Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes

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 dAmnionless and two cubilin paralogues Cubilin and Cubilin-2, 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, strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Noteworthy, 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 argue for a possible unexpected conserved role of this endocytic receptor in vertebrate SD remodelling.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction

<b><i>Introduction:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> 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.

VDR/Atg3 Axis Regulates Slit Diaphragm to Tight Junction Transition via p62-mediated Autophagy Pathway in Diabetic Nephropathy

Foot process effacement is an important feature of early diabetic nephropathy (DN) which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62 mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase 3 and caspase 8. Interestingly, the expression of VDR in podocytes was decreased under diabetic condition which impaired autophagic flux through down-regulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking p62-mediated autophagy pathway in DN.<br>

VDR/Atg3 Axis Regulates Slit Diaphragm to Tight Junction Transition via p62-mediated Autophagy Pathway in Diabetic Nephropathy

Foot process effacement is an important feature of early diabetic nephropathy (DN) which is closely related to the development of albuminuria. Under certain nephrotic conditions, the integrity and function of the glomerular slit diaphragm (SD) structure were impaired and replaced by the tight junction (TJ) structure, resulting in so-called SD-TJ transition, which could partially explain the effacement of foot processes at the molecular level. However, the mechanism underlying the SD-TJ transition has not been described in DN. Here, we demonstrated that impaired autophagic flux blocked p62 mediated degradation of ZO-1 (TJ protein) and promoted podocytes injury via activation of caspase 3 and caspase 8. Interestingly, the expression of VDR in podocytes was decreased under diabetic condition which impaired autophagic flux through down-regulating Atg3. Of note, we also found that VDR abundance was negatively associated with impaired autophagic flux and SD-TJ transition in the glomeruli from human renal biopsy samples with DN. Furthermore, VDR activation improved autophagic flux and attenuated SD-TJ transition in the glomeruli of diabetic animal models. In conclusion, our data provided the novel insight that VDR/Atg3 axis deficiency resulted in SD-TJ transition and foot processes effacement via blocking p62-mediated autophagy pathway in DN.<br>

PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes

Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated 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.

Phase Separation of MAGI2-Mediated Complex Underlies Formation of Slit Diaphragm Complex in Glomerular Filtration Barrier

BackgroundSlit diaphragm is a specialized adhesion junction between the opposing podocytes, establishing the final filtration barrier to urinary protein loss. At the cytoplasmic insertion site of each slit diaphragm there is an electron-dense and protein-rich cellular compartment that is essential for slit diaphragm integrity and signal transduction. Mutations in genes that encode components of this membrane-less compartment have been associated with glomerular diseases. However, the molecular mechanism governing formation of compartmentalized slit diaphragm assembly remains elusive.MethodsWe systematically investigated the interactions between key components at slit diaphragm, such as MAGI2, Dendrin, and CD2AP, through a combination of biochemical, biophysical, and cell biologic approaches.ResultsWe demonstrated that MAGI2, a unique MAGUK family scaffold protein at slit diaphragm, can autonomously undergo liquid-liquid phase separation. Multivalent interactions among the MAGI2-Dendrin-CD2AP complex drive the formation of the highly dense slit diaphragm condensates at physiologic conditions. The reconstituted slit diaphragm condensates can effectively recruit Nephrin. A nephrotic syndrome–associated mutation of MAGI2 interfered with formation of the slit diaphragm condensates, thus leading to impaired enrichment of Nephrin.ConclusionsKey components at slit diaphragm (e.g., MAGI2 and its complex) can spontaneously undergo phase separation. The reconstituted slit diaphragm condensates can be enriched in adhesion molecules and cytoskeletal adaptor proteins. Therefore, the electron-dense slit diaphragm assembly might form via phase separation of core components of the slit diaphragm in podocytes.

Evolutionary conservation of intrinsically unstructured regions in slit-diaphragm proteins

Vertebrate kidneys contribute to homeostasis by regulating electrolyte, acid-base balance, removing toxic metabolites from blood, and preventing protein loss into the urine. Glomerular podocytes constitute the blood-urine barrier, and podocyte slit-diaphragm (SD), a modified tight junction, contributes to the glomerular permselectivity. Nephrin, KIRREL1, podocin, CD2AP, and TRPC6 are crucial members of the SD that interact with each other and contribute to the SD’s structural and functional integrity. This study analyzed the distribution of these five essential SD proteins across the organisms for which the genome sequence is available. We found a diverse distribution of nephrin and KIRREL1 ranging from nematodes to higher vertebrates, whereas podocin, CD2AP, and TRPC6 are restricted to the vertebrates. Among invertebrates, nephrin and its orthologs consist of more immunoglobulin-3 domains, whereas in the vertebrates, CD80-like C2-set domains are predominant. In the case of KIRREL1 and its orthologs, more Ig domains were observed in invertebrates than vertebrates. Src Homology-3 (SH3) domain of CD2AP and SPFH domain of podocin are highly conserved among vertebrates. TRPC6 and its orthologs had conserved ankyrin repeats, TRP, and ion transport domains, except Chondrichthyes and Echinodermata, which do not possess the ankyrin repeats. Intrinsically unstructured regions (IURs) are conserved across the SD orthologs, suggesting IURs importance in the protein complexes that constitute the slit-diaphragm. For the first time, a study reports the evolutionary insights of vertebrate SD proteins and their invertebrate orthologs.

Downregulation of ephrin-B1 is a critical event of podocyte injury

Proteinuria in several glomerular diseases results from dysfunction of the slit diaphragm, a cell-cell junction of glomerular epithelial cells (podocytes). Ephrin-B1 and its related molecule NHERF 2 are novel essential components of the slit diaphragm. Ephrin-B1 interacts with nephrin via the extra-cellular domain and interacts with NHERF2 via the cytoplasmic site. In the proteinuric state induced by the stimulation to nephrin, nephrin and ephrin-B1 are phosphorylated, and NHERF2 is de- phosphorylated and consequent disruption of the linkage and downregulation of nephrin, ephrin-B1 and NHERF2 are a critical pathogenic event of podocyte injury. Keywords: Podocyte; Slit Diaphragm; Ephrin-B1; Nephrin; NHERF2