Figure - uploaded by Lauren Louise Ozimski
Content may be subject to copyright.
Figure S2. Rph is expressed in pericardial nephrocytes of Drosophila larvae. Representative confocal images of larvae expressing the GFP reporter (green in A, A'', B and B'') under the control of the HandGal4 driver. Immunostaining with anti-Rph antibody (A', A'', B' and B'') revealed Rph presence in pericardial nephrocytes of control larvae (A-A''), but not in larvae expressing a Rph RNA interference construct (B-B''). Scale bar = 10 μm.
Source publication
Drosophila nephrocyte 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 deve...
Similar publications
The podocyte slit diaphragm (SD) is an essential component of the glomerular filtration barrier and its disruption is a common cause of proteinuria and many types of kidney disease. Therefore, better understanding of the pathways and proteins that play key roles in SD formation and maintenance has been of great interest. Podocyte and SD biology hav...
Citations
... The IFM area of Drosophila thoraces was analyzed as previously described. 38,39 Briefly, six thoraces of adult females of each genotype, aged 7 and 15 days, were embedded in epoxy resin following standard procedures. After drying the resin, semi-thin cross sections of 1.5 µm were made using Reichert Jung Ultracut Ultramicrotome (Leica Microsystems, Wetzlar, Germany). ...
Limb‐girdle muscular dystrophy D2 (LGMDD2) is an ultrarare autosomal dominant myopathy caused by mutation of the normal stop codon of the TNPO3 nuclear importin. The mutant protein carries a 15 amino acid C‐terminal extension associated with pathogenicity. Here we report the first animal model of the disease by expressing the human mutant TNPO3 gene in Drosophila musculature or motor neurons and concomitantly silencing the endogenous expression of the fly protein ortholog. A similar genotype expressing wildtype TNPO3 served as a control. Phenotypes characterization revealed that mutant TNPO3 expression targeted at muscles or motor neurons caused LGMDD2‐like phenotypes such as muscle degeneration and atrophy, and reduced locomotor ability. Notably, LGMDD2 mutation increase TNPO3 at the transcript and protein level in the Drosophila model Upregulated muscle autophagy observed in LGMDD2 patients was also confirmed in the fly model, in which the anti‐autophagic drug chloroquine was able to rescue histologic and functional phenotypes. Overall, we provide a proof of concept of autophagy as a target to treat disease phenotypes and propose a neurogenic component to explain mutant TNPO3 pathogenicity in diseased muscles.
... An immunofluorescence assay with the human anti-Rabphilin antibody was performed with heart tubes of control flies to check the presence of Rph in this tissue. The UAS-Gal4 system was used to direct the silencing of Rph gene, with two different UAS-IR-Rabphilin lines, only in cardiomyocytes (GMH5-Gal4 > UAS-IR-Rabphilin in Fig. 1 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′). To demonstrate the specificity of the antibody, the same assay was performed in flies with combined Rph RNAi, with two different lines of Rph RNAi constructs, in cardiomyocytes and nephrocytes (Hand-Gal4 UAS-GFP > UAS-IR-Rabphilin), and an important reduction of Rph expression was observed both in the cardiac tube and in nephrocytes ( Fig. 1A-B′, G, Supplementary Figure 1A-B′, G, and Fig. 2A-B′, G). ...
... 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][22][23] , we counted the number of pericardial nephrocytes in 1-weekold 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. ...
... 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 . ...
Heart failure (HF) and the development of chronic kidney disease (CKD) have a direct association. Both can be cause and consequence of the other. Many factors are known, such as diabetes or hypertension, which can lead to the appearance and/or development of these two conditions. However, it is suspected that other factors, namely genetic ones, may explain the differences in the manifestation and progression of HF and CKD among patients. One candidate factor is Rph, a gene expressed in the nervous and excretory system in mammals and Drosophila, encoding a Rab small GTPase family effector protein implicated in vesicular trafficking. We found that Rph is expressed in the Drosophila heart, and the silencing of Rph gene expression in this organ had a strong impact in the organization of fibers and functional cardiac parameters. Specifically, we observed a significant increase in diastolic and systolic diameters of the heart tube, which is a phenotype that resembles dilated cardiomyopathy in humans. Importantly, we also show that silencing of Rabphilin (Rph) expression exclusively in the pericardial nephrocytes, which are part of the flies' excretory system, brings about a non-cell-autonomous effect on the Drosophila cardiac system. In summary, in this work, we demonstrate the importance of Rph in the fly cardiac system and how silencing Rph expression in nephrocytes affects the Drosophila cardiac system.
... In 2003, Rastaldi et al. investigated Rph3A and Rab3A as possible players in podocyte biology. Based on that Rph3A binds to Rab3A, it is a complex required for the docking of synaptic vesicles to their target membrane and that bind with cytoskeletal proteins in neurons, vesical and cytoskeletal are also important for podocyte homeostasis [47]. The complex Rab3A-Rph3A has been demonstrated in neurons and neuroendocrine structures as well as in normal kidneys from mice, rats, and humans in podocytes around vesicles contained in the foot processes [62,63]. ...
... The complex Rab3A-Rph3A has been demonstrated in neurons and neuroendocrine structures as well as in normal kidneys from mice, rats, and humans in podocytes around vesicles contained in the foot processes [62,63]. In addition, in human biopsies of proteinuric patients with focal and segmental glomeruloesclerosis, an increment of Rab3A-Rph3A expression was also identified [47]. The potential role of these molecules in renal damage was further studied by the same group in a model of Rab3A knockout mice with highglucose diet. ...
