Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis.

AIMS: Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply Translating Ribosome Affinity Purification sequencing (TRAP-Seq) to profile SMC-specific gene expression directly from tissue. METHODS AND RESULTS: To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, that are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, Itih4, Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease (CAD) through the colocalization of GWAS, splice-QTL, and protein-QTL signals. CONCLUSIONS: We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

Generation of novel in vitro flexible kidney organoid model to investigate the role of extracellular vesicles in induction of nephrogenesis.

BACKGROUND: During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis. RESULTS: Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs. CONCLUSION: Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.

Computational modelling of nephron progenitor cell movement and aggregation during kidney organogenesis.

During early kidney organogenesis, nephron progenitor (NP) cells move from the tip to the corner region of the ureteric bud (UB) branches in order to form the pretubular aggregate, the early structure giving rise to nephron formation. NP cells derive from metanephric mesenchymal cells and physically interact with them during the movement. Chemotaxis and cell-cell adhesion differences are believed to drive the cell patterning during this critical period of organogenesis. However, the effect of these forces to the cell patterns and their respective movements are known in limited details. We applied a Cellular Potts Model to explore how these forces and organizations contribute to directed cell movement and aggregation. Model parameters were estimated based on fitting to experimental data obtained in ex vivo kidney explant and dissociation-reaggregation organoid culture studies. Our simulations indicated that optimal enrichment and aggregation of NP cells in the UB corner niche requires chemoattractant secretion from both the UB epithelial cells and the NP cells themselves, as well as differences in cell-cell adhesion energies. Furthermore, NP cells were observed, both experimentally and by modelling, to move at higher speed in the UB corner as compared to the tip region where they originated. The existence of different cell speed domains along the UB was confirmed using self-organizing map analysis. In summary, we saw faster NP cell movements near aggregation. The applicability of Cellular Potts Model approach to simulate cell movement and patterning was found to be good during for this early nephrogenesis process. Further refinement of the model should allow us to recapitulate the effects of developmental changes of cell phenotypes and molecular crosstalk during further organ development.

Potential strategies to prevent encrustations on urinary stents and catheters - thinking outside the box: a European network of multidisciplinary research to improve urinary stents (ENIUS) initiative.

Introduction: Urinary stents have been around for the last 4 decades, urinary catheters even longer. They are associated with infections, encrustation, migration, and patient discomfort. Research efforts to improve them have shifted onto molecular and cellular levels. ENIUS brought together translational scientists to improve urinary implants and reduce morbidity.Methods & materials: A working group within the ENIUS network was tasked with assessing future research lines for the improvement of urinary implants.Topics were researched systematically using Embase and PubMed databases. Clinicaltrials.gov was consulted for ongoing trials.Areas covered: Relevant topics were coatings with antibodies, enzymes, biomimetics, bioactive nano-coats, antisense molecules, and engineered tissue. Further, pH sensors, biodegradable metals, bactericidal bacteriophages, nonpathogenic uropathogens, enhanced ureteric peristalsis, electrical charges, and ultrasound to prevent stent encrustations were addressed.Expert opinion: All research lines addressed in this paper seem viable and promising. Some of them have been around for decades but are yet to proceed to clinical application (i.e. tissue engineering). Others are very recent and, at least in urology, still only conceptual (i.e. antisense molecules). Perhaps the most important learning point resulting from this pan-European multidisciplinary effort is that collaboration between all stakeholders is not only fruitful but also truly essential.

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging.

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis.

Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.

Biodegradable Nanocarriers Resembling Extracellular Vesicles Deliver Genetic Material with the Highest Efficiency to Various Cell Types.

Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co-transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10-4 pmol of siRNA, and 1 × 10-3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.

Impact of Nanocapsules on Red Blood Cells Interplay Jointly Assessed by Optical Tweezers and Microscopy.

In the framework of novel medical paradigm the red blood cells (RBCs) have a great potential to be used as drug delivery carriers. This approach requires an ultimate understanding of the peculiarities of mutual interaction of RBC influenced by nano-materials composed the drugs. Optical tweezers (OT) is widely used to explore mechanisms of cells' interaction with the ability to trap non-invasively, manipulate and displace living cells with a notably high accuracy. In the current study, the mutual interaction of RBC with polymeric nano-capsules (NCs) is investigated utilizing a two-channel OT system. The obtained results suggest that, in the presence of NCs, the RBC aggregation in plasma satisfies the 'cross-bridges' model. Complementarily, the allocation of NCs on the RBC membrane was observed by scanning electron microscopy (SEM), while for assessment of NCs-induced morphological changes the tests with the human mesenchymal stem cells (hMSC) was performed. The combined application of OT and advanced microscopy approaches brings new insights into the conception of direct observation of cells interaction influenced by NCs for the estimation of possible cytotoxic effects.

