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Background: The prorenin receptor (PRR) plays a critical role in ureteric bud (UB) branching morphogenesis. DOT1 Like (DOT1L), a histone methyltransferase specific for Histone 3 lysine 79 (H3K79), is important for differentiation of the UB-derived renal collecting duct cells. In this study, we tested whether DOT1L/H3 dimethyl K79 (H3m2K79) are regulated by PRR deletion in the UB and UB-derived collecting ducts in the embryonic mouse kidneys., Methods: Mutant Hoxb7 Cre+ /PRR flox/flox (PRR UB-/- ) and control PRR UB+/+ , mice were studied on embryonic (E) day E17.5. DOT1L mRNA and protein expression in the kidney was examined by real-time qRT-PCR and immunohistochemistry, respectively. H3m2K79 protein expression was determined by immunohistochemistry and Western blot analysis., Results: DOT1L mRNA levels were decreased in mutant compared to control mice (0.68 ± 0.06 vs. 1.0 ± 0.01, p < 0.01). DOT1L and H3m2K79 immunostaining was reduced in the mutant vs. control kidneys (Dot1: 0.62 ± 0.03 vs. 1.0 ± 0.01, p < 0.05; H3m2K79: 0.64 ± 0.04 vs.1.1 ± 0.01. p < 0.05.). Western blot analysis revealed decreased H3m2K79 protein levels in mutant compared to control kidneys (1.0 ± 0.06 vs. 1.5 ± 0.02, p < 0.05)., Conclusion: Targeted deletion of the PRR in the UB and UB-derived collecting ducts results in reduced DOT1L gene/protein and H3m2K79 protein expression in the embryonic mouse metanephroi in vivo., Impact: The role of histone methylation in mediating the effect of the prorenin receptor on the ureteric bud branching (UB) morphogenesis and urine acidification during kidney development is unknown. We demonstrate that histone H3 lysine (K) 79 dimethylation by methyltransferase Dot1 is reduced in the embryonic kidney of mice that lack the prorenin receptor in the UB lineage., (© 2024. The Author(s).)

Collagen XVIII (ColXVIII) is a component of the extracellular matrix implicated in embryogenesis and control of tissue homoeostasis. We now provide evidence that ColXVIII has a specific role in renal branching morphogenesis as observed in analyses of total and isoform-specific knockout embryos and mice. The expression of the short and the two longer isoforms differ temporally and spatially during renal development. The lack of ColXVIII or its specific isoforms lead to congenital defects in the 3D patterning of the ureteric tree where the short isoform plays a prominent role. Moreover, the ex vivo data suggests that ColXVIII is involved in the kidney epithelial tree patterning via its N-terminal domains, and especially the Thrombospondin-1-like domain common to all isoforms. This morphogenetic function likely involves integrins expressed in the ureteric epithelium. Altogether, the results point to an important role for ColXVIII in the matrix-integrin-mediated functions regulating renal development., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oxidative metabolism in mitochondria regulates cellular differentiation and gene expression through intermediary metabolites and reactive oxygen species. Its role in kidney development and pathogenesis is not completely understood. Here we inactivated ubiquinone-binding protein QPC, a subunit of mitochondrial complex III, in two types of kidney progenitor cells to investigate the role of mitochondrial electron transport in kidney homeostasis. Inactivation of QPC in sine oculis-related homeobox 2 (SIX2)-expressing cap mesenchyme progenitors, which give rise to podocytes and all nephron segments except collecting ducts, resulted in perinatal death from severe kidney dysplasia. This was characterized by decreased proliferation of SIX2 progenitors and their failure to differentiate into kidney epithelium. QPC inactivation in cap mesenchyme progenitors induced activating transcription factor 4-mediated nutritional stress responses and was associated with a reduction in kidney tricarboxylic acid cycle metabolites and amino acid levels, which negatively impacted purine and pyrimidine synthesis. In contrast, QPC inactivation in ureteric tree epithelial cells, which give rise to the kidney collecting system, did not inhibit ureteric differentiation, and resulted in the development of functional kidneys that were smaller in size. Thus, our data demonstrate that mitochondrial oxidative metabolism is critical for the formation of cap mesenchyme-derived nephron segments but dispensable for formation of the kidney collecting system. Hence, our studies reveal compartment-specific needs for metabolic reprogramming during kidney development., (Copyright © 2022 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Congenital anomalies of the kidney and urinary tract (CAKUT) are a family of often-concurrent diseases with various anatomical spectra. Null-mutant Gen1 mice frequently develop multiple urinary phenotypes, most commonly duplex kidneys, and are ideal subjects for research on ectopic budding in CAKUT development. The upper and lower kidney poles of the Gen1 PB/PB mouse were examined by histology, immunofluorescence, and immunohistochemistry. The newborn Gen1 PB/PB mouse lower poles were significantly more hypoplastic than the corresponding upper poles, with significantly