Collagen IVα345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.

Biallelic pathogenic variants in roundabout guidance receptor 1 associate with syndromic congenital anomalies of the kidney and urinary tract.

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses with biallelic truncating or combined missense and truncating variants in ROBO1. Kidney and genitourinary manifestation included unilateral or bilateral kidney agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation, and increased kidney echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects. By in silico analysis, we determined the functional significance of identified missense variants and observed absence of kidney ROBO1 expression in both human and murine mutant tissues. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of the kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. Thus, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Hence, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for kidney involvement is indicated.

The underestimated burden of monogenic kidney disease in adults waitlisted for kidney transplantation.

PURPOSE: Chronic kidney disease (CKD) is a major health-care burden. Increasing evidence suggests that a considerable proportion of patients are affected by a monogenic kidney disorder. METHODS: In this study, the kidney transplantation waiting list at the Charité was screened for patients with undetermined cause of CKD. By next-generation sequencing (NGS) we targeted all 600 genes described and associated with kidney disease or allied disorders. RESULTS: In total, 635 patients were investigated. Of these, 245 individuals had a known cause of CKD (38.5%) of which 119 had a proven genetic disease (e.g., ADPKD, Alport). The other 340 patients (53.5%) were classified as undetermined diagnosis, of whom 87 had kidney failure (KF) onset <40 years. To this latter group genetic testing was offered as well as to those patients (n = 29) with focal segmental glomerulosclerosis (FSGS) and all individuals (n = 21) suspicious for thrombotic microangiopathy (TMA) in kidney biopsy. We detected diagnostic variants in 26 of 126 patients (20.6%) of which 14 of 126 (11.1%) were pathogenic or likely pathogenic. In another 12 of 126 (9.5%) patients, variants of unknown significance (VUS) were detected. CONCLUSION: Our study demonstrates the diagnostic value of comprehensive genetic testing among patients with undetermined CKD.

Spectral Properties of Stochastic Resonance in Quantum Transport.

We investigate theoretically and experimentally stochastic resonance in a quantum dot coupled to electron source and drain via time-dependent tunnel barriers. A central finding is a transition visible in the current noise spectrum as a bifurcation of a dip originally at zero frequency. The transition occurs close to the stochastic resonance working point and relates to quantized pumping. For the evaluation of power spectra from measured waiting times, we generalize a result from renewal theory to the ac-driven case. Moreover, we develop a master equation method to obtain phase-averaged current noise spectra for driven quantum transport.

Value of renal gene panel diagnostics in adults waiting for kidney transplantation due to undetermined end-stage renal disease.

End-stage renal disease (ESRD) of undetermined etiology is highly prevalent and constitutes a significant clinical challenge, particularly in the context of kidney transplantation (KT). Despite the identification of numerous rare hereditary nephropathies over the last few decades, patients with undetermined ESRD are not being systematically investigated for rare genetic causes in clinical practice. To address this, we utilized mutation analysis in patients on the kidney transplant waitlist and scrutinized underlying renal diagnoses of 142 patients in a single center KT-waitlist. This cohort was stratified into 85 cases of determined and 57 cases of undetermined ESRD. The latter patients were analyzed by a renal gene panel for mutations in 209 genes associated with ESRD. The most likely genetic diagnoses in 12% of the tested individuals with undetermined ESRD were established. All of these patients showed mutations in genes encoding components of the glomerular filtration barrier. Taken together, hereditary nephropathies, including autosomal dominant polycystic kidney disease, were identified in 35 of the 142 patients of the waitlist cohort. By significantly increasing the proportion of hereditary diagnoses from 29 to 35 patients, the rate of undetermined ESRD significantly decreased from 57 to 51 patients. This study demonstrates the beneficial use of genetic diagnostics in significantly unraveling undetermined ESRD cases prior to KT. Thus, in the absence of renal histology or the presence of unspecific histological conditions, such as hypertensive nephrosclerosis, focal segmental glomerulosclerosis or thrombotic microangiopathy, genetic analysis may provide a robust and specific renal diagnosis and allow for optimizing pre- and post-KT management.

Low density lipoprotein receptor-related protein 1 mediated endocytosis of ß1-integrin influences cell adhesion and cell migration.