Optical Studies of Nanodiamond-Tissue Interaction: Skin Penetration and Localization.

In this work, several optical-spectroscopic methods have been used to visualize and investigate the penetration of diamond nanoparticles (NPs) of various sizes (3-150 nm), surface structures and fluorescence properties into the animal skin in vitro. Murine skin samples have been treated with nanodiamond (ND) water suspensions and studied using optical coherence tomography (OCT), confocal and two-photon fluorescence microscopy and fluorescence lifetime imaging (FLIM). An analysis of the optical properties of the used nanodiamonds (NDs) enables the selection of optimal optical methods or their combination for the study of nanodiamond-skin interaction. Among studied NDs, particles of 100 nm in nominal size were shown to be appropriate for multimodal imaging using all three methods. All the applied NDs were able to cross the skin barrier and penetrate the different layers of the epidermis to finally arrive in the hair follicle niches. The results suggest that NDs have the potential for multifunctional applications utilizing multimodal imaging.

Time-Lapse Technologies and 4D Imaging of Kidney Development.

Time-lapse imaging is a technique of frequent imaging and following a course of a process. Because the development of the embryonic kidney can proceed ex vivo after dissection, it is possible to study the morphogenesis by culturing the kidney in the onstage incubator of a microscope and follow the developmental process by imaging. Confocal microscopes and other three-dimensional imaging systems offer the possibility for tracking the development process in four dimensions-3D and the time.

HNF1B controls epithelial organization and cell polarity during ureteric bud branching and collecting duct morphogenesis.

Kidney development depends crucially on proper ureteric bud branching giving rise to the entire collecting duct system. The transcription factor HNF1B is required for the early steps of ureteric bud branching, yet the molecular and cellular events regulated by HNF1B are poorly understood. We report that specific removal of Hnf1b from the ureteric bud leads to defective cell-cell contacts and apicobasal polarity during the early branching events. High-resolution ex vivo imaging combined with a membranous fluorescent reporter strategy show decreased mutant cell rearrangements during mitosis-associated cell dispersal and severe epithelial disorganization. Molecular analysis reveals downregulation of Gdnf-Ret pathway components and suggests that HNF1B acts both upstream and downstream of Ret signaling by directly regulating Gfra1 and Etv5 Subsequently, Hnf1b deletion leads to massively mispatterned ureteric tree network, defective collecting duct differentiation and disrupted tissue architecture, which leads to cystogenesis. Consistently, mRNA-seq analysis shows that the most impacted genes encode intrinsic cell-membrane components with transporter activity. Our study uncovers a fundamental and recurring role of HNF1B in epithelial organization during early ureteric bud branching and in further patterning and differentiation of the collecting duct system in mouse.

Renal carcinoma/kidney progenitor cell chimera organoid as a novel tumorigenesis gene discovery model.

Three-dimensional (3D) organoids provide a new way to model various diseases, including cancer. We made use of recently developed kidney-organ-primordia tissue-engineering technologies to create novel renal organoids for cancer gene discovery. We then tested whether our novel assays can be used to examine kidney cancer development. First, we identified the transcriptomic profiles of quiescent embryonic mouse metanephric mesenchyme (MM) and of MM in which the nephrogenesis program had been induced ex vivo The transcriptome profiles were then compared to the profiles of tumor biopsies from renal cell carcinoma (RCC) patients, and control samples from the same kidneys. Certain signature genes were identified that correlated in the developmentally induced MM and RCC, including components of the caveolar-mediated endocytosis signaling pathway. An efficient siRNA-mediated knockdown (KD) of Bnip3, Gsn, Lgals3, Pax8, Cav1, Egfr or Itgb2 gene expression was achieved in mouse RCC (Renca) cells. The live-cell imaging analysis revealed inhibition of cell migration and cell viability in the gene-KD Renca cells in comparison to Renca controls. Upon siRNA treatment, the transwell invasion capacity of Renca cells was also inhibited. Finally, we mixed E11.5 MM with yellow fluorescent protein (YFP)-expressing Renca cells to establish chimera organoids. Strikingly, we found that the Bnip3-, Cav1- and Gsn-KD Renca-YFP+ cells as a chimera with the MM in 3D organoid rescued, in part, the RCC-mediated inhibition of the nephrogenesis program during epithelial tubules formation. Altogether, our research indicates that comparing renal ontogenesis control genes to the genes involved in kidney cancer may provide new growth-associated gene screens and that 3D RCC-MM chimera organoids can serve as a novel model with which to investigate the behavioral roles of cancer cells within the context of emergent complex tissue structures.

Novel fixed z-direction (FiZD) kidney primordia and an organoid culture system for time-lapse confocal imaging.

