ABSTRACT
In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.
Subject(s)
Actin Cytoskeleton , Drosophila Proteins , Integrins , Podocytes , Animals , Actin Cytoskeleton/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Integrins/metabolism , Podocytes/metabolism , Drosophila melanogaster/metabolism , Drosophila/metabolism , Actins/metabolism , ImmunoglobulinsABSTRACT
Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.
Subject(s)
Drosophila Proteins , Phospholipid Transfer Proteins , Podocytes , Animals , Podocytes/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Phospholipid Transfer Proteins/metabolism , Phospholipid Transfer Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Membrane Microdomains/metabolism , Intercellular Junctions/metabolismABSTRACT
INTRODUCTION: Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. METHODS: Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. RESULTS: Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and ß-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. CONCLUSION: We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.
ABSTRACT
Introduction: Podocyte slit diaphragms are an important component of the glomerular filtration barrier. Podocyte injury frequently includes defects in slit diaphragms, and various mechanisms for these defects have been described, including altered endocytic trafficking of slit diaphragm proteins or oxidative stress. However, the potential relationship between endocytosis and oxidative stress in the context of slit diaphragm integrity has not been extensively considered. Methods: To examine the potential relationships between endocytosis, oxidative stress, and slit diaphragm integrity, we induced genetic or pharmacological disruption of endocytosis in Drosophila nephrocytes (the insect orthologue of podocytes) and cultured human podocytes. We then employed immunofluorescence microscopy to analyze protein localization and levels, and to quantify signal from reactive oxygen species (ROS) dyes. Immunoprecipitation from podocyte cell lysates was used to examine effects on slit diaphragm protein complex formation (i.e., nephrin/podocin and nephrin/ZO-1). Results: Disruption of endocytosis in nephrocytes and podocytes led to slit diaphragm defects, elevated levels of ROS (oxidative stress), and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway. In nephrocytes with defective endocytosis, perturbation of Nrf2 signaling exacerbated slit diaphragm defects. Conversely, overexpression of Nrf2 target genes catalase or glucose-6-phosphate dehydrogenase (G6PD) significantly ameliorated slit diaphragm defects caused by disruption of endocytosis. Conclusion: Oxidative stress is an important consequence of defective endocytosis and contributes to the defects in slit diaphragm integrity associated with disruption of endocytic trafficking.
ABSTRACT
The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Subject(s)
Nephritis, Hereditary , Podocytes , Humans , Mice , Animals , Integrin alpha1/genetics , Integrin alpha1/metabolism , Ramipril/pharmacology , Ramipril/metabolism , Longevity , Glomerular Basement Membrane/metabolism , Nephritis, Hereditary/drug therapy , Nephritis, Hereditary/genetics , Nephritis, Hereditary/metabolism , Podocytes/metabolism , Laminin/genetics , Laminin/metabolism , Mice, Knockout , Proteinuria/drug therapy , Proteinuria/genetics , Proteinuria/metabolism , Sequence Analysis, RNAABSTRACT
The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.
Subject(s)
Nephrotic Syndrome , Podocytes , Humans , Animals , Mice , Podocytes/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Nephrotic Syndrome/genetics , Nephrotic Syndrome/metabolism , RNA, Messenger/metabolismABSTRACT
Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.