The low density lipoprotein receptor-related protein 1 (LRP1) has been shown to interact with ß1-integrin and regulate its surface expression. LRP1 knock-out cells exhibit altered cytoskeleton organization and decreased cell migration. Here we demonstrate coupled endocytosis of LRP1 and ß1-integrin and the involvement of the intracellular NPxY2 motif of LRP1 in this process. Mouse embryonic fibroblasts harboring a knock in replacement of the NPxY2 motif of LRP1 by a multiple alanine cassette (AAxA) showed elevated surface expression of ß1-integrin and decreased ß1-integrin internalization rates. As a consequence, cell spreading was altered and adhesion rates were increased in our cell model. Cells formed more focal adhesion complexes, whereby in vitro cell migration rates were decreased. Similar results could be observed in a corresponding mouse model, the C57Bl6 LRP1 NPxYxxL knock in mice, therefore, the biochemistry of cellular adhesion was altered in primary cortical neurons. In vivo cell migration experiments demonstrated a disturbance of neuroblast cell migration along the rostral migratory stream. In summary, our results indicate that LRP1 interacts with ß1-integrin mediating integrin internalization and thus correlates with downstream signaling of ß1-integrin such as focal adhesion dynamics. Consequently, the disturbance of this interaction resulted in a dysfunction in in vivo and in vitro cell adhesion and cell migration.

Stx5 is a novel interactor of VLDL-R to affect its intracellular trafficking and processing.

We identified syntaxin 5 (Stx5), a protein involved in intracellular vesicle trafficking, as a novel interaction partner of the very low density lipoprotein (VLDL)-receptor (VLDL-R), a member of the LDL-receptor family. In addition, we investigated the effect of Stx5 on VLDL-R maturation, trafficking and processing. Here, we demonstrated mutual association of both proteins using several in vitro approaches. Furthermore, we detected a special maturation phenotype of VLDL-R resulting from Stx5 overexpression. We found that Stx5 prevented advanced Golgi-maturation of VLDL-R, but did not cause accumulation of the immature protein in ER, ER to Golgi compartments, or cis-Golgi ribbon, the main expression sites of Stx5. Rather more, abundantly present Stx5 was capable of translocating ER-/N-glycosylated VLDL-R to the plasma membrane, and thus was insensitive to BFA treatment and low temperature. Furthermore, abundant presence of Stx5 significantly interfered with VLDL-R reaching the trans-Golgi network. Based on our findings, we postulate that Stx5 can directly bind to the C-terminal domain of VLDL-R, thereby influencing the receptor's glycosylation, trafficking and processing characteristics. Resulting from that, we further suggest that Stx5 might play a role in modulating VLDL-R physiology by participating in an abrasively described or completely novel Golgi-bypass pathway.

The role of lipoprotein receptors on the physiological function of APP.

In this review, we will primarily focus on the role of members of the low-density lipoprotein receptor (LDL-R) family that are involved in trafficking and processing of the amyloid precursor protein (APP). We will discuss the role of the LDL-receptor family members, low-density lipoprotein receptor-related protein 1 (LRP1), LRP1b, apolipoprotein E receptor 2, sortilin-related receptor (SorLA/LR11) and megalin/LRP2 on the physiological function of APP and its cellular localization. Additionally, we will focus on adaptor proteins that have been shown to influence the physiological function of LDL-R family members in combination with APP processing. The results in this review emphasize that the physiological function of APP cannot be explained by the focus on the APP protein alone but rather in combination with various direct or indirect interaction partners within the cellular environment.

Loss of the collagen IV modifier prolyl 3-hydroxylase 2 causes thin basement membrane nephropathy.

The glomerular filtration barrier (GFB) produces primary urine and is composed of a fenestrated endothelium, a glomerular basement membrane (GBM), podocytes, and a slit diaphragm. Impairment of the GFB leads to albuminuria and microhematuria. The GBM is generated via secreted proteins from both endothelial cells and podocytes and is supposed to majorly contribute to filtration selectivity. While genetic mutations or variations of GBM components have been recently proposed to be a common cause of glomerular diseases, pathways modifying and stabilizing the GBM remain incompletely understood. Here, we identified prolyl 3-hydroxylase 2 (P3H2) as a regulator of the GBM in an a cohort of patients with albuminuria. P3H2 hydroxylates the 3' of prolines in collagen IV subchains in the endoplasmic reticulum. Characterization of a P3h2ΔPod mouse line revealed that the absence of P3H2 protein in podocytes induced a thin basement membrane nephropathy (TBMN) phenotype with a thinner GBM than that in WT mice and the development of microhematuria and microalbuminuria over time. Mechanistically, differential quantitative proteomics of the GBM identified a significant decrease in the abundance of collagen IV subchains and their interaction partners in P3h2ΔPod mice. To our knowledge, P3H2 protein is the first identified GBM modifier, and loss or mutation of P3H2 causes TBMN and focal segmental glomerulosclerosis in mice and humans.