Small Rab GTPases, the largest group of small monomeric GTPases, regulate vesicle trafficking in cells, which are integral to many cellular processes. Their role in neurological diseases, such as cancer and inflammation have been extensively studied, but their implication in kidney disease has not has not been researched in depth. Rab3a and its effector Rabphillin-3A (Rph3A) expression have been demonstrated to be present in the podocytes of normal kidneys of mice rats and humans, around vesicles contained in the foot processes, and they are overexpressed in diseases with proteinuria. In addition, the Rab3A knockout mice model induced profound cytoskeletal changes in podocytes of high glucose fed animals. Likewise, RphA interference in the Drosophila model produced structural and functional damage in nephrocytes with reduction in filtration capacities and nephrocyte number. Changes in the structure of cardiac fiber in the same RphA-interference model, open the question if Rab3A dysfunction would produce simultaneous damage in the heart and kidney cells, an attractive field that will require attention in the future.
Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi‐sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA‐sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early‐ and late‐stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post‐transcriptional regulators, miR‐1000‐5p, miR‐13b‐3p, miR‐263‐5p and miR‐252‐5p showed positive correlations, whereas miR‐315‐5p, miR‐92b‐3p, miR‐137‐3p, miR‐11‐3p and miR‐10‐3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)–mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna‐specific functions. Our data serve as a foundation for understanding stage‐specific antennal transcriptomes and large‐scale comparative analysis of transcriptomes in different insects.
Podocyte injury can disrupt the glomerular filtration barrier (GFB), leading to podocytopathies that emphasize podocytes as the glomerulus’s key organizer. The coordinated cytoskeleton is essential for supporting the elegant structure and complete functions of podocytes. Therefore, cytoskeleton rearrangement is closely related to the pathogenesis of podocytopathies. In podocytopathies, the rearrangement of the cytoskeleton refers to significant alterations in a string of slit diaphragm (SD) and focal adhesion proteins such as the signaling node nephrin, calcium influx via transient receptor potential channel 6 (TRPC6), and regulation of the Rho family, eventually leading to the disorganization of the original cytoskeletal architecture. Thus, it is imperative to focus on these proteins and signaling pathways to probe the cytoskeleton rearrangement in podocytopathies. In this review, we describe podocytopathies and the podocyte cytoskeleton, then discuss the molecular mechanisms involved in cytoskeleton rearrangement in podocytopathies and summarize the effects of currently existing drugs on regulating the podocyte cytoskeleton.
Biological and biomedical research using Drosophila melanogaster as a model organism has gained recognition through several Nobel prizes within the last 100 years. Drosophila exhibits several advantages when compared to other in vivo models such as mice and rats, as its life cycle is very short, animal maintenance is easy and inexpensive and a huge variety of transgenic strains and tools are publicly available. Moreover, more than 70% of human disease-causing genes are highly conserved in the fruit fly. Here, we explain the use of Drosophila in nephrology research and describe two kidney tissues, Malpighian tubules and the nephrocytes. The latter are the homologous cells to mammalian glomerular podocytes and helped to provide insights into a variety of signaling pathways due to the high morphological similarities and the conserved molecular make-up between nephrocytes and podocytes. In recent years, nephrocytes have also been used to study inter-organ communication as links between nephrocytes and the heart, the immune system and the muscles have been described. In addition, other tissues such as the eye and the reproductive system can be used to study the functional role of proteins being part of the kidney filtration barrier.
Cubilin (CUBN), the intrinsic factor-vitamin B12 receptor is a large endocytic protein involved in various physiological functions: vitamin B12 uptake in the gut; reabsorption of albumin and maturation of vitamin D in the kidney; nutrient delivery during embryonic development. Cubilin is an atypical receptor, peripherally associated to the plasma membrane. The transmembrane proteins amnionless (AMN) and Lrp2/Megalin are the currently known molecular partners contributing to plasma membrane transport and internalization of Cubilin. The role of Cubilin/Amn complex in the handling of vitamin B12 in health and disease has extensively been studied and so is the role of the Cubilin-Lrp2 tandem in renal pathophysiology. Accumulating evidence strongly supports a role of Cubilin in some developmental defects including impaired closure of the neural tube. Are these defects primarily caused by the dysfunction of a specific Cubilin ligand or are they secondary to impaired vitamin B12 or protein uptake? We will present the established Cubilin functions, discuss the developmental data and provide an overview of the emerging implications of Cubilin in the field of cardiovascular disease and cancer pathogenesis.
The podocyte slit diaphragm (SD) is an essential component of the glomerular filtration barrier and its disruption is a common cause of proteinuria and many types of kidney disease. Therefore, better understanding of the pathways and proteins that play key roles in SD formation and maintenance has been of great interest. Podocyte and SD biology have been mainly studied using mouse and other vertebrate models. However, vertebrates are limited by inherent properties and technically challenging in vivo access to the podocytes. Drosophila is a relatively new alternative model system but it has already made great strides. Past the initial obvious differences, mammalian podocytes and fly nephrocytes are remarkably similar at the genetic, molecular and functional levels. This review discusses SD formation and maintenance, and their dependence on cell polarity, the cytoskeleton, and endo- and exocytosis, as learned from studies in fly nephrocytes and mammalian podocytes. In addition, it reflects on the remaining gaps in our knowledge, the physiological implications for glomerular diseases and how we can leverage the advantages Drosophila has to offer to further our understanding.
