Nek8 Mutation Causes Overexpression of Galectin-1, Sorcin, and Vimentin and Accumulation of the Major Urinary Protein in Renal Cysts of jck Mice

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. Molecular & Cellular Proteomics 4:1009–1020, 2005. fluid-filled cysts in the kidneys and other organs. The renal cysts originate from a single layer of epithelial cells lining the nephrons and renal collecting ducts. Cells from cystogenic epithelium have a higher rate of cellular proliferation and are less differentiated than normal collecting duct cells. The autosomal dominant form of the disease (ADPKD) occurs at a rate of 1 in 500–1,000 individuals, whereas the autosomal recessive form (ARPKD) occurs at a rate of 1 in 6,000–40,000 live births (1). Mutations in the genes PKD1 or PKD2 are responsible for ADPKD, whereas the gene PKHD1 is mutated in ARPKD. The proteins encoded by the PKD1 and PKD2 genes, polycystin-1 and polycystin-2, are both integral membrane proteins and associate to function as receptors or cation channels, affecting cytosolic Ca 2 concentration. The protein fibrocystin/polyductin, encoded by the gene PKHD1, is also a membrane protein, and although its function is unknown, its large extracellular domain suggests receptor activity (1). The proteins polycystin-1 and polycystin-2, as well as fibrocystin/polyductin, have been localized to the primary cilia, an organelle associated with the basal bodies and predominantly observed in epithelial cells, and have been found to interact directly with each other to form a complex (2–4). A number of murine models have been developed to study the mechanism of PKD. These models were generated by spontaneous mutation or gene-targeted engineering and closely resemble human PKD with regards to cyst morphology, cyst localization, and disease progression (5). Several models resemble ADPKD, characterized by the formation of cysts originating from most parts of the nephron, whereas other models resemble ARPKD with cyst formation predominantly originating in the collecting duct. The proteins mutated in several murine models have been identified, specifically the protein cystin in the cpk model, Bicaudal C in the bpk and jcpk models, polaris in the orpk model, inversin in the inv model, and Nek1 in the kat model. Most of the murine PKD models,