Nephrocytes Remove Microbiota-Derived Peptidoglycan from Systemic Circulation to Maintain Immune Homeostasis.

Preventing aberrant immune responses against the microbiota is essential for the health of the host. Microbiota-shed pathogen-associated molecular patterns translocate from the gut lumen into systemic circulation. Here, we examined the role of hemolymph (insect blood) filtration in regulating systemic responses to microbiota-derived peptidoglycan. Drosophila deficient for the transcription factor Klf15 (Klf15NN) are viable but lack nephrocytes-cells structurally and functionally homologous to the glomerular podocytes of the kidney. We found that Klf15NN flies were more resistant to infection than wild-type (WT) counterparts but exhibited a shortened lifespan. This was associated with constitutive Toll pathway activation triggered by excess peptidoglycan circulating in Klf15NN flies. In WT flies, peptidoglycan was removed from systemic circulation by nephrocytes through endocytosis and subsequent lysosomal degradation. Thus, renal filtration of microbiota-derived peptidoglycan maintains immune homeostasis in Drosophila, a function likely conserved in mammals and potentially relevant to the chronic immune activation seen in settings of impaired blood filtration.

Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing.

Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

Endocytosis at the Crossroad of Polarity and Signaling Regulation: Learning from Drosophila melanogaster and Beyond.

Cellular trafficking through the endosomal-lysosomal system is essential for the transport of cargo proteins, receptors and lipids from the plasma membrane inside the cells and across membranous organelles. By acting as sorting stations, vesicle compartments direct the fate of their content for degradation, recycling to the membrane or transport to the trans-Golgi network. To effectively communicate with their neighbors, cells need to regulate their compartmentation and guide their signaling machineries to cortical membranes underlying these contact sites. Endosomal trafficking is indispensable for the polarized distribution of fate determinants, adaptors and junctional proteins. Conversely, endocytic machineries cooperate with polarity and scaffolding components to internalize receptors and target them to discrete membrane domains. Depending on the cell and tissue context, receptor endocytosis can terminate signaling responses but can also activate them within endosomes that act as signaling platforms. Therefore, cell homeostasis and responses to environmental cues rely on the dynamic cooperation of endosomal-lysosomal machineries with polarity and signaling cues. This review aims to address advances and emerging concepts on the cooperative regulation of endocytosis, polarity and signaling, primarily in Drosophila melanogaster and discuss some of the open questions across the different cell and tissue types that have not yet been fully explored.

Nephrocytes are part of the spectrum of filtration epithelial diversity.

The excretory system produces urine by ultrafiltration via a filtration epithelium. Podocytes are widely found as filtration epithelial cells in eucoelomates. In some animal taxa, including insects and crustaceans, nephrocytes serve to separate toxic substances from the body fluid, in addition to podocytes. Drosophila nephrocytes have been recently utilized as a model system to study podocyte function and disease. However, functionality and cellular architecture are strikingly different between Drosophila nephrocytes and eucoelomate podocytes, and the phylogenetic relationship between these cells remains enigmatic. In this study, using focused-ion beam-scanning electron microscopy (FIB-SEM) tomography, we revealed three-dimensional architecture of decapod nephrocytes with unprecedented accuracy-they filled an enormous gap, which can be called "missing link," in the evolutionary diversity of podocytes and nephrocytes. Thus, we concluded that nephrocytes are part of the spectrum of filtration epithelial diversity in animal phylogeny.

Conformational and functional effects of MPA-CdTe quantum dots on SOD: Evaluating the mechanism of oxidative stress induced by quantum dots in the mouse nephrocytes.

