Whole mount X-gal staining. ...................................................... 38 2.11.2 OCT embedding ......................................................................... 38 2.12 Histology methods. ........................................................................... 39 vi 2.12.1 Brain MRI Imaging. .................................................................... 39 2.12.2 Rehydrating kidney tissues embedded in paraffin. ..................... 39 2.12.3 H+E staining and Mounting coverslips. ...................................... 40 2.12.4 Trichrome Masson Staining ....................................................... 40 2.12.5 Sirius Red Staining .................................................................... 40 2.12.6 Nuclear Fast Red Staining ......................................................... 41 2.13 Light Microscopy ............................................................................... 41 2.14 Electron Microscopy Methods ........................................................... 41 2.14.1 Scanning Electron Microscopy (SEM) ........................................ 41 2.14.2 Transmission Electron Microscopy (TEM).