TRANSMEMBRANE PROTEIN 2: A NOVEL PROTEIN IN POLYCYSTIC KIDNEY DISEASE

Abstract

ABSTRACT Background: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common hereditary cause of end stage renal disease. Affected individuals have mutations in PKD1 or PKD2 genes, generating defective polycystin -1 (PC1) or polycystin -2 (PC2) protein, respectively. Transmembrane protein 2 (TMEM2) is a novel protein with extensive homology to fibrocystin, the product of the polycystic the polycystic kidney and hepatic disease (PKHD1) gene. Mutations in PKHD1 cause Autosomal Recessive Polycystic Kidney Disease. It has been shown that urinary exosomes from individuals with PKD1 mutations have decreased levels of PC1 and increased levels of TMEM2 when compared with individuals with no mutations in the PKD genes. We sought to determine the expression status of TMEM2 in ADPKD and whether TMEM2 interacts with PC1. Methods: Immunohistochemistry (IHC): Sections from ADPKD human kidney tissues and normal human kidney (NHK) tissues were probed for presence of TMEM2 and PC1. Immunofluorescence: Cells from ADPKD and normal human kidneys were grown until confluent and probed with TMEM2 and tubulin. Immunoprecipitations (IP): HEK293T cells were co-transfected with full length V5 tagged TMEM2 construct, C terminus FLAG tagged PC1 construct and various FLAG tagged N terminus PC1 constructs. Co-IPs were performed on membrane preparations derived from the transfectants. Results: Increased TMEM2 expression was detected on the apical aspect of cyst epithelial cells in ADPKD kidneys when compared to normal human kidneys. There was an increase in cellular expression and colocalization of TMEM2 to ciliary structures in ADPKD cells compared to NHK cells. Co-IPs showed that there was an interaction between PC1 and TMEM2. The interaction was mapped to the N-terminal extra-cellular portion of PC1 but not the C terminus. TMEM2 appeared to have high affinity for the PKD and REJ domains within N terminal portion of PC1. Conclusion: Increased TMEM2 expression in ADPKD kidneys and urinary exosomes combined with the interaction of TMEM2 with PC1 suggests that TMEM2 is a novel protein implicated in the pathogenesis of ADPKD. The exosomal PC1/TMEM2 ratio may have utility in the diagnosis of pre-cystic disease as well as in monitoring the disease progression and response to treatment.