Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes

The kidneys generate about 180 liters of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment

ERAP1 allotypes shape the epitope repertoire of virus-specific CD8+ T cell responses in acute hepatitis C virus infection

Background & Aims Endoplasmic reticulum aminopeptidase 1 (ERAP1) polymorphisms are linked with human leukocyte antigen (HLA) class I-associated autoinflammatory disorders, including ankylosing spondylitis and Behçet’s disease. Disease-associated ERAP1 allotypes exhibit distinct functional properties, but it remains unclear how differential peptide trimming in vivo affects the repertoire of epitopes presented to CD8+ T cells. The aim of this study was to determine the impact of ERAP1 allotypes on the virus-specific CD8+ T cell epitope repertoire in an HLA-B*27:05+ individual with acute hepatitis C virus (HCV) infection. Methods We performed genetic and functional analyses of ERAP1 allotypes and characterized the HCV-specific CD8+ T cell repertoire at the level of fine epitope specificity and HLA class I restriction, in a patient who had acquired an HCV genotype 1a infection through a needle-stick injury. Results Two hypoactive allotypic variants of ERAP1 were identified in an individual with acute HCV infection. The associated repertoire of virus-derived epitopes recognized by CD8+ T cells was uncommon in a couple of respects. Firstly, reactivity was directed away from classically immunodominant epitopes, preferentially targeting either novel or subdominant epitopes. Secondly, reactivity was biased towards longer epitopes (10–11-mers). Despite the patient exhibiting favorable prognostic indicators, these atypical immune responses failed to clear the virus and the patient developed persistent low-level infection with HCV. Conclusions ERAP1 allotypes modify the virus-specific CD8+ T cell epitope repertoire in vivo, leading to altered immunodominance patterns that may contribute to the failure of antiviral immunity after infection with HCV

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Abstract: Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Publisher Copyright: © 2019, The Author(s).Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria.Peer reviewe

Genome-wide association meta-analyses and fine-mapping elucidate pathways influencing albuminuria

Increased levels of the urinary albumin-to-creatinine ratio (UACR) are associated with higher risk of kidney disease progression and cardiovascular events, but underlying mechanisms are incompletely understood. Here, we conduct trans-ethnic (n = 564,257) and European-ancestry specific meta-analyses of genome-wide association studies of UACR, including ancestry- and diabetes-specific analyses, and identify 68 UACR-associated loci. Genetic correlation analyses and risk score associations in an independent electronic medical records database (n = 192,868) reveal connections with proteinuria, hyperlipidemia, gout, and hypertension. Fine-mapping and trans-Omics analyses with gene expression in 47 tissues and plasma protein levels implicate genes potentially operating through differential expression in kidney (including TGFB1, MUC1, PRKCI, and OAF), and allow coupling of UACR associations to altered plasma OAF concentrations. Knockdown of OAF and PRKCI orthologs in Drosophila nephrocytes reduces albumin endocytosis. Silencing fly PRKCI further impairs slit diaphragm formation. These results generate a priority list of genes and pathways for translational research to reduce albuminuria