Subject(s)
Cytokinesis , Drosophila , Animals , Cell Division , Cerebral Cortex , DiaphragmABSTRACT
Introduction: The unique architecture of glomerular podocytes is integral to kidney filtration. Interdigitating foot processes extend from the podocyte cell body, wrap around fenestrated capillaries, and form specialized junctional complexes termed slit diaphragms to create a molecular sieve. However, the full complement of proteins which maintain foot process integrity, and how this localized proteome changes with disease, remain to be elucidated. Methods: Proximity-dependent biotin identification (BioID) enables the identification of spatially localized proteomes. To this end, we developed a novel in vivo BioID knock-in mouse model. We utilized the slit diaphragm protein podocin (Nphs2) to create a podocin-BioID fusion. Podocin-BioID localizes to the slit diaphragm, and biotin injection leads to podocyte-specific protein biotinylation. We isolated the biotinylated proteins and performed mass spectrometry to identify proximal interactors. Results and Discussion: Gene ontology analysis of 54 proteins specifically enriched in our podocin-BioID sample revealed 'cell junctions,' 'actin binding,' and 'cytoskeleton organization' as top terms. Known foot process components were identified, and we further uncovered two novel proteins: the tricellular junctional protein Ildr2 and the CDC42 and N-WASP interactor Fnbp1l. We confirmed that Ildr2 and Fnbp1l are expressed by podocytes and partially colocalize with podocin. Finally, we investigated how this proteome changes with age and uncovered a significant increase in Ildr2. This was confirmed by immunofluorescence on human kidney samples and suggests altered junctional composition may preserve podocyte integrity. Together, these assays have led to new insights into podocyte biology and support the efficacy of utilizing BioID in vivo to interrogate spatially localized proteomes in health, aging, and disease.
ABSTRACT
Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Here, using Drosophila melanogaster garland nephrocytes and cultured human podocytes, we aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. We found that ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, our data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and our data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases.
Subject(s)
Kidney Diseases , Podocytes , Animals , Humans , Podocytes/pathology , Transcription Factors/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation , Kidney Diseases/genetics , Kidney Diseases/pathology , Endoplasmic Reticulum Stress/genetics , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolismABSTRACT
Podocyte injury is a common hallmark of chronic kidney disease (CKD). The podocin-nephrin complex localized in lipid rafts of podocyte is vital to reduce podocyte injury and proteinuria, however, the mechanism underlying its localization remains unclear. This study uncovers an important role of Flot2 in stabilizing the podocin-nephrin complex localized in lipid rafts. We first confirmed that Flot2 was expressed in podocyte and demenstrated that podocyte-specific Flot2 deletion worsen albuminuria, podocyte injury and glomerular pathology in LPS/ADR-induced nephropathy mouse models. Meanwhile, podocyte injury, albuminuria and pathologic aberrance were prevented in podocyte-specific Flot2 overexpression transgenic mice when challenged with LPS or ADR. Further found that Flot2 was vital to recruit podocin and nephrin into rafts and ameliorated podocyte injury. Flot2 and podocin directly interacted with each other via their SPFH domain. Meanwhile, we also showed that Flot-2 is a direct target of Krüppel-like factor (KLF15). Importanly, we observed that Flot2 was downregulated in renal biopsies from patients with podocytopathies and its expression negatively correlated with proteinuria and positively correlated with eGFR, indicating that Flot2 may be a novel therapeutic target for proteinuric kidney disease.
Subject(s)
Albuminuria , Podocytes , Renal Insufficiency, Chronic , Animals , Mice , Albuminuria/metabolism , Albuminuria/pathology , Lipopolysaccharides , Mice, Transgenic , Podocytes/pathology , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathologyABSTRACT
Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Nephropathies , Podocytes , Humans , Mice , Animals , Diabetic Nephropathies/pathology , Autophagy , Diabetes Mellitus, Experimental/metabolism , Streptozocin/adverse effects , Streptozocin/metabolism , Albuminuria/metabolism , Albuminuria/pathology , Phosphatidylinositol 3-Kinases/metabolism , Tumor Necrosis Factors/adverse effects , Tumor Necrosis Factors/metabolism , Glycation End Products, Advanced/adverse effects , Glycation End Products, Advanced/metabolism , Glucose/pharmacology , Glucose/metabolism , Cytokines/metabolismABSTRACT
Both mTOR signaling and autophagy are important modulators of podocyte homeostasis, regeneration, and aging and have been implicated in glomerular diseases. However, the mechanistic role of these pathways for the glomerular filtration barrier remains poorly understood. We used Drosophila nephrocytes as an established podocyte model and found that inhibition of mTOR signaling resulted in increased spacing between slit diaphragms. Gain-of-function of mTOR signaling did not affect spacing, suggesting that additional cues limit the maximal slit diaphragm density. Interestingly, both activation and inhibition of mTOR signaling led to decreased nephrocyte function, indicating that a fine balance of signaling activity is needed for proper function. Furthermore, mTOR positively controlled cell size, survival, and the extent of the subcortical actin network. We also showed that basal autophagy in nephrocytes is required for survival and limits the expression of the sns (nephrin) but does not directly affect slit diaphragm formation or endocytic activity. However, using a genetic rescue approach, we demonstrated that excessive, mTOR-dependent autophagy is primarily responsible for slit diaphragm misspacing. In conclusion, we established this invertebrate podocyte model for mechanistic studies on the role of mTOR signaling and autophagy, and we discovered a direct mTOR/autophagy-dependent regulation of the slit diaphragm architecture.