fewer glomeruli. On embryonic day 14.5, immediately before first urine formation, the upper pole kidney was already larger than the lower pole kidney. In vivo and in vitro, embryonic kidney upper poles had more ureteric buds than lower poles. Gen1 PB/PB embryos exhibited ectopic ureteric buds, usually near the original budding site, occasionally far away, or, rarely, derived from the primary budding site. Therefore, ectopia of the ureteric buds is the core of CAKUT formation. Further studies will be needed to investigate the regulatory roles of these genes in initial ureteric budding and subsequent ontogenesis during metanephros development., Competing Interests: Declaration of competing interest This study has no competing financial interests exist., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed the binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18 and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The patterned array of basal, intermediate and superficial cells in the urothelium of the mature ureter arises from uncommitted epithelial progenitors of the distal ureteric bud. Urothelial development requires signaling input from surrounding mesenchymal cells, which, in turn, depend on cues from the epithelial primordium to form a layered fibro-muscular wall. Here, we have identified FGFR2 as a crucial component in this reciprocal signaling crosstalk in the murine ureter. Loss of Fgfr2 in the ureteric epithelium led to reduced proliferation, stratification, intermediate and basal cell differentiation in this tissue, and affected cell survival and smooth muscle cell differentiation in the surrounding mesenchyme. Loss of Fgfr2 impacted negatively on epithelial expression of Shh and its mesenchymal effector gene Bmp4. Activation of SHH or BMP4 signaling largely rescued the cellular defects of mutant ureters in explant cultures. Conversely, inhibition of SHH or BMP signaling in wild-type ureters recapitulated the mutant phenotype in a dose-dependent manner. Our study suggests that FGF signals from the mesenchyme enhance, via epithelial FGFR2, the SHH-BMP4 signaling axis to drive urothelial and mesenchymal development in the early ureter., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The kidneys are essential organs that filter the blood, removing urinary waste while maintaining fluid and electrolyte homeostasis. Current conventional research models such as static cell cultures and animal models are insufficient to grasp the complex human in vivo situation or lack translational value. To accelerate kidney research, novel research tools are required. Recent developments have allowed the directed differentiation of induced pluripotent stem cells to generate kidney organoids. Kidney organoids resemble the human kidney in vitro and can be applied in regenerative medicine and as developmental, toxicity, and disease models. Although current studies have shown great promise, challenges remain including the immaturity, limited reproducibility, and lack of perfusable vascular and collecting duct systems. This review gives an overview of our current understanding of nephrogenesis that enabled the generation of kidney organoids. Next, the potential applications of kidney organoids are discussed followed by future perspectives. This review proposes that advancement in kidney organoid research will be facilitated through our increasing knowledge on nephrogenesis and combining promising techniques such as organ-on-a-chip models.

Development of the mammalian kidney is orchestrated by reciprocal interactions of stromal and nephrogenic mesenchymal cells with the ureteric bud epithelium. Previous work showed that the transcription factor Wilms tumor 1 (WT1) acts in the nephrogenic lineage to maintain precursor cells, to drive the epithelial transition of aggregating precursors into a renal vesicle and to specify and maintain the podocyte fate. However, WT1 is expressed not only in the nephrogenic lineage but also transiently in stromal progenitors in the renal cortex. Here we report that specific deletion of Wt1 in the stromal lineage using the Foxd1 cre driver line results at birth in cryptorchidism and hypoplastic kidneys that harbour fewer and enlarged ureteric bud tips and display an expansion of capsular stroma into the cortical region. In vivo and ex vivo analysis at earlier stages revealed that stromal loss of Wt1 reduces stromal proliferation, and delays and alters branching morphogenesis, resulting in a variant architecture of the collecting duct tree with an increase of single at the expense of bifurcated ureteric bud tips. Molecular analysis identified a transient reduction of Aldh1a2 expression and of retinoic acid signalling activity in stromal progenitors, and of Ret in ureteric bud tips. Administration of retinoic acid partly rescued the branching defects of mutant kidneys in culture. We propose that WT1 maintains retinoic acid signalling in the cortical stroma, which, in turn, assures proper levels and dynamics of Ret expression in the ureteric bud tips, and thus normal ramification of the ureteric tree. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland., (© 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.)

