A missense mutation in Ehd1 associated with defective spermatogenesis and male infertility.

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7-2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.

Impact of interferon-γ on the target cell tropism of nanoparticles.

Interferon-γ (IFN-γ) is well known to reduce the infectivity of viral pathogens by altering their tissue tropism. This effect is induced by upregulation of cholesterol 25-hydroxylase (CH25H). Given the similarity of viral pathogens and ligand-functionalized nanoparticles in the underlying strategy of receptor-mediated cell recognition, it appears conceivable that IFN-γ exceeds similar effects on nanoparticles. Concretely, IFN-γ-induced activation of CH25H could decrease nanoparticle avidity for target cells via depletion of clathrin-coated pits. We hypothesized that this effect would cause deterioration of target-cell specific accumulation of nanoparticles. To prove our hypothesis, we investigated the cell tropism of angiotensin II functionalized nanoparticles (NPLys-Ang II) in a co-culture system of angiotensin II subtype 1 receptor (AT1R) positive rat mesangial target cells (rMCs) and AT1R-negative HeLa off-target cells. In the presence of IFN-γ we observed an up to 5-fold loss of target cell preference for NPLys-Ang II. Thus, our in vitro results suggest a strong influence of IFN-γ on nanoparticle distribution, which is relevant in the context of nanotherapeutic approaches to cancer treatment, as IFN-γ is strongly expressed in tumors. For the target cell tropism of viruses, our results provide a conclusive hypothesis for the underlying mechanism behind non-directed viral distribution in the presence of IFN-γ.

ATG8-dependent LMX1B-autophagy crosstalk shapes human midbrain dopaminergic neuronal resilience.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

Elemental Compositions of Enamel or Dentin in Human and Bovine Teeth Differ from Murine Teeth.

Teeth with different chemical compositions can show vastly different physical properties, so knowledge of elemental composition is required to use animal teeth as substitutes for human teeth in research. In vitro, energy dispersive X-ray spectroscopy (EDX), improved by calibration standards and Si3N4-window material, enables determining local elemental compositions of inorganic and organic compounds without sample destruction. Six human molars, bovine incisors, murine incisors, and murine molars were analyzed. EDX-field scans and line scans were analyzed for elements Ca, P, O, C, N, F, Na, Mg, Fe, Cl, and S. Furthermore, Ca/P- and Ca/N-Ratios were calculated. The presence of iron in murine incisor enamel was investigated using additional wavelength dispersive X-ray spectroscopy measurements (WDX) near the enamel surface. Bovine and human enamel and dentin revealed close similarities regarding elemental composition. The median (25-75% percentiles) of At%Ca was 21.1 (20.8-21.3) in human enamel, 21.0 (20.7-21.2) in bovine enamel, and in murine enamel, 18.3 (17.85-18.88) for molars and 18.35 (18.00-18.75) for incisors. In dentin, murine teeth revealed a higher At%Ca compared to human and bovine teeth. Significant differences between human and bovine teeth were found for nitrogen in dentin, with a median of 4.5 (3.3-5) At%N for human dentin and 2.7 (2.3-3.2) At%N for bovine dentin. The Ca/P-Ratio was the highest in human and bovine enamel, which did not differ significantly. Enamel from murine molars had a higher Ca/P-Ratio than murine incisors and the highest Ca/P-Ratio in dentin was observed for human teeth and murine molars (1.49). WDX revealed iron in the outer 10 µm of pre- and post-eruptive enamel of murine incisors. Pre- and post-eruptive enamel on murine incisors only differed significantly in At%Ca (p = 0.041) and At%P (p = 0.026) with both At% higher in the pre-eruptive enamel. Murine teeth differ significantly from human and bovine teeth in terms of the elemental composition of enamel and dentin.

CLN7/MFSD8 may be an important factor for SARS-CoV-2 cell entry.

