Skip to main content

Role of Polycystic Kidney disease proteins in establishing and maintaining tubular structure

Final Activity Report Summary - PKD IN TUBULOGENESIS (Role of Polycystic Kidney Disease proteins in establishing and maintaining tubular structure)

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic diseases with an incidence of 1 in 1 000 in the general population affecting more than 12 million people worldwide. The hallmark of this slowly progressive disease is bilateral renal cysts formation. Cysts develop by massive enlargement of every segment of the nephron throughout an individual's lifetime and approximately 50 % of the patients progress to renal failure by age 50, requiring dialysis or transplant. There are no specific therapies to date to cure ADPKD and dyalisis and organ replacement therapy are the only available treatments to date. About 10 % of all patients undergoing renal transplantation are ADPKD patients generating an incredible economical burden and a serious social problem.

Mutations in two genes have been to date associated with the disease: PKD1 and PKD2. The former is responsible for 85 % of all cases, while the latter for the remaining 15 %. Both genes are developmentally regulated and are believed to play a key role in normal renal tubular differentiation, but both proteins' functions are largely unkown. PKD1 encodes a large (520kDa) transmembrane receptor (polycystin-1, PC-1) involved in cell / cell or cell / matrix interactions, while the PKD2 gene product (Polycystin-2, PC-2) is a non-selective cation channel.

The goal of this project was to implement a newly established lab entirely focused on understanding the molecular basis of the disease with a particular interest in understanding the function of polycystin-1. The four major achievements of this EXT grant are as follow:
1. Using a series of gain-and loss-of- function cellular systems we were able to better define the function of polycystin-1. In particular, we demonstrated that polycystin-1 is able to control cell migration, the actin cytoskeleton and cell-cell adhesion through regulation of GSK3beta. In addition, we found that PC-1 is able to regulate polarised migration through regulation of the microtubular cytoskeleton.
2 .We have identified a new domain in the C-tail of PC-1, performed an interaction screening and identified several potential interactors. Of these two have been followed closely and were verified by in vitro GST pull-down assays and by use of NMR spectroscopy in collaboration with the group of Dr G. Musco. Furthermore, both interactors were shown to interact with full-length polycystin-1 and we are currently performing functional studies to understand the biological significance of such interactions.
3. We were able to demonstrate that polycystin-1 plays a role in renal development by developing a renal organ culture technique. Using cultures of metanephric explants from E11.5 or E12.5 Pkd1-/- kidneys no branching defects could be visualized as compared to controls. However, culturing of organ cultures at day E14.5 showed defective growth and enhancement of apoptosis, have defective nephrogenesis and fail to elongate the mesenchymal-derived renal tubule properly.
4. We have generated knock-in mice carrying a tagged version of endogenous PC-1 in which we could remove the whole C-terminus by the use of the Cre-Lox system. Highly immunogenic commercially available antibodies could thus be employed for staining the protein and wild-type tissues could be easily employed as negative controls. Our strategy was successful and after performing a number of controls to make sure that the protein generated by our manipulations is fully functional, we have performed a full characterization of endogenous polycystin-1. Furthermore, we were able to achieve tissue-specific inactivation of the gene.