Previously, urinary exosomes have been identified as excreted vesicles (50-100nm) that contain a selection of kidney proteins, and hence are an accessible source of kidney biomarkers. Analysis of rodent models of ARPKD and patient samples revealed the accumulation of exosome-like vesicles (ELVs) around primary cilia, an organelle central to the pathogenesis in all forms of PKD. Further analysis of urinary ELVs showed they are highly enriched for three PKD-related proteins, polycystin-1 and -2 (ADPKD) and fibrocystin-polyductin (ARPKD), suggesting a functional role for the PKD proteins in kidney ELVs. Proteomic analysis confirmed the presence of the three major human cystogene products and supplied a list of 552 proteins, some with intriguing biological functions (for example smoothened protein was relatively abundant). In addition we were able to show physical interaction between ELVs and primary cilia, using cellular and whole tubule systems. We propose here to follow up on these preliminary observations to fully characterize the structure and function of these vesicles and determine their importance to the development of PKD.
The first aim will investigate the role of membrane proteins with large extracellular regions that are present on PKD-ELVs and that are implicated in cystic disease, in the PKD-ELV/primary cilium interaction. We will use the Pkhd1pk(+) pk(+) mouse as a source of epitope tagged PKD-ELVs to follow the interaction and Fc-chimeras containing domains derived from PC1, MEGF8, fibrocystin/polyductin and FAT4 to block the interaction. In the second aim we will analyze the effect of PKD-ELVs on cell signaling by surveying the mRNA and miRNA profiles of cells which can integrate a PKD-ELV signal with those that are blocked. We will use Fc-chimeras capable of blocking the interaction, such as the LRR_WSC domain from polycystin-1, to inhibit PKD-ELV binding to the primary cilium and we will also make paired primary cell populations from a mouse with a Cre removable transcriptional STOP cassette in intron 2 of the Pkhd1 gene. One half of the isolated cells will be treated with a Cre adenovirus and the other with an enzymically inactive Cre adenovirus. mRNA and miRNA will be isolated, from Fc-chimera treated cells and controls and the two paired cell populations (in separate experiments), the mRNA will be analyzed using long SAGE and high throughput DNA sequencing, the miRNA will be used to make a library again for high throughput sequencing. We will therefore be able to identify genes regulated by loss of PKD-ELV/primary cilium interactions (Fc-chimera block) and confirm these in the cells which regain their fibrocystin function (and hence PKD-ELV/primary cilium interactions) after the deletion of the transcriptional STOP cassette.
Human urine contains small membrane vesicles, called exosome-like vesicles (ELVs). Some of these ELVs contain proteins that are known to be intrinsically involved in the three major forms of human polycystic kidney disease. There are high levels of polycystin-1, the product of the autosomal dominant polycystic kidney disease 1 gene (PKD1) (the commonest genetic cause of renal failure), polycystin-2 the product of the PKD2 gene and fibrocystin the product of the autosomal recessive polycystic kidney disease gene (ARPKD). We will investigate the role of these proteins in ELVs and study the interaction of these vesicles with the primary cilium, a small cellular extension found in kidney tubules. We believe that the polycystins and fibrocystin may have a role in the attachment of ELVs to primary cilia and that ELVs transmit important biological signals. We believe that these studies will generate important insights into polycystic kidney disease which affects 250, 000 Americans.
|Lea, Wendy A; Parnell, Stephen C; Wallace, Darren P et al. (2018) Human-Specific Abnormal Alternative Splicing of Wild-Type PKD1 Induces Premature Termination of Polycystin-1. J Am Soc Nephrol 29:2482-2492|
|Gainullin, Vladimir G; Hopp, Katharina; Ward, Christopher J et al. (2015) Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner. J Clin Invest 125:607-20|
|Rossol-Allison, Jessica; Ward, Christopher J (2015) Exosomes to the Rescue. J Am Soc Nephrol 26:2303-4|
|Hogan, Marie C; Johnson, Kenneth L; Zenka, Roman M et al. (2014) Subfractionation, characterization, and in-depth proteomic analysis of glomerular membrane vesicles in human urine. Kidney Int 85:1225-37|
|Chen, Christopher Y; Hogan, Marie C; Ward, Christopher J (2013) Purification of exosome-like vesicles from urine. Methods Enzymol 524:225-41|