The SARS-CoV-2 virus has triggered a worldwide pandemic. According to the BioGrid database, CLN7 (MFSD8) is thought to interact with several viral proteins. The aim of this work was to investigate a possible involvement of CLN7 in the infection process. Experiments on a CLN7-deficient HEK293T cell line exhibited a 90% reduced viral load compared to wild-type cells. This observation may be linked to the finding that CLN7 ko cells have a significantly reduced GM1 content in their cell membrane. GM1 is found highly enriched in lipid rafts, which are thought to play an important role in SARS-CoV-2 infection. In contrast, overexpression of CLN7 led to an increase in viral load. This study provides evidence that CLN7 is involved in SARS-CoV-2 infection. This makes it a potential pharmacological target for drug development against COVID-19. Furthermore, it provides insights into the physiological function of CLN7 where still only little is known about.

A Founder Mutation in EHD1 Presents with Tubular Proteinuria and Deafness.

BACKGROUND: The endocytic reabsorption of proteins in the proximal tubule requires a complex machinery and defects can lead to tubular proteinuria. The precise mechanisms of endocytosis and processing of receptors and cargo are incompletely understood. EHD1 belongs to a family of proteins presumably involved in the scission of intracellular vesicles and in ciliogenesis. However, the relevance of EHD1 in human tissues, in particular in the kidney, was unknown. METHODS: Genetic techniques were used in patients with tubular proteinuria and deafness to identify the disease-causing gene. Diagnostic and functional studies were performed in patients and disease models to investigate the pathophysiology. RESULTS: We identified six individuals (5-33 years) with proteinuria and a high-frequency hearing deficit associated with the homozygous missense variant c.1192C>T (p.R398W) in EHD1. Proteinuria (0.7-2.1 g/d) consisted predominantly of low molecular weight proteins, reflecting impaired renal proximal tubular endocytosis of filtered proteins. Ehd1 knockout and Ehd1R398W/R398W knockin mice also showed a high-frequency hearing deficit and impaired receptor-mediated endocytosis in proximal tubules, and a zebrafish model showed impaired ability to reabsorb low molecular weight dextran. Interestingly, ciliogenesis appeared unaffected in patients and mouse models. In silico structural analysis predicted a destabilizing effect of the R398W variant and possible inference with nucleotide binding leading to impaired EHD1 oligomerization and membrane remodeling ability. CONCLUSIONS: A homozygous missense variant of EHD1 causes a previously unrecognized autosomal recessive disorder characterized by sensorineural deafness and tubular proteinuria. Recessive EHD1 variants should be considered in individuals with hearing impairment, especially if tubular proteinuria is noted.

A polycystin-2 protein with modified channel properties leads to an increased diameter of renal tubules and to renal cysts.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.

On-section correlative light and electron microscopy of large cellular volumes using STEM tomography.

The application of both fluorescence and electron microscopy results in a powerful combination of imaging modalities called "correlative light and electron microscopy" (CLEM). Whereas conventional transmission electron microscopy (TEM) tomography is only able to image sections up to a thickness of ~300nm, scanning transmission electron microscopy (STEM) tomography at 200kV allows the analysis of sections up to a thickness of 900nm in three dimensions. In the current study we have successfully integrated STEM tomography into CLEM as demonstrated for human retinal pigment epithelial 1 (RPE1) cells expressing various fluorescent fusion proteins which were high-pressure frozen and then embedded in Lowicryl HM20. Fluorescently labeled gold nanoparticles were applied onto resin sections and imaged by fluorescence and electron microscopy. STEM tomograms were recorded at regions of interest, and overlays were generated using the eC-CLEM software package. Through the nuclear staining of living cells, the use of fluorescently labeled gold fiducials for the generation of overlays, and the integration of STEM tomography we have markedly extended the application of the Kukulski protocol (Kukulski et al., 2011, 2012). Various fluorescently tagged proteins localizing to different cellular organelles could be assigned to their ultrastructural compartments. By combining STEM tomography with on-section CLEM, fluorescently tagged proteins can be localized in three-dimensional ultrastructural environments with a volume of at least 2.7×2.7×0.5µm.

Mesangial cells regulate the single nephron GFR and preserve the integrity of the glomerular filtration barrier: An intravital multiphoton microscopy study.

