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MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle’s loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.

Wingless/Wnts are signalling molecules, traditionally considered to pattern tissues as long-range morphogens. However, more recently the spread of Wingless was shown to be dispensable in diverse developmental contexts in Drosophila and vertebrates. Here we demonstrate that release and spread of Wingless is required to pattern the proximo-distal (P-D) axis of Drosophila Malpighian tubules. Wingless signalling, emanating from the midgut, directly activates odd skipped expression several cells distant in the proximal tubule. Replacing Wingless with a membrane-tethered version that is unable to diffuse from the Wingless producing cells results in aberrant patterning of the Malpighian tubule P-D axis and development of short, deformed ureters. This work directly demonstrates a patterning role for a released Wingless signal. As well as extending our understanding about the functional modes by which Wnts shape animal development, we anticipate this mechanism to be relevant to patterning epithelial tubes in other organs, such as the vertebrate kidney.

The kidney arises from two types of progenitors; here, the signalling conditions that induce the production of collecting ducts and functional nephrons from human pluripotent stem cells are determined, and organoids that recapitulate the functional regionalization of the kidney are produced. Human kidney organoids with all renal components The development of the human kidney in the embryo depends on two different stem cell types, one to generate collecting ducts and the other to generate functional nephrons. Melissa Little, Minoru Takasato and colleagues showed previously that human pluripotent stem cells (hPSCs) can differentiate into both types of progenitors. They have now identified the signalling conditions required to induce not only these structures but also the surrounding cell types including interstitium and blood vessels. Using this approach, they have grown kidney organoids that recapitulate the functional regionalization of the embryonic kidney. The tissue complexity and degree of functionalization achieved in these organoids are not on a par with a working kidney, but replicate the normal human embryonic kidney. Importantly, they provide evidence of their potential in screening drugs for toxicity, modelling genetic kidney disease or perhaps to provide specific kidney cell types for cellular therapy. The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells 1 . Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney’s collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney’s collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system. Here, the authors model the collecting duct system in kidneys by taking ureteric bud (UB) progenitor cells from both mouse and human primary tissues, as well as from hESC and hiPSC to generate organoids, which can model congenital anomalies of the kidney and urinary tract.

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.

Multicellular organisms rely on cell adhesion molecules to coordinate cell–cell interactions, and to provide navigational cues during tissue formation. In Drosophila , Fasciclin 2 (Fas2) has been intensively studied due to its role in nervous system development and maintenance; yet, Fas2 is most abundantly expressed in the adult renal (Malpighian) tubule rather than in neuronal tissues. The role Fas2 serves in this epithelium is unknown. Here we show that Fas2 is essential to brush border maintenance in renal tubules of Drosophila . Fas2 is dynamically expressed during tubule morphogenesis, localizing to the brush border whenever the tissue is transport competent. Genetic manipulations of Fas2 expression levels impact on both microvilli length and organization, which in turn dramatically affect stimulated rates of fluid secretion by the tissue. Consequently, we demonstrate a radically different role for this well-known cell adhesion molecule, and propose that Fas2-mediated intermicrovillar homophilic adhesion complexes help stabilize the brush border. In Drosophila , Fasciclin 2 (Fas2) has been mainly studied in the nervous system, yet this adhesion protein is more abundant in the adult renal tubule. Here the authors show that Fas2 is essential for brush border maintenance in renal tubules through regulation of microvilli length and organization.

Polycystin-1, which is encoded by a gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), is involved in cell-matrix interactions as well as in ciliary signaling. The precise mechanisms by which it functions, however, remain unclear. Here we find that polycystin-1 undergoes a proteolytic cleavage that releases its C-terminal tail (CTT), which enters the nucleus and initiates signaling processes. The cleavage occurs in vivo in association with alterations in mechanical stimuli. Polycystin-2, the product of the second gene mutated in ADPKD, modulates the signaling properties of the polycystin-1 CTT. These data reveal a novel pathway by which polycystin-1 transmits messages directly to the nucleus.

Recent advances in renal tissue engineering have shown that dissociated, early renogenic tissue from the developing embryo can self-assemble into morphologically accurate kidney-like organs arranged around a central collecting duct tree. In order for such self-assembled kidneys to be useful therapeutically or as models for drug screening, it is necessary to demonstrate that they are functional. One of the main functional characteristics of mature kidneys is transport of organic anions and cations into and out of the proximal tubule. Here, we show that the transport function of embryonic kidneys allowed to develop in culture follows a developmental time-course that is comparable to embryonic kidney development in vivo . We also demonstrate that serially-reaggregated engineered kidneys can transport organic anions and cations through specific uptake and efflux channels. These results support the physiological relevance of kidneys grown in culture, a commonly used model for kidney development and research and suggest that serially-reaggregated kidneys self-assembled from separated cells have some functional characteristics of intact kidneys.

The kidney vasculature facilitates the excretion of wastes, the dissemination of hormones, and the regulation of blood chemistry. To carry out these diverse functions, the vasculature is regionalized within the kidney and along the nephron. However, when and how endothelial regionalization occurs remains unknown. Here, we examine the developing kidney vasculature to assess its 3-dimensional structure and transcriptional heterogeneity. First, we observe that endothelial cells (ECs) grow coordinately with the kidney bud as early as E10.5, and begin to show signs of specification by E13.5 when the first arteries can be identified. We then focus on how ECs pattern and remodel with respect to the developing nephron and collecting duct epithelia. ECs circumscribe nephron progenitor populations at the distal tips of the ureteric bud (UB) tree and form stereotyped cruciform structures around each tip. Beginning at the renal vesicle (RV) stage, ECs form a continuous plexus around developing nephrons. The endothelial plexus envelops and elaborates with the maturing nephron, becoming preferentially enriched along the early distal tubule. Lastly, we perform transcriptional and immunofluorescent screens to characterize spatiotemporal heterogeneity in the kidney vasculature and identify novel regionally enriched genes. A better understanding of development of the kidney vasculature will help instruct engineering of properly vascularized ex vivo kidneys and evaluate diseased kidneys.

Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.