Anti-BICC1 (N-terminal half) monoclonal antibody, clone 3E0.2

IntroductionNephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6.Objectives/methodsWe investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i5(2) and mIMCD3 krab shANKS6 i1(2).ResultsIn combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i5(2) knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i5(2) cells, but not in tet treated mIMCD3 krab shANKS6 i1(2) cells. However, yH2AX was found to be increased in the latter.ConclusionIn combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.

Bicc1 is a mouse homologue of Drosophila Bicaudal-C (dBic-C), which encodes an RNA-binding protein. Orthologs of dBic-C have been identified in many species, from C. elegans to humans. Bicc1-mutant mice exhibit a cystic phenotype in the kidney that is very similar to human polycystic kidney disease. Even though many studies have explored the gene characteristics and its functions in multiple species, the developmental profile of the Bicc1 gene product (Bicc1) in mammal has not yet been completely characterized. To this end, we generated a polyclonal antibody against Bicc1 and examined its spatial and temporal expression patterns during mouse embryogenesis and organogenesis. Our results demonstrated that Bicc1 starts to be expressed in the neural tube as early as embryonic day (E) 8.5 and is widely expressed in epithelial derivatives including the gut and hepatic cells at E10.5, and the pulmonary bronchi at E11.5. In mouse kidney development, Bicc1 appears in the early ureteric bud and mesonephric tubules at E11.5 and is also expressed in the metanephros at the same stage. During postnatal kidney development, Bicc1 expression gradually expands from the cortical to the medullary and papillary regions, and it is highly expressed in the proximal tubules. In addition, we discovered that loss of the Pkd1 gene product, polycystin-1 (PC1), whose mutation causes human autosomal dominant polycystic kidney disease (ADPKD), downregulates Bicc1 expression in vitro and in vivo. Our findings demonstrate that Bicc1 is developmentally regulated and reveal a new molecular link between Bicc1 and Pkd1.