Linus A. Völker

Mutations in NEK8 link multiple organ dysplasia with altered Hippo signalling and increased c-MYC expression

Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.

Light microscopic visualization of podocyte ultrastructure demonstrates oscillating glomerular contractions.

Podocytes, the visceral epithelial cells of the kidney glomerulus, elaborate primary and interdigitating secondary extensions to enwrap the glomerular capillaries. A hallmark of podocyte injury is the loss of unique ultrastructure and simplification of the cell shape, called foot process effacement, which is a classic feature of proteinuric kidney disease. Although several key pathways have been identified that control cytoskeletal regulation, actin dynamics, and polarity signaling, studies into the dynamic regulation of the podocyte structure have been hampered by the fact that ultrastructural analyses require electron microscopic imaging of fixed tissue. We developed a new technique that allows for visualization of podocyte foot processes using confocal laser scanning microscopy. The combination of inducible and mosaic expression of membrane-tagged fluorescent proteins in a small subset of podocytes enabled us to acquire light microscopic images of podocyte foot processes in unprecedented detail, even in living podocytes of freshly isolated glomeruli. Moreover, this technique visualized oscillatory glomerular contractions and confirmed the morphometric evaluations obtained in static electron microscopic images of podocyte processes. These data suggest that the new technique will provide an extremely powerful tool for studying the dynamics of podocyte ultrastructure.

Comparative analysis of Neph gene expression in mouse and chicken development

Neph proteins are evolutionarily conserved members of the immunoglobulin superfamily of adhesion proteins and regulate morphogenesis and patterning of different tissues. They share a common protein structure consisting of extracellular immunoglobulin-like domains, a transmembrane region, and a carboxyl terminal cytoplasmic tail required for signaling. Neph orthologs have been widely characterized in invertebrates where they mediate such diverse processes as neural development, synaptogenesis, or myoblast fusion. Vertebrate Neph proteins have been described first at the glomerular filtration barrier of the kidney. Recently, there has been accumulating evidence suggesting a function of Neph proteins also outside the kidney. Here we demonstrate that Neph1, Neph2, and Neph3 are expressed differentially in various tissues during ontogenesis in mouse and chicken. Neph1 and Neph2 were found to be amply expressed in the central nervous system while Neph3 expression remained localized to the cerebellum anlage and the spinal cord. Outside the nervous system, Neph mRNAs were also differentially expressed in branchial arches, somites, heart, lung bud, and apical ectodermal ridge. Our findings support the concept that vertebrate Neph proteins, similarly to their Drosophila and C. elegans orthologs, provide guidance cues for cell recognition and tissue patterning in various organs which may open interesting perspectives for future research on Neph1-3 controlled morphogenesis.

A flexible, multilayered protein scaffold maintains the slit in between glomerular podocytes.

Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.

Survival and distribution of injected haematopoietic stem cells in acute kidney injury

BACKGROUND Endogenous bone marrow-derived cells are known to incorporate into renal epithelium at a low rate. Haematopoietic stem cells (HSCs) rather than mesenchymal stem cells (MSC) are responsible for this phenomenon. MSCs have the potential to ameliorate kidney function after acute kidney injury (AKI) without directly repopulating the tubules. However, little is known about the short-term effect of HSCs. METHODS In this article, we analysed the survival rate and organ distribution of isolated rat HSCs injected into the renal artery after ischaemic renal injury, using quantitative real-time PCR, as well as their impact on renal function and histomorphology. RESULTS Intra-arterially injected Lin(-)CD90(+) HSCs were detected in the kidney at significant amounts only within the first 24 h after injection and were virtually absent by Day 2. Compared with control animals, no differences were seen after HSC administration with respect to kidney function or histomorphologic changes of AKI. At Day 7 HSCs were again readily detectable in the kidney suggesting a redistribution of cells at later time points. Of note, HSCs did not seem to have an exclusive tropism for the injured kidney but were detectable in the lungs, liver, spleen, heart and brain at all time points. CONCLUSIONS Injected HSCs do not appear to significantly contribute to tubular repair or ameliorate renal damage in ischaemic AKI although they may show considerable engraftment in various organs. These data further challenge the concept that injection of HSCs may be used as a therapeutic approach in treating AKI.