Programmed cell death of fetal erythrocytes

Eryptose stellt die besondere Form der Apoptose der Erythrozyten dar, die wie die Apoptose kernhaltiger Zellen zu Phosphatidylserin-Exposition und Zellschrumpfung führt. Über durchflusszytometrische Untersuchung der Bindung FITC-markierten Annexins und des forward scatters kann dies nachgewiesen werden. Die Eryptose lässt sich in vitro durch die Entfernung von Chlorid oder Glucose aus dem Medium sowie durch Hyperosmolarität und oxidativen Stress auslösen. Hyperosmolarität und Chloridwegnahme führen durch PGE2-Bildung zur Öffnung unselektiver Kationenkanäle, was einen Calcium-Einstrom in die Zelle verursacht. Die für die Phosphatidylserin-Exposition verantwortliche Scramblase wird durch den Calcium-Einstrom aktiviert. Die Leitfähigkeit der am Ca2+-Einstrom beteiligten Kanäle kann mittels patch-clamp untersucht werden, der Calcium-Einstrom selbst wird durch Färbung mit Ca2+-sensitivem Fluo-3/AM nachgewiesen. Die unselektiven Kationenkanäle reagieren weiterhin auf oxidativen Stress (hier ausgelöst durch Zugabe von tert-Butylhydroperoxid). Hyperosmolarer Schock führt zusätzlich über PAF-Freisetzung zu Ceramid-Bildung, was die Scramblase für Calcium sensitiviert. Fetale Erythrozyten unterscheiden sich deutlich von adulten Zellen und diese besondere Erythrozytenpopulation wird bald nach der Geburt aus der Blutbahn entfernt. Diese Arbeit setzte sich zum Ziel, die Eryptose fetaler Erythrozyten zu untersuchen. Dabei zeigte sich eine geringere Empfindlichkeit der fetalen Zellen gegenüber Hyperosmolarität und Chloridwegnahme. Dementsprechend wurde eine geringere Bildung sowie Empfindlichkeit gegenüber PGE2 nachgewiesen. Weiterhin konnten die vermutlich daraus resultierende geringere Kationenkanal-Leitfähigkeit und der dadurch abgeschwächte Calcium-Anstieg in der Zelle beobachtet werden. Darüber hinaus wurde auch eine geringere Empfindlichkeit gegenüber PAF festgestellt. Schließlich wurde etwas weniger spontane Ceramid-Bildung beobachtet. Andererseits sind fetale Erythrozyten empfindlicher gegenüber Glucose-Depletion und oxidativem Stress, was auch im forward scatter zu stärkerer Zellschrumpfung führt. Fetale Erythrozyten zeigen somit wie adulte Zellen Eryptose, wobei je nach Stimulus eine geringere oder höhere Resistenz beobachtet wurde. Diese Ergebnisse liefern einen möglichen Erklärungsansatz für die Trimenonreduktion und die Anämie des Frühgeborenen.Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of non-selective Ca2+-permeable cation channels with subsequent entry of Ca2+ and stimulation of Ca2+-sensitive scrambling of the cell membrane. Apart from increased cytosolic Ca2+ activity enhanced ceramide levels also lead to membrane scrambling with subsequent phosphatidylserine exposure. Eryptosis is triggered by Cl- removal, osmotic shock, oxidative stress or glucose deprivation. Fetal erythrocytes show distinctive characteristics. Postpartum they are rapidly cleared from the peripheral blood-stream. The present work compares susceptibility to eryptosis in fetal and adult erythrocytes. Channel activity was determined utilizing patch clamp, cytosolic Ca2+ activity by Fluo3 fluorescence, phosphatidylserine exposure by FITC-labelled annexin-V binding, and cell shrinkage by decrease of forward scatter in FACS analysis. PGE2 formation, cation channel activity, Ca2+ entry, annexin-V binding and decrease of forward scatter were triggered by removal of Cl- in both, adult and fetal erythrocytes. The effects were, however, significantly blunted in fetal erythrocytes. Osmotic shock, PGE2 and PAF similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in fetal erythrocytes. On the other hand, spontaneous and oxidative stress (addition of tertbutylperoxide)-induced eryptosis was significantly larger in fetal erythrocytes. Eryptosis triggered by glucose deprivation was enhanced in fetal erythrocytes. Ceramide formation seems to be less apparent in fetal erythrocytes, albeit there was no significant difference. In conclusion, cation channel activity, Ca2+ leak and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous, oxidative stress- and glucose deprivation-induced eryptosis is more pronounced in fetal erythrocytes. These findings render possible explanations for the physiological anemia of the newborn and the anemia of prematurity

Using the Drosophila Nephrocyte to Model Podocyte Function and Disease

Glomerular disorders are a major cause of end-stage renal disease and effective therapies are often lacking. Nephrocytes are considered to be part of the Drosophila excretory system and form slit diaphragms across cellular membrane invaginations. Nehphrocytes have been shown to share functional, morphological, and molecular features with podocytes, which form the glomerular filter in vertebrates. Here, we report the progress and the evolving tool-set of this model system. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in Drosophila, the nephrocyte has the potential to greatly advance our understanding of the glomerular filtration barrier in health and disease