Investigating the molecular mechanisms of endocytosis in the renal proximal tubule

Abstract

The high endocytic activity of the renal proximal tubule is essential for maintaining kidney health through the removal of low molecular weight proteins and other biological solutes from the renal filtrate. The scavenger receptor megalin drives the reabsorption of ~90% of filtered protein, and the mis-regulation of megalin trafficking is associated with a number of human diseases. We have used zebrafish as a model to study the molecular mechanisms involved in megalin trafficking in vivo and have identified Pacsin2 and Sorting nexin-27a (Snx27a) as novel components of the trafficking machinery. Pacsin2 is a member of the F-BAR family of proteins that dimerise and bind to negatively charged phospholipids in membranes to aid in membrane deformation or act as a scaffold for recruitment of effector proteins. Pacsin2 has been shown to participate in numerous trafficking events in cultured cells such as clathrin-mediated endocytosis, bi-directional transport between early endosomes and the trans-Golgi network, and endocytic recycling events. We have found that zebrafish Pacsin2 colocalises to clathrincoated structures, early endosomes and recycling endosomes in cultured zebrafish cells, in line with the mammalian protein, as well as with megalin, suggestive of a possible role for Pacsin2 in megalin trafficking. This was confirmed through knockout and knockdown of Pacsin2 in zebrafish larvae, which have a decreased endocytic capacity in the proximal tubule epithelium indicative of a megalin trafficking defect. Immunofluorescence microscopy reveals a decrease in the abundance of endocytic markers and megalin in Pacsin2 depleted larvae, further supporting the hypothesis that Pacsin2 is contributing to the normal trafficking of megalin in vivo. Furthermore, electron microscopy reveals a significant reduction in the apical endocytic apparatus in proximal tubule epithelial cells, with a significant decrease in the abundance of apical vacuolar endosomes, suggestive of a decrease in clathrin-mediated endocytosis in this tissue. Finally, human SNX27 is known to bind both membrane-bound cargo proteins and the retromer complex to direct membrane-bound cargo protein recycling from early endosomes to the plasma membrane. Our preliminary data suggests Snx27a is also participating in normal megalin trafficking in the proximal tubule in vivo. Morpholino knockdown of snx27a in zebrafish larvae severely impairs tracer endocytosis into the proximal tubule. We predict that Snx27a is needed for the efficient recycling of megalin to the plasma membrane from the early/recycling endosome of proximal tubule epithelial cells.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Martin Lowe (Supervisor) & Shane Herbert (Supervisor)