Controlled emission time statistics of a dynamic single-electron transistor.

Quantum technologies involving qubit measurements based on electronic interferometers rely critically on accurate single-particle emission. However, achieving precisely timed operations requires exquisite control of the single-particle sources in the time domain. Here, we demonstrate accurate control of the emission time statistics of a dynamic single-electron transistor by measuring the waiting times between emitted electrons. By ramping up the modulation frequency, we controllably drive the system through a crossover from adiabatic to nonadiabatic dynamics, which we visualize by measuring the temporal fluctuations at the single-electron level and explain using detailed theory. Our work paves the way for future technologies based on the ability to control, transmit, and detect single quanta of charge or heat in the form of electrons, photons, or phonons.

Human C-terminal CUBN variants associate with chronic proteinuria and normal renal function.

BACKGROUNDProteinuria is considered an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear whether all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS), which is characterized by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding.METHODSWe used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling, and epidemiological methods.RESULTSWe identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene were associated with chronic isolated proteinuria and early childhood onset. Since the proteinuria in these patients had a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and the use of proteinuria-lowering treatments. However, renal function was normal in all cases. By contrast, we did not found any biallelic CUBN variants in proteinuric patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that 4 C-terminal CUBN variants are associated with albuminuria and slightly increased GFR in meta-analyses of large population-based cohorts.CONCLUSIONCollectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsy.FUNDINGATIP-Avenir program, Fondation Bettencourt-Schueller (Liliane Bettencourt Chair of Developmental Biology), Agence Nationale de la Recherche (ANR) Investissements d'avenir program (ANR-10-IAHU-01) and NEPHROFLY (ANR-14-ACHN-0013, to MS), Steno Collaborative Grant 2018 (NNF18OC0052457, to TSA and MS), Heisenberg Professorship of the German Research Foundation (KO 3598/5-1, to AK), Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Centre (SFB) KIDGEM 1140 (project 246781735, to CB), and Federal Ministry of Education and Research (BMB) (01GM1515C, to CB).

Strong suppression of shot noise in a feedback-controlled single-electron transistor.

Feedback control of quantum mechanical systems is rapidly attracting attention not only due to fundamental questions about quantum measurements, but also because of its novel applications in many fields in physics. Quantum control has been studied intensively in quantum optics but progress has recently been made in the control of solid-state qubits as well. In quantum transport only a few active and passive feedback experiments have been realized on the level of single electrons, although theoretical proposals exist. Here we demonstrate the suppression of shot noise in a single-electron transistor using an exclusively electronic closed-loop feedback to monitor and adjust the counting statistics. With increasing feedback response we observe a stronger suppression and faster freezing of charge current fluctuations. Our technique is analogous to the generation of squeezed light with in-loop photodetection as used in quantum optics. Sub-Poisson single-electron sources will pave the way for high-precision measurements in quantum transport similar to optical or optomechanical equivalents.

Partitioning of on-demand electron pairs.

The on-demand generation and separation of entangled photon pairs are key components of quantum information processing in quantum optics. In an electronic analogue, the decomposition of electron pairs represents an essential building block for using the quantum state of ballistic electrons in electron quantum optics. The scattering of electrons has been used to probe the particle statistics of stochastic sources in Hanbury Brown and Twiss experiments and the recent advent of on-demand sources further offers the possibility to achieve indistinguishability between multiple sources in Hong-Ou-Mandel experiments. Cooper pairs impinging stochastically at a mesoscopic beamsplitter have been successfully partitioned, as verified by measuring the coincidence of arrival. Here, we demonstrate the splitting of electron pairs generated on demand. Coincidence correlation measurements allow the reconstruction of the full counting statistics, revealing regimes of statistically independent, distinguishable or correlated partitioning, and have been envisioned as a source of information on the quantum state of the electron pair. The high pair-splitting fidelity opens a path to future on-demand generation of spin-entangled electron pairs from a suitably prepared two-electron quantum-dot ground state.