While polycystin-1 (PC1) and polycystin-2 (PC2) are postulated to function as a receptor-channel complex, it is unknown what they sense nor what they signal. Fibrocystin (FPC) is a ciliary protein with Notch-like properties but its functional properties also remain uncertain. The PKD Section is tackling this problem in a variety of ways. As noted last year, we have used an agnostic, systems-based approach to examine the effects of acquired loss of Pkd1 in mouse kidney as they transition from normal to cystic state. Surprisingly, we found that the transcriptional profiles of normal male and female kidneys differed almost as much as those of normal and cystic kidneys and that differentially expressed gene modules were greatly enriched for genes involved in lipid metabolism. Using WGNA, we found that the gene networks were mostly preserved between sexes and between genotypes, suggesting that the signaling pathways are likely conserved across conditions. While mutant male kidneys had more differentially expressed genes at earlier time points, the number of differentially expressed genes was similar in male and female mutant samples at more severe stages with very few differentially expressed genes unique to either sex. Gene ontology of the differentially expressed genes common to both sexes showed enrichment for metabolic pathways, consistent with our previous findings in the early onset model, and confirmed global metabolic differences in cystic kidneys. We also found that the adult-onset and early onset models share common transcriptional signatures, suggesting the two forms of the disease share similar mechanisms of cyst initiation and growth. Finally, we showed that a very modest change in the composition of diet could significantly affect the progression of disease. Our study provides new evidence that metabolic pathways are an integral part of PKD and identifies sex as an important modifier, perhaps also through metabolic pathways. It has been long known that sex can be an important determinant of kidney disease severity, with male sex associated with accelerated progression of many renal diseases. It is therefore surprising that sex effects have not been considered in either of the two mouse models that are orthologous to human ADPKD, Pkd1 and Pkd2. This is particularly notable given that human studies suggest that males with autosomal dominant PKD have worse renal cystic disease with an earlier mean age of end stage renal disease but less severe liver cystic disease . Our studies unequivocally establish the sex-discordant effects of acquired inactivation of Pkd1 in the mouse and fully recapitulate what is observed in humans. These findings validate the model as a useful pre-clinical tool for studying the effects of hormone status on the pathogenesis of renal and biliary cystic disease and provide new opportunities for identifying potential therapies. Our data also illustrate the importance of sexual dimorphism as a confounding variable that can lead to irreproducible results or incorrect conclusions about the benefit or ineffectiveness of any intervention if not properly controlled. These findings highlight the importance of considering both sexes in preclinical studies as recently required by the National Institutes of Health. Progress has also been made on a number of other fronts. In one set of studies, we are seeking to develop cell models that be can used to screen for PKD function. Not only can they be used to determine mechanisms of action, they can be highly useful for screening for compounds that can rescue function of mutant cells, as exemplified by work in the CFTR field. Unfortunately, the PKD field has lacked good methods and high-throughput assay systems that can be used for this purpose. As noted last year, we have generated a number of LTL+ and DBA+ renal epithelial cell lines derived from our Pkd1 conditional mice and from these derived subclones with induced Pkd1 loss. After several years of previously unsuccessful searches, we have recently identified a phenotypic property that differs between the lines with and without Pkd1. We have also shown that we can reproduce the property in an mCCD cell line in which Pkd1 is silenced by shRNA. We are currently investigating the underlying mechanisms and looking for ways to test in vivo. We also have initiated a collaboration with Dr. Will Prinz to study the subcellular localization of Pkd1 by EM, and we have worked with collaborators to help determine how PC1 traffics in the cell. We also have generated a mutant mouse line by CRISPR as part of a collaboration with the Watnick lab to examine the functional consequences of loss of a factor identified in a Drosophila screen that disrupts PC2 trafficking. Finally, we have continued to evaluate our Pkhd1 mouse models. A manuscript describing the floxed exon 67 line with its knocked-in HA epitope tag is in final stages of preparation, and we continue to examine the functional consequences of complete deletion of the gene as we have identified a phenotype not previously described associated with this locus.
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