Congenital anomalies of external genitalia affect approximately 1 in 125 live male births. Development of the genital tubercle, the precursor of the penis and clitoris, is regulated by the urethral plate epithelium, an endodermal signaling center. Signaling activity of the urethral plate is mediated by Sonic hedgehog (SHH), which coordinates outgrowth and patterning of the genital tubercle by controlling cell cycle kinetics and expression of downstream genes. The mechanisms that govern Shh transcription in urethral plate cells are largely unknown. Here we show that deletion of Foxa1 and Foxa2 results in persistent cloaca, an incomplete separation of urinary, genital, and anorectal tracts, and severe hypospadias, a failure of urethral tubulogenesis. Loss of Foxa2 and only one copy of Foxa1 results in urethral fistula, an additional opening of the penile urethra. Foxa1/a2 participate in an autoregulatory feedback loop with Shh, in which FOXA1 and FOXA2 positively regulate transcription of Shh in the urethra, and SHH feeds back to negatively regulate Foxa1 and Foxa2 expression. These findings reveal novel roles for Foxa genes in development of the urethral tube and in division of the embryonic cloaca., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Congenital anomalies of the urinary tract are a significant cause of morbidity in infancy, and many congenital anomalies are linked to ureter development; however, the mechanism by which congenital anomalies control ureter development remains unknown. The loss of Robo2 can cause ureter defects and vesicoureteral reflux. However, how Robo2 impacts ureter development is unclear. We found that ROBO2 is expressed in the common nephric duct (CND) and primitive bladder, and impacts CND migration and fusion with the primitive bladder via its novel binding partner retinaldehyde dehydrogenase-2 (RALDH2). Delayed apoptosis that is due to the failure of CND fusion with the primitive bladder in the Robo2 -/- embryo results in an abnormal ureter connection to the CND, which is required for ureter development. We define a novel pathway in which the CND is remodeled by ROBO2 and retinoic acid rescued the ureter anomalies in the Robo2 -/- embryo. These findings may be relevant to diverse disease conditions that are associated with altered signaling in the primitive bladder., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously reported that maternal nutrient restriction (NR) inhibited ureteric branching, metanephric growth, and nephrogenesis in the rat. Here we examined whether folic acid, a methyl-group donor, rescues the inhibition of kidney development induced by NR and whether DNA methylation is involved in it. The offspring of dams given food ad libitum (CON) and those subjected to 50% food restriction (NR) were examined. NR significantly reduced ureteric tip number at embryonic day 14, which was attenuated by folic acid supplementation to nutrient restricted dams. At embryonic day 18, glomerular number, kidney weight, and global DNA methylation were reduced by NR, and maternal folic acid supplementation again alleviated them. Among DNA methyltransferases (DNMTs), DNMT1 was strongly expressed at embryonic day 15 in CON but was reduced in NR. In organ culture, an inhibitor of DNA methylation 5-aza-2 '-deoxycytidine as well as medium lacking methyl donors folic acid, choline, and methionine, significantly decreased ureteric tip number and kidney size mimicking the effect of NR. In conclusion, global DNA methylation is necessary for normal kidney development. Folic acid supplementation to nutrient restricted dams alleviated the impaired kidney development and DNA methylation in the offspring., Competing Interests: The authors have declared that no competing interests exist.

The ureteric epithelial progenitor (UEP) population within the embryonic kidney generates the arborized epithelial network of the kidney's collecting system and plays a critical role in the expansion and induction of the surrounding nephron progenitor pool. Adamts18 shows UEP- restricted expression in the kidney and progenitor tip-restricted expression in several other organs undergoing branching epithelial growth. Adamts18 is encoded by 23 exons. Genetic removal of genomic sequence spanning exons 1 to 3 led to a specific loss of Adamts18 expression in UEPs, suggesting this region may encode a UEP-specific enhancer. Intron 2 (3 ​kb) was shown to have enhancer activity driving expression of the doxycycline inducible tet-on transcriptional regulator (rtTA) in an Adamts18en-rtTA transgenic mouse strain. Crossing Adamts18en-rtTA mice to a doxycycline dependent GFP reporter mouse enabled the live imaging of embryonic kidney explants. This facilitated the analysis of ureteric epithelial branching events at the cellular level. Ablation of UEPs at the initiation of ureteric bud outgrowth through the doxycycline-mediated induction of Diphtheria Toxin A (DTA) generated a range of phenotypes from complete kidneys agenesis, to duplex kidneys with double ureters. The latter outcome points to the potential of regulative processes to restore UEPs. In contrast, overexpression of YAP prior to ureteric bud outgrowth led to a complete failure of kidney development. Elevating YAP levels at later stages retarded branching growth. A similar phenotype was observed with the overexpression of MYC within the branch-tip localized UEP population. These experiments showcase the utility of the Adamts18en-rtTA transgenic model to the investigation of cellular and molecular events specific to branch tip progenitors within the mammalian kidney complementing existing CRE-dependent genetic tools. Further, the illustrative examples point to areas where new insight may be gained into the regulation of UEP programs., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The establishment of the peristaltic machinery of the ureter is precisely controlled to cope with the onset of urine