AIM: The intraglomerular mesangial cells are located between the glomerular capillaries. Here we hypothesized that mesangial cells regulate the single nephron glomerular filtration rate (snGFR) and that mesangial cells support the integrity of the glomerular filtration barrier. METHODS: We assessed the function of mesangial cells in vivo by multiphoton microscopy. Mesangial cells were depleted in Munich Wistar Froemter rats using the Thy1.1 antibody model. RESULTS: The Thy1.1 antibody caused the cell-specific loss of 82 ± 3% of mesangial cells. After mesangial cell depletion, the baseline snGFR was reduced to 12.0 ± 1.2 vs 32.4 ± 3.2 nL/min in controls. In control rats, the snGFR decreased after angiotensin II infusion by 61 ± 3% (P = .004), whereas it remained unchanged in Thy1.1-treated rats. The changes in the snGFR after angiotensin II infusion in control rats were accompanied by the marked rotation of the capillary loops within Bowman's space. This phenomenon was absent in anti-Thy1.1-treated rats. The glomerular sieving coefficient (GSCA ) for albumin, used as a measure of the integrity of the glomerular filtration barrier, was low in control rats (0.00061 ± 0.00004) and increased after angiotensin II infusion (0.00121 ± 0.00015). In Thy1.1-treated rats, the GSC was elevated (0.0032 ± 0.00059) and did not change in response to angiotensin II. Electron microscopy revealed the increased thickness of the glomerular basement membrane after mesangial cell depletion. CONCLUSION: Our data suggest that mesangial cells actively contribute to the regulation of the snGFR. Furthermore, mesangial cells are crucially involved in maintaining the integrity of the glomerular filtration barrier, in part by modulating the thickness of the glomerular basement membrane.

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced In Vitro/In Vivo Cell Identification.

Viral infection patterns often rely on precisely coordinated sequences of distinct ligand-receptor interactions, leading in many cases to an outstanding target cell specificity. A successful mimicry of viral targeting strategies to create more site-specific nanoparticles (NPs) would therefore require particle-cell interactions to also be adequately controllable. In the present study, hetero-multivalent block-copolymer NPs present their attached ligands in a sterically controlled manner to create a sequential NP-cell interaction similar to the cell infiltration strategy of human adenovirus type 2. Targeting renal mesangial cells, particles therefore initially bind angiotensin II receptor type 1 (AT1r) on the cell surface via a structurally flexible AT1r antagonist. After a mandatory spatial approach, particle endocytosis is realized via binding of immobile αVß3 integrins with a previously concealed secondary ligand, thereby creating a stepwise particle-cell interplay of primary NP attachment and subsequent uptake. Manufactured adenovirus-mimetic NPs show great avidity for both target motifs in vitro, leading to a substantial binding as well as subsequent cell uptake into target mesangial cells. Additionally, steric shielding of secondary ligand visibility leads to a highly controllable, sequential ligand-receptor interaction, whereby hetero-functional NPs activate mesangial cell surface integrins only after a successful prior binding to the AT1r. This stepwise cell identification significantly enhances mesangial cell specificity in co-culture assays with different off-target cells. Additionally, described NPs display excellent in vivo robustness by efficiently accumulating in the mesangium upon injection, thereby opening new paths for possible drug delivery applications.

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells.

Poor drug availability in the tissue of interest is a frequent cause of therapy failure. While nanotechnology has developed a plethora of nanocarriers for drug transport, their ability to unequivocally identify cells of interest remains moderate. Viruses are the ideal nanosized carriers as they are able to address their embedded nucleic acids with high specificity to their host cells. Here, it is reported that particles endowed with a virus-like ability to identify cells by three consecutive checks have a superior ability to recognize mesangial cells (MCs) in vivo compared to conventional nanoparticles. Mimicking the initial viral attachment followed by a stepwise target cell recognition process leads to a 5- to 15-fold higher accumulation in the kidney mesangium and extensive cell uptake compared to particles lacking one or both of the viral traits. These results highlight the relevance that the viral cell identification process has on specificity and its application on the targeting strategies of nanomaterials. More so, these findings pave the way for transporting drugs into the mesangium, a tissue that is pivotal in the development of diabetic nephropathy and for which currently no efficient pharmacotherapy exists.

