Analysis of inversin in mouse kidney and cultured renal epithelial cells.

摘要

Autosomal recessive inheritance of INVS gene mutations causes renal cyst formation in children with infantile nephronophthisis (NPH2) and in inv/inv mice. Inversin, the INVS gene product, interacts with multiple proteins and localizes to diverse sub-cellular compartments. The precise function of inversin in normal kidneys and resulting alterations in affected kidneys are not well defined. Proteomic analysis of newborn inv/inv and control mouse kidney lysates revealed ∼1000 differentially expressed proteins on two-dimensional gels. Ninety-six of the most differentially expressed proteins were identified using MALDI-TOF MS. Data analysis using pathway generation software revealed seventeen canonical signaling pathways and seven metabolic pathways that may be affected by the loss of functional inversin. Proteomic analysis prompted further investigation of adhesion proteins, and immunoblot studies revealed that adhesion protein expression was altered in inv/inv mouse kidney compared to control. INVS mutations alter or extinguish internal sequences encoding functional protein motifs including ankyrin repeats, D-boxes, IQ domains, and nuclear localization signals. Motifs were deleted from Invs-GFP reporter plasmids to redirect inversin localization and function. Plasmids were transfected into cultured kidney cells and imaged using confocal microscopy. Full-length inversin-GFP localized to primary cilium, centrioles, and nuclei, in agreement with published studies. Cells transfected with a plasmid lacking D-box-1 showed multiple nuclei after 24 hours of growth, indicating that D-box-1 loss altered cytokinesis. To determine how targeted knockdown of Invs altered renal morphology, embryonic mouse kidneys were treated with anti-Invs oligodeoxynucleotides (ODN) or scrambled ODN controls and cultured for 24 to 144 hours. Rudiments were fixed, stained with fluorescent lectins, and imaged using two-photon microscopy. Images were rendered to examine the three-dimensional morphology and branching pattern of the developing ureteric bud. No significant difference was identified between treated and control kidneys. Data analysis indicated that a stimulus not present in a cultured system may be required for cystogenesis. In conclusion, these studies indicated that aberrant inversin protein motifs may affect progression of cellular division and identified protein interactions and networks not previously implicated in the pathogenesis of NPH2.