Sandra Habbig

NPHP4, a cilia-associated protein, negatively regulates the Hippo pathway

The conserved Hippo signaling pathway regulates organ size in Drosophila melanogaster and mammals and has an essential role in tumor suppression and the control of cell proliferation. Recent studies identified activators of Hippo signaling, but antagonists of the pathway have remained largely elusive. In this paper, we show that NPHP4, a known cilia-associated protein that is mutated in the severe degenerative renal disease nephronophthisis, acts as a potent negative regulator of mammalian Hippo signaling. NPHP4 directly interacted with the kinase Lats1 and inhibited Lats1-mediated phosphorylation of the Yes-associated protein (YAP) and TAZ (transcriptional coactivator with PDZ-binding domain), leading to derepression of these protooncogenic transcriptional regulators. Moreover, NPHP4 induced release from 14-3-3 binding and nuclear translocation of YAP and TAZ, promoting TEA domain (TEAD)/TAZ/YAP-dependent transcriptional activity. Consistent with these data, knockdown of NPHP4 negatively affected cellular proliferation and TEAD/TAZ activity, essentially phenocopying loss of TAZ function. These data identify NPHP4 as a negative regulator of the Hippo pathway and suggest that NPHP4 regulates cell proliferation through its effects on Hippo signaling.

Long-term follow-up after rituximab for steroid-dependent idiopathic nephrotic syndrome

BACKGROUND In patients with refractory steroid-sensitive nephrotic syndrome (SSNS), treatment with rituximab has shown encouraging results; however, long-term follow-up data are not available. METHODS We performed a retrospective analysis of 37 patients (25 boys) with steroid-dependent nephrotic syndrome who were treated with rituximab (375 mg/m(2) given weekly for one to four courses). Long-term follow-up data (>2 years, median 36, range 24-92.8 months) are available for 29 patients (12 boys). RESULTS Twenty-six of 37 (70.3%) patients remained in remission after 12 months. Relapses occurred in 24 (64.8%) patients after a median of 9.6 (range 5.2-64.1) months. Time to first relapse was significantly shorter in patients receiving one or two compared to three or four initial infusions. In the 29 patients with long-term follow-up for >2 years, 12 (41%) patients remained in remission after the initial rituximab course for >24 months, 7 (24.1%) patients without further maintenance immunosuppression. Nineteen children received two to four repeated courses of rituximab increasing the total number of patients with long-term remission to 20 (69%), remission including 14 (48%) patients off immunosuppression. The proportion of patients with long-term remission was not related to the number of initial rituximab applications. No serious side effects were noted. CONCLUSION Rituximab is an effective treatment option in the short- and long-term control of treatment refractory SSNS. Further controlled studies are needed to address optimal patient selection, dose and safety of rituximab infusions.

C3 deposition glomerulopathy due to a functional Factor H defect

Factor H defect Sandra Habbig, Michael J. Mihatsch, Stefan Heinen, Bodo Beck, Mathias Emmel, Christine Skerka, Michael Kirschfink, Bernd Hoppe, Peter F. Zipfel and Christoph Licht Division of Pediatric Nephrology, Children’s Hospital of the University of Cologne, Cologne, Germany; Institute of Pathology, Basel, Switzerland; Leibniz Institute for Natural Products Research and Infection Biology, Jena, Germany; Department of Pediatric Cardiology, Children’s Hospital of the University of Cologne, Cologne, Germany; Institute of Immunology, University of Heidelberg, Heidelberg, Germany; Friedrich Schiller University, Jena, Germany; Division of Pediatric Nephrology, The Hospital for Sick Children; University of Toronto, Toronto, Ontario, Canada

Platelet-associated complement factor H in healthy persons and patients with atypical HUS