Dual-axis STEM tomography at 200 kV: Setup, performance, limitations.

The interpretation of cell biological processes hinges on the elucidation of the underlying structures. Their three-dimensional analysis using electron tomography has extended our understanding of cellular organelles tremendously. The investigations depend on the availability of appropriate instruments for data recording. So far, such investigations have been done to a great extent on 300 keV transmission electron microscopes. Here we show the implementation of STEM tomography on a 200 kV FEG transmission electron microscope, including the tuning of the condenser for forming a beam with a small illumination aperture, dual-axis data recording, and evaluation of the maximum sample thickness and quality of the data. Our results show that the approach is accomplishable and promising, with high reliability, and reaching excellent data quality from plastic sections with a thickness of at least 900 nm.

Subcellular localization of the chemotherapeutic agent doxorubicin in renal epithelial cells and in tumor cells using correlative light and electron microscopy.

BACKGROUND: Doxorubicin is a cytostatic drug from the group of anthracycline antibiotics that is widely used as a chemotherapeutic agent. Side effects of the active substance include cardiotoxicity and nephrotoxicity. Doxorubicin-treated renal epithelial cells and (sarcoma) tumors are examined by correlative light and electron microscopy (CLEM) to investigate the subcellular localization of doxorubicin. METHODS: The kidney epithelial cell line MDCK II (Madin-Darby Canine Kidney) grown on culture dishes were treated with doxorubicin. Subsequently, the cells are analyzed by means of fluorescence and transmission electron microscopy (TEM). In vivo, alveolar rhabdomyosarcoma (RH 30) tumor cells are transferred to the chorioallantoic membrane (CAM) of the chicken embryo. Doxorubicin is injected into a vein of the chicken embryo. After 24 hours, the tumor is removed and examined using CLEM. RESULTS: The kidney epithelial cells and the doxorubicin-injected tumors show a clear staining of the cell nucleus, which correlates with electron-dense regions (heterochromatin). High-resolution TEM shows that doxorubicin treatment leads to an enormous stress situation with an increased formation of membrane blebbings. CONCLUSIONS: CLEM is a promising new method to visualize the pattern of fluorescing drugs (e.g. doxorubicin) in renal epithelial cells and tumors, and to localize the drug in its subcellular context combined with high resolution.

Glycine Amidinotransferase (GATM), Renal Fanconi Syndrome, and Kidney Failure.

Background For many patients with kidney failure, the cause and underlying defect remain unknown. Here, we describe a novel mechanism of a genetic order characterized by renal Fanconi syndrome and kidney failure.Methods We clinically and genetically characterized members of five families with autosomal dominant renal Fanconi syndrome and kidney failure. We performed genome-wide linkage analysis, sequencing, and expression studies in kidney biopsy specimens and renal cells along with knockout mouse studies and evaluations of mitochondrial morphology and function. Structural studies examined the effects of recognized mutations.Results The renal disease in these patients resulted from monoallelic mutations in the gene encoding glycine amidinotransferase (GATM), a renal proximal tubular enzyme in the creatine biosynthetic pathway that is otherwise associated with a recessive disorder of creatine deficiency. In silico analysis showed that the particular GATM mutations, identified in 28 members of the five families, create an additional interaction interface within the GATM protein and likely cause the linear aggregation of GATM observed in patient biopsy specimens and cultured proximal tubule cells. GATM aggregates-containing mitochondria were elongated and associated with increased ROS production, activation of the NLRP3 inflammasome, enhanced expression of the profibrotic cytokine IL-18, and increased cell death.Conclusions In this novel genetic disorder, fully penetrant heterozygous missense mutations in GATM trigger intramitochondrial fibrillary deposition of GATM and lead to elongated and abnormal mitochondria. We speculate that this renal proximal tubular mitochondrial pathology initiates a response from the inflammasome, with subsequent development of kidney fibrosis.

