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

Abstract

Drosophila 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.

Rabphilin silencing causes dilated cardiomyopathy in a Drosophila model of nephrocyte damage

“…1 and Supplementary Figure 1A–B′). Previously, we observed Rph expression in nephrocytes 9 , and in this study, we report that Rph is also expressed in the Drosophila heart tube with a punctate pattern in the cytoplasm of cardiomyocytes (Fig. 1 and Supplementary Figure 1A, A′, C, C′, E and E′).…”

“…Specifically, we show that Rph, a well-conserved Rab effector protein, is expressed in the Drosophila heart. Similarly, we have previously reported Rph expression in pericardial nephrocytes 9 and human podocytes 4 .…”

“…To account for this strong effect in median survival, and because the loss of nephrocytes is known to promote defects in cardiogenesis and cardiac function 21 – 23 , we counted the number of pericardial nephrocytes in 1-week-old adult females. The total average number of pericardial nephrocytes was the same for both genotypes, but when focusing on functional nephrocytes only, cells highly differentiated with strong capacity to endocytose and filtrate, which are characterized by Hand -driven GFP signal and intact nuclei as it is described in Selma-Soriano et al 9 . The number of functional nephrocytes in IR-Rph1 flies was significantly reduced compared to controls.…”

“…We have also shown previously that Rabphilin ( Rph ), the Drosophila ortholog of human Rabphilin-3A , participates in the endocytic pathway in nephrocytes and is necessary for the filtration of toxins from the hemolymph in this cell type. We also demonstrated that reduced levels of Rph caused structural alterations that had a negative impact on endocytosis and filtration rates 9 .…”

“…We previously demonstrated alterations in the ultrastructure of nephrocytes upon the interference of Rph expression 9 using the Hand-Gal4 driver. As it is known that Hand-Gal4 drives expression more strongly in cardiomy ocytes than in pericardial nephrocytes 15 , this driver was used to study the effect of Rph interference on Drosophila heart tube structure as well on pericardial nephrocytes.…”