Subject(s)
Drosophila Proteins , Podocytes , Animals , Autophagy , Drosophila/metabolism , Drosophila Proteins/metabolism , Podocytes/metabolism , TOR Serine-Threonine Kinases/metabolismABSTRACT
Selective glomerular filtration relies on the membrane separating the glomerular arterioles from the Bowman space. As a major component of the glomerular filtration barrier, podocytes form foot processes by the actin cytoskeleton, which dynamically adjusts in response to environmental changes to maintain filtration barrier integrity. The slit diaphragms bridge the filtration slits between neighboring foot processes and act as signaling hubs interacting with the actin cytoskeleton. Focal adhesions relay signals to regulate actin dynamics while allowing podocyte adherence to the basement membrane. Mutations in actin regulatory and signaling proteins may disrupt the actin cytoskeleton, resulting in foot process retraction, effacement, and proteinuria. Large-scale gene expression profiling platforms, transgenic animal models, and other in vivo gene delivery methods now enhance our understanding of the interactions among podocyte focal adhesions, slit diaphragms, and actin dynamics. In addition, our team found that at least 66% of idiopathic nephrotic syndrome (INS) children have podocyte autoantibodies, which was defined as a new disease subgroup-, autoimmune podocytopathies. This review outlines the pathophysiological mechanisms of podocyte cytoskeleton protein interactions in proteinuria and glomerular podocytopathy.
Subject(s)
Nephrotic Syndrome , Podocytes , Actins , Animals , Cytoskeleton/metabolism , Nephrotic Syndrome/metabolism , Podocytes/metabolism , Proteinuria/metabolismABSTRACT
Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Endocytosis , Mammals/metabolism , Phosphatidylinositols/metabolismABSTRACT
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.
ABSTRACT
A structural abnormality or dysfunction of podocytes is the major cause of nephrotic syndrome (NS). The TBC1D8B protein interacts with nephrin, a podocyte slit diaphragm protein, regulates vesicle transport, and functions in the pathogenesis of NS. We report a novel potentially pathogenic variant in the TBC1D8B gene in a 6-month-old boy with NS. A 6-month-old boy was admitted to the hospital because of edema and fever. Our systematic examination led to a diagnosis of NS. Because of the early age of onset, we performed trio whole-exome sequencing of him and his parents. The results showed a new potentially pathogenic variant in the TBC1D8B gene on the X chromosome, c.2717A>G (p.His906Arg). After routine glucocorticoid therapy, his urine protein turned negative, indicating steroid-sensitive NS. The new TBC1D8B variant identified here, c.2717A>G (p.His906Arg), may be associated with early-onset NS in children. Although NS due to pathogenic variants in this gene is more commonly steroid-resistant, our patient had steroid-sensitive NS.
ABSTRACT
The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of Amnionless and two cubilin paralogues, Cubilin and Cubilin2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, which is strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Of note, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes suggest a possible unexpected conserved role for this endocytic receptor in vertebrate SD remodelling.
