Rabphilin involvement in filtration and molecular uptake in Drosophila nephrocytes suggests a similar role in human podocytes.

Estela Selma-Soriano,Rubén Artero,Juan Manuel Fernández-Costa,Beatriz Llamusi,Lauren Louise Ozimski,Josep Redón

doi:10.1242/dmm.041509

Abstract

ABSTRACTDrosophila nephrocytes share functional, structural and molecular similarities with human podocytes. It is known that podocytes express the rabphilin 3A (RPH3A)-RAB3A complex, and its expression is altered in mouse and human proteinuric disease. Furthermore, we previously identified a polymorphism that suggested a role for RPH3A protein in the development of urinary albumin excretion. As endocytosis and vesicle trafficking are fundamental pathways for nephrocytes, the objective of this study was to assess the role of the RPH3A orthologue in Drosophila, Rabphilin (Rph), in the structure and function of nephrocytes. We confirmed that Rph is required for the correct function of the endocytic pathway in pericardial Drosophila nephrocytes. Knockdown of Rph reduced the expression of the cubilin and stick and stones genes, which encode proteins that are involved in protein uptake and filtration. We also found that reduced Rph expression resulted in a disappearance of the labyrinthine channel structure and a reduction in the number of endosomes, which ultimately leads to changes in the number and volume of nephrocytes. Finally, we demonstrated that the administration of retinoic acid to IR-Rph nephrocytes rescued some altered aspects, such as filtration and molecular uptake, as well as the maintenance of cell fate. According to our data, Rph is crucial for nephrocyte filtration and reabsorption, and it is required for the maintenance of the ultrastructure, integrity and differentiation of the nephrocyte.

Highlights

Rabphilin 3A (RPH3A) gene encodes a Rab small GTPase family effector protein implicated in vesicle docking/fusion reactions and the regulation of exo- and endocytosis processes in the central nervous system[1]
Following the same immunofluorescence assay, we demonstrated that Rph is expressed in nephrocytes from control larvae, and its signal is decreased in nephrocytes from Rph knock-down larvae (Fig. S2)
To assess whether the loss of nephrocytes in the IR-Rph flies was anticipated by defective differentiation during development, we studied the expression of the Drosophila ortholog of KLF15, which is critical for the development and differentiation of Drosophila nephrocytes and can be used as a terminal differentiation marker[17,18,23]

Summary

Introduction

Rabphilin 3A (RPH3A) gene encodes a Rab small GTPase family effector protein implicated in vesicle docking/fusion reactions and the regulation of exo- and endocytosis processes in the central nervous system[1]. Other data that support the importance of RPH3A in human proteinuric diseases are combined genomic and metabolomic analyses, which have previously associated increased urinary albumin excretion (UAE) in the general population[3,4] with a RPH3A gene polymorphism. Drosophila provides a suitable experimental model since it combines podocyte and renal proximal tubule functions in the same cell, the nephrocyte[5] This makes Drosophila nephrocytes interesting for in vivo studies that can be difficult to perform in mammals due to a lack of accessibility. Cubilin and Amnionless are located in the innermost part of the labyrinthine channels, where these proteins have an important role in endocytosis[15,16] Another requirement for the differentiation and maintenance of nephrocytes is the expression of the transcription factor Krüppel-like factor 15 (Klf15)[17,18]. The role of Rph in the maintenance of the structure and function of nephrocytes opens a window to explore the contribution of RPH3A gene polymorphisms to the risk of developing chronic kidney disease

Methods

Results

Conclusion