The application of quantum dots (QDs) is restricted by the biosafety issue. QDs contribute to the adverse effects of organisms probably because of the ability to induce oxidative stress via changing the activity of antioxidant enzyme, for example, superoxide dismutase (SOD). But the underlying molecular mechanisms still remain unclear. This study investigated the harmful effects of oxidative stress induced by mercaptopropionic acid capped CdTe QDs (MPA-CdTe QDs) on the mouse primary nephrocytes as well as the structure and function of SOD molecule and explored the underlying molecular mechanism. After 24-hour MPA-CdTe QD exposure, the activation level of extracellular regulated protein kinase (ERK) signaling pathway and cysteinyl-directed aspartate-specific proteases (Caspases) significantly increased, which led to the increasing level of reactive oxygen species (ROS) and cell apoptosis; the group pretreated with ROS scavenger N-acetyl-L-cysteine (NAC) significantly reduced the apoptotic cell percentage, indicating that ROS played a critical role in QD-induced cytotoxicity. Further molecular experiments showed that the interacting processes between the MPA-CdTe QDs and SOD were spontaneous which changed the conformation, secondary structure of SOD. The interaction significantly resulted in the tightening of polypeptide chains and the shrinkage of SOD, leading to the inhibition of molecular SOD activity. This study demonstrates the adverse effects of QDs, revealing their potential risk in biomedical applications.

Three-dimensional architecture of pericardial nephrocytes in Drosophila melanogaster revealed by FIB/SEM tomography.

Nephrocytes are similar in structure to podocytes and play a role in the isolation of toxic substances from hemolymph in insects. Drosophila melanogaster nephrocytes have recently been used to study podocyte function and disease. However, the three-dimensional ultrastructure of nephrocytes is not clearly understood because their surrounding basement membrane makes it difficult to observe using conventional scanning electron microscopy. We reconstructed the three-dimensional ultrastructure of Drosophila pericardial nephrocytes using serial focused-ion beam/scanning electron microscopy (FIB/SEM) images. The basal surfaces were occupied by foot processes and slit-like spaces between them. The slit-like spaces corresponded to the podocyte filtration slits and were formed by longitudinal infolding/invagination of the basal plasma membrane. The basal surface between the slit-like spaces became the foot processes, which ran almost linearly, and had a "washboard-like" appearance. Both ends of the foot processes were usually anastomosed to neighboring foot processes and thus free ends were rarely observed. We demonstrated that FIB/SEM is a powerful tool to better understand the three-dimensional architecture of nephrocytes.

A brief overview on IRM function across evolution.

The description of the Rst protein by Karl-Friedrich Fischbach and colleagues was a milestone in the discovery of the irre cell recognition module (IRM). IRM proteins represent a family of immunoglobulin superfamily cell adhesion proteins that orchestrate intercellular adhesion and signaling events necessary for the development of various tissues. This review briefly summarizes the fundamental role of IRM proteins for neuronal wiring and filtration in organisms spanning the evolutionary distance from Drosophila (nephrocyte diaphragm) to humans (slit diaphragm).

Dysfunction of Mitochondrial Dynamics Induces Endocytosis Defect and Cell Damage in Drosophila Nephrocytes.

Mitochondria are crucial for cellular ATP production. They are highly dynamic organelles, whose morphology and function are controlled through mitochondrial fusion and fission. The specific roles of mitochondria in podocytes, the highly specialized cells of the kidney glomerulus, remain less understood. Given the significant structural, functional, and molecular similarities between mammalian podocytes and Drosophila nephrocytes, we employed fly nephrocytes to explore the roles of mitochondria in cellular function. Our study revealed that alterations in the Pink1-Park (mammalian PINK1-PRKN) pathway can disrupt mitochondrial dynamics in Drosophila nephrocytes. This disruption led to either fragmented or enlarged mitochondria, both of which impaired mitochondrial function. The mitochondrial dysfunction subsequently triggered defective intracellular endocytosis, protein aggregation, and cellular damage. These findings underscore the critical roles of mitochondria in nephrocyte functionality.

A SNARE protective pool antagonizes APOL1 renal toxicity in Drosophila nephrocytes.