A Disease-causing Mutation Illuminates the Protein Membrane Topology of the Kidney-expressed Prohibitin Homology (PHB) Domain Protein Podocin

Background: Mutations in the stomatin family protein podocin are the most common genetic cause of proteinuria. Results: A conserved proline residue of podocin is essential for its membrane topology. Conclusion: This study confirms a hairpin-like structure of the membrane-attached PHB domain protein and its significance for cholesterol recruitment. Significance: PodocinP118L elucidates the pathogenic implication in kidney disease and identifies a novel family of PHB domain proteins. Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocinP118L) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2P134S). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. PodocinP118L and MEC-2P134S did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

Characterization of a short isoform of the kidney protein podocin in human kidney

BackgroundSteroid resistant nephrotic syndrome is a severe hereditary disease often caused by mutations in the NPHS2 gene. This gene encodes the lipid binding protein podocin which localizes to the slit diaphragm of podocytes and is essential for the maintenance of an intact glomerular filtration barrier. Podocin is a hairpin-like membrane-associated protein that multimerizes to recruit lipids of the plasma membrane. Recent evidence suggested that podocin may exist in a canonical, well-studied large isoform and an ill-defined short isoform. Conclusive proof of the presence of this new podocin protein in the human system is still lacking.MethodsWe used database analyses to identify organisms for which an alternative splice variant has been annotated. Mass spectrometry was employed to prove the presence of the shorter isoform of podocin in human kidney lysates. Immunofluorescence, sucrose density gradient fractionation and PNGase-F assays were used to characterize this short isoform of human podocin.ResultsMass spectrometry revealed the existence of the short isoform of human podocin on protein level. We cloned the coding sequence from a human kidney cDNA library and showed that the expressed short variant was retained in the endoplasmic reticulum while still associating with detergent-resistant membrane fractions in sucrose gradient density centrifugation. The protein is partially N-glycosylated which implies the presence of a transmembranous form of the short isoform.ConclusionsA second isoform of human podocin is expressed in the kidney. This isoform lacks part of the PHB domain. It can be detected on protein level. Distinct subcellular localization suggests a physiological role for this isoform which may be different from the well-studied canonical variant. Possibly, the short isoform influences lipid and protein composition of the slit diaphragm complex by sequestration of lipid and protein interactors into the endoplasmic reticulum.

A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population

Background Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. Methods We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as “Severe” or “Mild”, based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. Results and conclusions Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients’ kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a “rare variant-strong effect” role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.

N-Degradomic Analysis Reveals a Proteolytic Network Processing the Podocyte Cytoskeleton

Regulated intracellular proteostasis, controlled in part by proteolysis, is essential in maintaining the integrity of podocytes and the glomerular filtration barrier of the kidney. We applied a novel proteomics technology that enables proteome-wide identification, mapping, and quantification of protein N-termini to comprehensively characterize cleaved podocyte proteins in the glomerulus in vivo We found evidence that defined proteolytic cleavage results in various proteoforms of important podocyte proteins, including those of podocin, nephrin, neph1, α-actinin-4, and vimentin. Quantitative mapping of N-termini demonstrated perturbation of protease action during podocyte injury in vitro, including diminished proteolysis of α-actinin-4. Differentially regulated protease substrates comprised cytoskeletal proteins as well as intermediate filaments. Determination of preferential protease motifs during podocyte damage indicated activation of caspase proteases and inhibition of arginine-specific proteases. Several proteolytic processes were clearly site-specific, were conserved across species, and could be confirmed by differential migration behavior of protein fragments in gel electrophoresis. Some of the proteolytic changes discovered in vitro also occurred in two in vivo models of podocyte damage (WT1 heterozygous knockout mice and puromycin aminonucleoside-treated rats). Thus, we provide direct and systems-level evidence that the slit diaphragm and podocyte cytoskeleton are regulated targets of proteolytic modification, which is altered upon podocyte damage.

Neph2/Kirrel3 regulates sensory input, motor coordination, and home-cage activity in rodents

Adhesion molecules of the immunoglobulin superfamily (IgSF) are essential for neuronal synapse development across evolution and control various aspects of synapse formation and maturation. Neph2, also known as Kirrel3, is an IgSF adhesion molecule implicated in synapse formation, synaptic transmission and ultrastructure. In humans, defects in the NEPH2 gene have been associated with neurodevelopmental disorders such as Jacobsen syndrome, intellectual disability, and autism‐spectrum disorders. However, the precise role in development and function of the nervous system is still unclear. Here, we present the histomorphological and phenotypical analysis of a constitutive Neph2‐knockout mouse line. Knockout mice display defects in auditory sensory processing, motor skills, and hyperactivity in the home‐cage analysis. Olfactory, memory and metabolic testing did not differ from controls. Despite the wide‐spread expression of Neph2 in various brain areas, no gross anatomic defects could be observed. Neph2 protein could be located at the cerebellar pinceaux. It interacted with the pinceau core component neurofascin and other synaptic proteins thus suggesting a possible role in cerebellar synapse formation and circuit assembly. Our results suggest that Neph2/Kirrel3 acts on the synaptic ultrastructural level and neuronal wiring rather than on ontogenetic events affecting macroscopic structure. Neph2‐knockout mice may provide a valuable rodent model for research on autism spectrum diseases and neurodevelopmental disorders.