Abstract
The jck murine model, which results from a double point mutation in the nek8 gene, has been used to study the mechanism of autosomal recessive polycystic kidney disease (ARPKD). The renal proteome of jck mice was characterized by two-dimensional gel electrophoresis combined with mass spectrometry (MALDI-TOF/TOF). Four newly identified proteins were found to accumulate in the kidneys of jck mice with polycystic kidney disease (PKD) compared with their wild-type littermates. The proteins galectin-1, sorcin, and vimentin were found to be induced 9-, 9-, and 25-fold, respectively, in the PKD proteome relative to the wild type. The identity of these proteins was established by peptide mass fingerprinting and de novo MS/MS sequencing of selected peptides. Up-regulation of these three proteins may be due to the nek8 mutation, and their function may be related to the signaling and structural processes in the primary cilium. Additionally a series of protein isoforms observed only in the ARPKD kidney was identified as the major urinary protein (MUP). Peptide sequencing demonstrated that the isoforms MUP1, MUP2, and MUP6 are contained in this series. The MUP series showed a number of male-specific isoforms and a phosphorylation of the entire series with an increasing degree of phosphorylation of the acidic isoforms. In addition, the MUP series was localized to the cyst fluid of PKD mice, and a cellular mislocalization of galectin-1, sorcin, and vimentin in PKD tubular epithelial cells was shown. The abnormal and extremely high accumulation of the MUPs in the ARPKD kidney may be linked to a defect in protein transport and secretion. The discovery of these proteins will provide new information on the molecular and cellular processes associated with the mechanism of ARPKD.
Highlights
The jck murine model, which results from a double point mutation in the nek8 gene, has been used to study the mechanism of autosomal recessive polycystic kidney disease (ARPKD)
Mutations in the genes PKD1 or PKD2 are responsible for ADPKD, whereas the gene PKHD1 is mutated in ARPKD
The jck murine model of cystic disease closely parallels the phenotype of human ARPKD in that it is a juvenile form of PKD and in cyst formation originating in the collecting ducts
Summary
Animals and Genotyping—The jck mice were obtained from The Jackson Laboratory (Bar Harbor, ME). IPG dry strips (24 cm, pH 3–10 nonlinear, Amersham Biosciences) were rehydrated with 450 l of sample containing 1.5 mg of protein, and isoelectric focusing was carried out in an IPGphor unit for 64,000 V-h (Amersham Biosciences). IPG strips were frozen at Ϫ80 °C and equilibrated for 10 min in equilibration buffer (375 mM Tris1⁄7HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 100 mM DTT and for another 10 min in equilibration buffer containing 250 mM iodoacetamide They were loaded on 25 ϫ 20-cm slab gels (11% polyacrylamide), and proteins were separated according to molecular weight in an EttanDaltSix unit (Amersham Biosciences). Slides were incubated in blocking solution (PBS containing 2% BSA (Roche Applied Science)) for 1 h at room temperature. Controls using rabbit IgG and goat IgG instead of primary antibodies were processed in parallel and were negative in each case
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