Atypical hemolytic uremic syndrome (aHUS) is associated with complement system dysregulation, and more than 25% of pediatric aHUS cases are linked to mutations in complement factor H (CFH) or CFH autoantibodies. The observation of thrombocytopenia and platelet-rich thrombi in the glomerular microvasculature indicates that platelets are intimately involved in aHUS pathogenesis. It has been reported that a releasable pool of platelet CFH originates from alpha-granules. We observed that platelet CFH can arise from endogenous synthesis in megakaryocytes and that platelets constitutively lacking alpha-granules contain CFH. Electron and high-resolution laser fluorescence confocal microscopy revealed that CFH was present throughout the cytoplasm and on the surface of normal resting platelets with no evident concentration in alpha-granules, lysosomes, or dense granules. Therapeutic plasma transfusion in a CFH-null aHUS patient revealed that circulating platelets take up CFH with similar persistence of CFH in platelets and plasma in vivo. Washed normal platelets were also observed to take up labeled CFH in vitro. Exposure of washed normal platelets to plasma of an aHUS patient with CFH autoantibodies produced partial platelet aggregation or agglutination, which was prevented by preincubation of platelets with purified CFH. This CFH-dependent response did not involve P-selectin mobilization, indicating a complement-induced platelet response distinct from alpha-granule secretion.

Nephrocalcinosis and urolithiasis in children.

The incidence of adult urolithiasis has increased significantly in industrialized countries over the past decades. Sound incidence rates are not available for children, nor are they known for nephrocalcinosis, which can appear as a single entity or together with urolithiasis. In contrast to the adult kidney stone patient, where environmental factors are the main cause, genetic and/or metabolic disorders are the main reason for childhood nephrocalcinosis and urolithiasis. While hypercalciuria is considered to be the most frequent risk factor, several other metabolic disorders such as hypocitraturia or hyperoxaluria, as well as a variety of renal tubular diseases, e.g., Dents disease or renal tubular acidosis, have to be ruled out by urine and/or blood analysis. Associated symptoms such as growth retardation, intestinal absorption, or bone demineralization should be evaluated for diagnostic and therapeutic purposes. Preterm infants are a special risk population with a high incidence of nephrocalcinosis arising from immature kidney, medication, and hypocitraturia. In children, concise evaluation will reveal an underlying pathomechanism in >75% of patients. Early treatment reducing urinary saturation of the soluble by increasing fluid intake and by providing crystallization inhibitors, as well as disease-specific medication, are mandatory to prevent recurrent kidney stones and/or progressive nephrocalcinosis, and consequently deterioration of renal function.

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.

Label-free quantitative proteomic analysis of the YAP/TAZ interactome

The function of an individual protein is typically defined by protein-protein interactions orchestrating the formation of large complexes critical for a wide variety of biological processes. Over the last decade the analysis of purified protein complexes by mass spectrometry became a key technique to identify protein-protein interactions. We present a fast and straightforward approach for analyses of interacting proteins combining a Flp-in single-copy cellular integration system and single-step affinity purification with single-shot mass spectrometry analysis. We applied this protocol to the analysis of the YAP and TAZ interactome. YAP and TAZ are the downstream effectors of the mammalian Hippo tumor suppressor pathway. Our study provides comprehensive interactomes for both YAP and TAZ and does not only confirm the majority of previously described interactors but, strikingly, revealed uncharacterized interaction partners that affect YAP/TAZ TEAD-dependent transcription. Among these newly identified candidates are Rassf8, thymopoetin, and the transcription factors CCAAT/enhancer-binding protein (C/EBP)β/δ and core-binding factor subunit β (Cbfb). In addition, our data allowed insights into complex stoichiometry and uncovered discrepancies between the YAP and TAZ interactomes. Taken together, the stringent approach presented here could help to significantly sharpen the understanding of protein-protein networks.