BACKGROUND: People of Sub-Saharan African ancestry are at higher risk of developing chronic kidney disease (CKD), attributed to the Apolipoprotein L1 (APOL1) gene risk alleles (RA) G1 and G2. The underlying mechanisms by which the APOL1-RA precipitate CKD remain elusive, hindering the development of potential treatments. RESULTS: Using a Drosophila genetic modifier screen, we found that SNARE proteins (Syx7, Ykt6, and Syb) play an important role in preventing APOL1 cytotoxicity. Reducing the expression of these SNARE proteins significantly increased APOL1 cytotoxicity in fly nephrocytes, the equivalent of mammalian podocytes, whereas overexpression of Syx7, Ykt6, or Syb attenuated their toxicity in nephrocytes. These SNARE proteins bound to APOL1-G0 with higher affinity than APOL1-G1/G2, and attenuated APOL1-G0 cytotoxicity to a greater extent than either APOL1-RA. CONCLUSIONS: Using a Drosophila screen, we identified SNARE proteins (Syx7, Ykt6, and Syb) as antagonists of APOL1-induced cytotoxicity by directly binding APOL1. These data uncovered a new potential protective role for certain SNARE proteins in the pathogenesis of APOL1-CKD and provide novel therapeutic targets for APOL1-associated nephropathies.

Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover.

Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. In this work we aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans.

Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm.

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.

dCubilin- or dAMN-mediated protein reabsorption in Drosophila nephrocytes modulates longevity.

Aging is a multifaceted process regulated by multiple cellular pathways, including the proteostasis network. Pharmacological or genetic enhancement of the intracellular proteostasis network extends lifespan and prevents age-related diseases. However, how proteostasis is regulated in different tissues throughout the aging process remains unclear. Here, we show that Drosophila homologs of Cubilin- and Amnionless (dCubilin and dAMN, respectively)-mediated protein reabsorption (CAMPR) from hemolymph insect blood by nephrocytes modulate longevity through regulating proteostasis in muscle and brain tissues. We find that overexpression of dAMN receptor in nephrocytes extends lifespan, whereas nephrocyte-specific dCubilin or dAMN RNAi knockdown shortens lifespan. We also show that CAMPR in nephrocytes regulates proteostasis in hemolymph and improves healthspan. In addition, we show that enhanced CAMPR in nephrocytes slows down the aging process in muscle and brain by maintaining the proteostasis network in these tissues. Altogether, our work has revealed an inter-organ communication network across nephrocytes and muscle/neuronal tissue that is essential for maintaining proteostasis, and to delay senescence in these organs. These findings provide insight into the role of renal protein reabsorption in the aging process via this tele-proteostasis network.

Drosophila melanogaster and its nephrocytes: A versatile model for glomerular research.

Glomerular disorders are a predominant cause of chronic kidney diseases and end-stage renal failure. Especially podocytes, epithelial cells which represent the outermost part of the filtration barrier, are affected by disease and experience a gradual loss of function. Despite recent advances in identifying potential pathways underlying podocyte injury, treatment remains challenging. It is therefore desirable to employ suitable model organisms in order to study glomerular disease and elucidate affected pathways. Due to its diverse ways of genetic manipulation and high genomic conservation, Drosophila melanogaster is a powerful model organism for biomedical research. The fly was recently used to assess podocytopathies by exploiting the nephrocyte system. Nephrocytes are spherical cells within the body cavity of the fly responsible for detoxification and clearance of unwanted substances. More importantly, they share many characteristics with mammalian podocytes. Here, we summarize how to use Drosophila as a model organism for podocyte research. We discuss examples of techniques that can be used to genetically manipulate nephrocytes and provide protocols for nephrocyte isolation and for morphological as well as functional analysis.

Drosophila Renal Organ as a Model for Identification of Targets and Screening of Potential Therapeutic Agents for Diabetic Nephropathy.

Diabetic nephropathy is one of the major causes of kidney failure, accounting for ~44% of all cases. In spite of the significant mortality rate of diabetic nephropathy, specific and effective treatment is still eluding. Identification of genetic determinants and understanding their role in the progression of disease are essential for developing diagnostic tools and effective therapy. Drosophila melanogaster is a genetically tractable model organism and is being used for understanding the genetic basis of several human diseases. Drosophila has a well developed renal system and shares conserved developmental and functional processes with humans. Apart from similarities in renal system, type 1 and type 2 diabetes can be induced in Drosophila following mechanisms similar to those in human. This review discusses the current therapies available for diabetic nephropathy and examines the potential of Drosophila renal system as a model for identifying drug targets for diabetic nephropathy and screening of the potential drugs for their efficacy.

