The role of PKD proteins in regulating tubular morphology

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

As previously reported, we had developed a mouse line with eGPF knocked into the c-terminus of the Pkd1 locus and used it to identify the PC1 interactome in 2-day old mouse head. In the past year, we have continued to refine conditions for determining the in vivo interactome in normal kidney. While we had developed methods that reproducibly isolate PC1 and PC2 as top targets, we have had inconsistent sets of other binding partners. Improving the reliability of the results continues to be a work in progress. In parallel, we have established kidney cell lines expressing PC1 CTT-AirID and a control expressing a neon-green-AirID protein with the PC1 mitochondrial targeting sequence. We have determined conditions that yield reproducible results and completed multiple independent replicates. The set of reproducibly identified interactors include mitochondrial proteins and other proteins listed as related to cystic phenotypes in OMIM and JAX-Mammalian phenotype ontology. There also is considerable overlap in the set of proteins that have been enriched in all AirID and in vivo “2-day old head” interactome experiments (N=234). We are currently working out strategies for validating targets, and we are generating a mouse line that can be used for in vivo AirID validation. In other studies, we have extended our studies of the Pkhd1del3-67 eye phenotype to include RNAscope Tfap2b hybridization data that in combination with our earlier immunolocalization studies show that both Tfap2b gene and AP2beta protein expression are greatly decreased in NCC-derived cells in the developing eye. A manuscript describing these findings is still under review. Since last year, we have successfully imported combinatorial sequencing methods based on the Shendure method to increase both the number of samples and cells that can be affordably analyzed. This is imperative for studies of cholangiocytes from Pkhd1 and Pkd1 livers since they comprise a very small fraction of all cells within the organ (1-2%). We have been refining methods for isolating cholangiocytes and begun investigating the snRNA profile of WT and Pkhd1 and Pkd1 mutant livers. We have also expanded use of the method to include nephron organoids and started generating Pkd1 and Pkd2 mutant embryos for the same purpose. In other updates, we have been investigating genetic interaction between Pkd1 and Itgb1, an integrin that had previously been implicated in PKD. Previous work by others using Aqp2–Cre–driven deletions reported that Itgb1 inactivation nearly prevented Pkd1-associated cystogenesis.. Given the profound benefits this afforded, we initiated our study to better understand the functional link between Pkd1 and Itgb1. Contrary to prior reports, we found that Itgb1 loss did not suppress cyst formation in Pkd1-deficient mice, though kidney/body weight ratios were modestly reduced in KspCre+ Pkd1cko/cko; Itgb1cko/cko compared to Pkd1cko/cko; Itgb1cko/wt littermates. Unexpectedly, KspCre-driven deletion of Itgb1 caused considerable perinatal lethality in Pkd1cko/wt; Itgb1cko/cko, which was profoundly mitigated by the homozygous deletion of Pkd1. A manuscript describing these and other findings is in final stages of preparation.