Casein kinase 1ε and 1α as novel players in polycystic kidney disease and mechanistic targets for (R)-roscovitine and (S)-CR8.

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

Golgi bypass of ciliary proteins.

Primary cilia represent small, yet distinct compartments of the plasma membrane. They are speculated to exercise chemo- and mechanosensory functions and to serve as signaling hubs for crucial pathways such as the Wnt and hedgehog cascades. It is therefore necessary that specific integral membrane proteins, in particular sensors and receptors, are sorted to the cilium and not to the surrounding somatic plasma membrane upon being synthesized at the rough endoplasmic reticulum. Apparently no singular "zip code" for the primary cilium exists but rather several ciliary targeting signals whose biochemical and cell biological implications are just about being unravelled. Among the better understood proteins residing in the primary cilium is polycystin-2 which is mutated in patients suffering from autosomal-dominant polycystic kidney disease. A special case in the context of this review concerns the connecting cilium which serves as the trafficking pathway for proteins involved in visual sensation of retinal photoreceptor cells. In order to efficiently capture photons, the photopigments are organized in discs or membrane invaginations. Mutations in certain proteins involved in these processes lead to retinal degeneration and ultimately to blindness. One example is peripherin/rds which is mutated in the rds (retinal degeneration slow) mouse. The trafficking of peripherin/rds from the inner to the outer segment of photoreceptor cells by way of the connecting cilium also seems to diverge at the Golgi apparatus, and the routes of polycystin-2 and peripherin/rds may represent paradigms of ciliary proteins for the type IV pathway of unconventional protein "secretion". This review is part of a special issue of Seminars in Cell and Developmental Biology edited by Walter Nickel and Catherine Rabouille.

Electron microscopy of Drosophila garland cell nephrocytes: Optimal preparation, immunostaining and STEM tomography.

Due to its structural and molecular similarities to mammalian podocytes, the Drosophila nephrocyte emerged as a model system to study podocyte development and associated diseases. Similar to podocytes, nephrocytes establish a slit diaphragm between foot process-like structures in order to filter the hemolymph. One major obstacle in nephrocyte research is the distinct visualization of this subcellular structure to assess its integrity. Therefore, we developed a specialized dissection and fixation protocol, including high pressure freezing and freeze substitution techniques, to improve the preservation of the intricate ultrastructural details necessary for electron microscopic assessment. By means of scanning transmission electron microscopy (STEM) tomography, a three-dimensional dataset was generated to further understand the complex architecture of the nephrocyte channel system. Moreover, a staining protocol for immunolabeling of ultrathin sections of Epon-embedded nephrocytes is discussed, which allows the reliable detection of GFP-tagged fusion proteins combined with superior sample preservation. Due to the growing number of available GFP-trap fly lines, this approach is widely applicable for high resolution localization studies in wild type and mutant nephrocytes.

Nail-patella syndrome.

The pathognomonic symptoms of patients with nail-patella syndrome are their small or absent patellae and dysplastic or absent finger- and toenails. Many of the patients suffer from renal symptoms which also affect their prognosis. In 1998, mutations in the gene encoding the transcription factor LMX1B were identified as underlying this autosomal-dominant disease. The LMX1B gene is expressed in a variety of tissues, and the symptoms are reflected nicely by its expression pattern. LMX1B is essential for dorso-ventral pattern formation in the limbs, for differentiation of the anterior portions of the eyes, for development of certain neuron populations in the central nervous system, and for the differentiation and maintenance of podocytes. Accordingly, kidney biopsies of patients with nail-patella syndrome show an altered podocyte structure and defects in the glomerular basement membrane. Recent evidence suggests that LMX1B regulates genes which encode proteins associated with the actin cytoskeleton.

Distinct functions of Crumbs regulating slit diaphragms and endocytosis in Drosophila nephrocytes.

Mammalian podocytes, the key determinants of the kidney's filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical-basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.