Investigating nephronophthisis using a novel murine and cell model

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.