Drosophila pericardial nephrocyte ultrastructure changes during ageing.

Here we show that a labyrinth channel compartment and slit diaphragms, which are the histological structures enabling insect nephrocytes ultrafiltration, are established during embryogenesis first by the garland nephrocytes (GCNs). The later pericardial nephrocytes, which represent the majority of functional nephrocytes in larvae and adults, lack these characteristic features at the embryonic stage. During larval development, a subpopulation of the pericardial cells survives and matures into functional nephrocytes (PCNs) displaying a fully differentiated slit diaphragm and a labyrinth channel compartment. Likely the embryonic pericardial cells have primary functions other than ultrafiltration (e.g. in production and secretion of ECM constituents). We also show, for the first time, that PCNs in the adult fly undergo dramatic histological degeneration upon ageing. The slit diaphragms disappear, the labyrinth channel system degenerates and the lysosomal compartment becomes highly enriched with electron-dense material. When using nephrocytes as a model for genetic screening purposes or to investigate the specific role of genes involved in endocytosis, histological changes occurring upon ageing need to be taken into account when interpreting structural data.

Drosophila tools and assays for the study of human diseases.

Many of the internal organ systems of Drosophila melanogaster are functionally analogous to those in vertebrates, including humans. Although humans and flies differ greatly in terms of their gross morphological and cellular features, many of the molecular mechanisms that govern development and drive cellular and physiological processes are conserved between both organisms. The morphological differences are deceiving and have led researchers to undervalue the study of invertebrate organs in unraveling pathogenic mechanisms of diseases. In this review and accompanying poster, we highlight the physiological and molecular parallels between fly and human organs that validate the use of Drosophila to study the molecular pathogenesis underlying human diseases. We discuss assays that have been developed in flies to study the function of specific genes in the central nervous system, heart, liver and kidney, and provide examples of the use of these assays to address questions related to human diseases. These assays provide us with simple yet powerful tools to study the pathogenic mechanisms associated with human disease-causing genes.

Molecular mechanism of copper-zinc superoxide dismutase activity change exposed to N-acetyl-L-cysteine-capped CdTe quantum dots-induced oxidative damage in mouse primary hepatocytes and nephrocytes.

Quantum dots (QDs) are engineered semiconductor nanocrystals with promising application in biomedicine, which have potential toxic effect on biomacromolecules by direct interaction and indirect impact in the body. In this work, the effect of N-acetyl-L-cysteine-capped CdTe quantum dots with fluorescence emission peak at 612 nm (QDs-612) on copper-zinc superoxide dismutase (Cu/ZnSOD) at molecular and cellular level was investigated using isothermal titration calorimetry, spectroscopic techniques, cell counting kit-8, and total SOD assay. The hydrophobic interaction between Cu/ZnSOD and QDs-612 caused static fluorescence quenching of the protein, which was spontaneous with binding constant calculated to be 3.28 × 10(5) L mol(-1). The microenvironment of tyrosine residues, skeleton, and secondary structure of Cu/ZnSOD were changed with adding QDs-612. The molecular Cu/ZnSOD activity was inhibited at different concentrations of QDs-612 as well as the intracellular Cu/ZnSOD activity after 2-h exposure. Compared with the cell viability of hepatocytes and nephrocytes (decreased markedly of the initial level) with higher concentrations of QDs-612 in the absence of vitamin C, the cell viability of these two primary cells increased in the presence of vitamin C, indicating the oxidative damage induced by QDs-612. Therefore, the inhibition of Cu/ZnSOD activity in these two primary cells may be caused by the oxidative damage of massive ROS or direct interaction with QDs-612. This work establishes a new approach to investigate the biological toxicity of CdTe QDs to biomacromolecule from both molecular and cellular perspectives and obtains experimental evidence to thoroughly study the toxicity of CdTe QDs in vivo.