Tissue, organ and organoid cultures provide suitable models for developmental studies, but our understanding of how the organs are assembled at the single-cell level still remains unclear. We describe here a novel fixed z-direction (FiZD) culture setup that permits high-resolution confocal imaging of organoids and embryonic tissues. In a FiZD culture a permeable membrane compresses the tissues onto a glass coverslip and the spacers adjust the thickness, enabling the tissue to grow for up to 12 days. Thus, the kidney rudiment and the organoids can adjust to the limited z-directional space and yet advance the process of kidney morphogenesis, enabling long-term time-lapse and high-resolution confocal imaging. As the data quality achieved was sufficient for computer-assisted cell segmentation and analysis, the method can be used for studying morphogenesis ex vivo at the level of the single constituent cells of a complex mammalian organogenesis model system.

CD146(+) cells are essential for kidney vasculature development.

The kidney vasculature is critical for renal function, but its developmental assembly mechanisms remain poorly understood and models for studying its assembly dynamics are limited. Here, we tested whether the embryonic kidney contains endothelial cells (ECs) that are heterogeneous with respect to VEGFR2/Flk1/KDR, CD31/PECAM, and CD146/MCAM markers. Tie1Cre;R26R(YFP)-based fate mapping with a time-lapse in embryonic kidney organ culture successfully depicted the dynamics of kidney vasculature development and the correlation of the process with the CD31(+) EC network. Depletion of Tie1(+) or CD31(+) ECs from embryonic kidneys, with either Tie1Cre-induced diphtheria toxin susceptibility or cell surface marker-based sorting in a novel dissociation and reaggregation technology, illustrated substantial EC network regeneration. Depletion of the CD146(+) cells abolished this EC regeneration. Fate mapping of green fluorescent protein (GFP)-marked CD146(+)/CD31(-) cells indicated that they became CD31(+) cells, which took part in EC structures with CD31(+) wild-type ECs. EC network development depends on VEGF signaling, and VEGF and erythropoietin are expressed in the embryonic kidney even in the absence of any external hypoxic stimulus. Thus, the ex vivo embryonic kidney culture models adopted here provided novel ways for targeting renal EC development and demonstrated that CD146(+) cells are critical for kidney vasculature development.

Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract.

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4(EGFPCre)-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4(monomeric cherry/monomeric cherry) (Wnt4(mCh/mCh)) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4(mCh/mCh) females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4(mCh/mCh) females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development.

Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

Titania nanofibers in gypsum composites: an antibacterial and cytotoxicology study.

Further developments of antibacterial coatings based on photocatalytic nanomaterials could be a promising route towards potential environmentally friendly applications in households, public buildings and health care facilities. Hereby we describe a simple chemical approach to synthesize photocatalytic nanomaterial-embedded coatings using gypsum as a binder. Various types of TiO2 nanofiber-based photocatalytic materials (nitrogen-doped and/or palladium nanoparticle decorated) and their composites with gypsum were characterized by means of scanning (SEM) and transmission (TEM) electron microscopy as well as electron and X-ray diffraction (XRD) and energy-dispersive X-ray spectroscopy (EDX) techniques. These gypsum-based composites can be directly applied as commercially available paints on indoor walls. Herein we report that surfaces coated with photocatalytic composites exhibit excellent antimicrobial properties by killing both methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) under blue light. In the case of MSSA cells, the palladium nanoparticle-decorated and nitrogen-doped TiO2 composites demonstrated the highest antimicrobial activity. For the MRSA strain even pure gypsum samples were proven to be efficient in eradicating Gram-positive human pathogens. The cytotoxicity of freestanding TiO2 nanofibers was revealed by analyzing the viability of HeLa cells using MTT and fluorescent cell assays.

Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo.

Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low-density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin-expressing cells that have been transduced by a Lodavin-containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock-in Lodavin mouse to develop biotin-based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed ßgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line-derived R26RLodavin mouse line was generated. Efficient removal of the floxed ßgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7-Cre, Wnt4-Cre, or Tie1-Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction.

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development.

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.

Novel perspectives for investigating congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the commonest cause of chronic kidney disease in children. Structural anomalies within the CAKUT spectrum include renal agenesis, kidney hypo-/dysplasia, multicystic kidney dysplasia, duplex collecting system, posterior urethral valves and ureter abnormalities. While most CAKUT cases are sporadic, familial clustering of CAKUT is common, emphasizing a strong genetic contribution to CAKUT origin. Animal experiments demonstrate that alterations in genes crucial for kidney development can cause experimental CAKUT, while expression studies implicate mislocalization and/or aberrant levels of the encoded proteins in human CAKUT. Further insight into the pathogenesis of CAKUT will improve strategies for early diagnosis, follow-up and treatment. Here, we outline a collaborative approach to identify and characterize novel factors underlying human CAKUT. This European consortium will share the largest collection of CAKUT patients available worldwide and undertake multidisciplinary research into molecular and genetic pathogenesis, with extension into translational studies to improve long-term patient outcomes.