Subject(s)
Drosophila Proteins/genetics , Endocytosis/genetics , Low Density Lipoprotein Receptor-Related Protein-2/genetics , Receptors, Cell Surface/genetics , rab5 GTP-Binding Proteins/genetics , Animals , Diaphragm/growth & development , Diaphragm/metabolism , Drosophila melanogaster/genetics , Intercellular Junctions/genetics , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Morphogenesis/genetics , Multiprotein Complexes/genetics , Podocytes/metabolismABSTRACT
Background: Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria. Methods: The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin. Results: Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes. (AU)
Antecedentes: El topiroxostat, un inhibidor de la xantina oxidorreductasa (XOR), mostró reducir la excreción de albúmina en la orina de pacientes hiperuricémicos con enfermedad renal crónica. Sin embargo, su mecanismo farmacológico no se conoce con exactitud. En este estudio examinamos los efectos del topiroxostat en los podocitos glomerulares. El podocito se caracteriza por unas prolongaciones en forma de pie y un diafragma de hendidura de unión célula-célula único que funciona como barrera final en la prevención de la proteinuria. Métodos: Se analizaron los efectos del topiroxostat en las expresiones de las moléculas funcionales de los podocitos en ratones db/db, en un modelo de nefropatía diabética, en un modelo de lesión podocitaria inducida por anticuerpos antinefrina en ratas y en podocitos cultivados tratados con adriamicina. Resultados: El tratamiento con topiroxostat mejoró la albuminuria en ratones db/db. La expresión de la desmina, un marcador de lesión podocitaria, estaba aumentada, y la nefrina y la podocina, moléculas clave del diafragma de hendidura, y la podoplanina, una molécula esencial en el mantenimiento de las prolongaciones en forma de pie, estaban atenuadas en los ratones db/db. El tratamiento con topiroxostat evitó alteraciones en las expresiones de estas moléculas en los ratones db/db. La actividad de la XOR en el riñón se incrementó en ratas con lesión podocitaria inducida por anticuerpos antinefrina. El tratamiento con topiroxostat redujo la actividad de la XOR y restauró la disminución de la expresión de nefrina, podocina y podoplanina en la lesión podocitaria. Además, el topiroxostat aumentó la expresión de podoplanina en podocitos humanos cultivados lesionados. (AU)
Subject(s)
Humans , Xanthines/antagonists & inhibitors , Xanthines/adverse effects , Podocytes/pathology , Oxidoreductases , Podocytes/metabolismABSTRACT
INTRODUCTION: Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. METHODS: The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. RESULTS: Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. CONCLUSION: Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.
Subject(s)
Down-Regulation , Kidney Diseases , Kidney Glomerulus , Podocytes , Animals , Female , Rats , Down-Regulation/physiology , Kidney Diseases/metabolism , Kidney Glomerulus/metabolism , Podocytes/metabolism , Rats, WistarABSTRACT
Despite increasing awareness and therapeutic options chronic kidney disease (CKD) is still and important health problem and glomerular diseases constitute and important percentage of CKD. Proteinuria/albuminuria is not just a marker; but it also plays a direct pathogenic role in renal disease progression of CKD. Glomerular filtration barrier (GFB) which consists of fenestrated endothelial cells, fused basal membrane and interdigitating podocyte foot process and filtration slits between foot process is the major barrier for proteinuria/albuminuria. Many glomerular diseases are characterized by disruption of GFB podocytes, foot process and slit diaphragm. Many proteinuric diseases are non-specifically targeted by therapeutic agents such as steroids and calcineurin inhibitors with systemic side effects. Thus, there is unmet need for more efficient and less toxic therapeutic options to treat glomerular diseases. In recent years, modification of dietary intake, has been gained to treat pathologic processes introducing the concept of 'food as a medicine'. The effect of various nutritional products on podocyte function and structure is also trending, especially in recent years. In the current review, we summarized the effect of nutritional interventions on podocyte function and structure.