Novel findings in patients with primary hyperoxaluria type III and implications for advanced molecular testing strategies

Identification of mutations in the HOGA1 gene as the cause of autosomal recessive primary hyperoxaluria (PH) type III has revitalized research in the field of PH and related stone disease. In contrast to the well-characterized entities of PH type I and type II, the pathophysiology and prevalence of type III is largely unknown. In this study, we analyzed a large cohort of subjects previously tested negative for type I/II by complete HOGA1 sequencing. Seven distinct mutations, among them four novel, were found in 15 patients. In patients of non-consanguineous European descent the previously reported c.700+5G>T splice-site mutation was predominant and represents a potential founder mutation, while in consanguineous families private homozygous mutations were identified throughout the gene. Furthermore, we identified a family where a homozygous mutation in HOGA1 (p.P190L) segregated in two siblings with an additional AGXT mutation (p.D201E). The two girls exhibiting triallelic inheritance presented a more severe phenotype than their only mildly affected p.P190L homozygous father. In silico analysis of five mutations reveals that HOGA1 deficiency is causing type III, yet reduced HOGA1 expression or aberrant subcellular protein targeting is unlikely to be the responsible pathomechanism. Our results strongly suggest HOGA1 as a major cause of PH, indicate a greater genetic heterogeneity of hyperoxaluria, and point to a favorable outcome of type III in the context of PH despite incomplete or absent biochemical remission. Multiallelic inheritance could have implications for genetic testing strategies and might represent an unrecognized mechanism for phenotype variability in PH.

The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling.

Background: Deregulation of Wnt signaling contributes to the development of cystic kidney diseases such as nephronophthisis (NPH). Results: The NPH protein NPHP4 stabilizes Jade-1, a negative Wnt regulator, and translocates Jade-1 to the nucleus. Conclusion: NPHP4 and Jade-1 additively decrease canonical Wnt signaling. Significance: Loss of NPHP4-mediated Wnt repression via Jade-1 may contribute to cystogenesis in NPH. Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease and represents the most common genetic cause for end-stage renal disease in children and adolescents. It can be caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs). All NPHPs localize to primary cilia, classifying this disease as a “ciliopathy.” The primary cilium is a critical regulator of several cell signaling pathways. Cystogenesis in the kidney is thought to involve overactivation of canonical Wnt signaling, which is negatively regulated by the primary cilium and several NPH proteins, although the mechanism remains unclear. Jade-1 has recently been identified as a novel ubiquitin ligase targeting the canonical Wnt downstream effector β-catenin for proteasomal degradation. Here, we identify Jade-1 as a novel component of the NPHP protein complex. Jade-1 colocalizes with NPHP1 at the transition zone of primary cilia and interacts with NPHP4. Furthermore, NPHP4 stabilizes protein levels of Jade-1 and promotes the translocation of Jade-1 to the nucleus. Finally, NPHP4 and Jade-1 additively inhibit canonical Wnt signaling, and this genetic interaction is conserved in zebrafish. The stabilization and nuclear translocation of Jade-1 by NPHP4 enhances the ability of Jade-1 to negatively regulate canonical Wnt signaling. Loss of this repressor function in nephronophthisis might be an important factor promoting Wnt activation and contributing to cyst formation.

Hyperoxaluria and systemic oxalosis: an update on current therapy and future directions

Introduction: The primary hyperoxalurias (PH) are rare, but underdiagnosed disorders where the loss of enzymatic activity in key compounds of glyoxylate metabolism results in excessive endogenous oxalate generation. Clinically, they are characterized by recurrent urolithiasis and/or nephrocalcinosis. PH type I is the most frequent and most devastating subtype often leading to early end-stage renal failure. Areas covered: Profound overview of clinical, diagnostic, and currently available treatment options with a focus on PH I at different stages of the disease. Discussion of future therapeutic avenues including pharmacological chaperones (small molecules rescuing protein function), gene therapy with safer adenoviral vectors, and potential application of cell-based transplantation strategies is provided. Expert opinion: Due to lack of familiarity with PH and its heterogeneous clinical expression, diagnosis is often delayed until advanced disease is present, a condition, requiring intensive hemodialysis and timely transplantation. Achieving the most beneficial outcome largely depends on the knowledge of the clinical spectrum, early diagnosis, and initiation of treatment before renal failure ensues. A number of preconditions required for substantial improvement in the care of orphan disease like PH have now been achieved or soon will come within reach, so new treatment options can be expected in the near future.