production in the fetal kidney. Retinoic acid (RA) has been identified as a signal that maintains the mesenchymal progenitors of the contractile smooth muscle cells (SMCs), while WNTs, SHH, and BMP4 induce their differentiation. How the activity of the underlying signalling pathways is controlled in time, space, and quantity to activate coordinately the SMC programme is poorly understood. Here, we provide evidence that the Zn-finger transcription factor GATA2 is involved in this crosstalk. In mice, Gata2 is expressed in the undifferentiated ureteric mesenchyme under control of RA signalling. Conditional deletion of Gata2 by a Tbx18 cre driver results in hydroureter formation at birth, associated with a loss of differentiated SMCs. Analysis at earlier stages and in explant cultures revealed that SMC differentiation is not abrogated but delayed and that dilated ureters can partially regain peristaltic activity when relieved of urine pressure. Molecular analysis identified increased RA signalling as one factor contributing to the delay in SMC differentiation, possibly caused by reduced direct transcriptional activation of Cyp26a1, which encodes an RA-degrading enzyme. Our study identified GATA2 as a feedback inhibitor of RA signalling important for precise onset of ureteric SMC differentiation, and suggests that in a subset of cases of human congenital ureter dilatations, temporary relief of urine pressure may ameliorate the differentiation status of the SMC coat. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Recent advances in the generation of kidney organoids and the culture of primary nephron progenitors from mouse and human have been based on knowledge of the molecular basis of kidney development in mice. Although gene expression during kidney development has been intensely investigated, single cell profiling provides new opportunities to further subsect component cell types and the signalling networks at play. Here, we describe the generation and analysis of 6732 single cell transcriptomes from the fetal mouse kidney [embryonic day (E)18.5] and 7853 sorted nephron progenitor cells (E14.5). These datasets provide improved resolution of cell types and specific markers, including subdivision of the renal stroma and heterogeneity within the nephron progenitor population. Ligand-receptor interaction and pathway analysis reveals novel crosstalk between cellular compartments and associates new pathways with differentiation of nephron and ureteric epithelium cell types. We identify transcriptional congruence between the distal nephron and ureteric epithelium, showing that most markers previously used to identify ureteric epithelium are not specific. Together, this work improves our understanding of metanephric kidney development and provides a template to guide the regeneration of renal tissue., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The etiology of ureterovesical junction obstruction (UVJO) and ureteropelvic junction obstruction (UPJO) is obscure with an adynamic narrow segment causing the obstruction. In this study, the authors compared interstitial cells of Cajal (ICC) and collagen-to-muscle ratio (CM ratio) between UVJO, UPJO, and fetal ureters to investigate whether a maturational arrest of the fetal ureter could explain both clinical pathologies., Methods: Group 1 (control) involved specimens of the normal ureter (nephrectomy for trauma/tumor; n = 20), while group 2, specimens of UVJO (n = 14); group 2 was further divided into group 2a, the dilated megaureter above UVJO, and group 2b, UVJO narrow segment; group 3, UPJO narrow segment excised during pyeloplasty (n = 31); and group 4, normal fetal ureters (n = 12). The specimens were analyzed for ICC using immunohistochemistry and CM ratio on Masson's trichrome (stains collagen in blue and muscle in red)., Results: The median ICC/10 high-power field was 16.1 (8.3) in the normal and 17.3 (7.9) in the dilated segment of the megaureter, with no significant difference, but was significantly less in the narrow segment of UVJO at 4.5 (2.0), narrow segment of UPJO at 5.1 (2.3), and fetal ureter at 5.0 (2.3). The median CM ratio was 0.75 (0.29) in the normal and 0.65 (0.2) in the dilated segment of the megaureter, with no significant difference between them (figure), but was significantly higher in the narrow segment of UVJO at 3.0 (0.8), narrow segment of UPJO at 2.5 (0.71), and fetal ureter at 3.1 (0.61). Overall UVJO, UPJO, and fetal ureter segment had significantly less ICC density and more collagen compared with the normal ureter (P < 0.001 by Mann-Whitney U test)., Discussion: There are conflicting reports on the etiopathogenesis of UVJO and UPJO, with several authors showing decreased ICC and increased collagen in the narrow segment. In this study, the authors found that the pathological changes at UVJ and UPJ segments resemble fetal ureter morphology. We also found that in fetal ureters, as the gestation progressed, there was an increase in the ICC density/smooth muscle, whereas the collagen content decreased. While the entire ureter has uniform embryological origin, it essentially remains an epithelial tube until the late gestation. The maturational process involves differentiation of smooth muscles cells/ICC to establish the peristaltic machinery required to functionally connect the ureter at both ends. This process, probably, starts at the mid ureter during fetal life and extends toward the UPJ and UVJ, and its failure, probably, results in UPJO or UVJO. The study's limitations are small numbers, and further larger studies are required to validate this hypothesis., (Copyright © 2019 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf    hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3'UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.

Recently, the cellular origin of the connecting tubule (CNT) has been genetically characterized. The CNT is a segment between two embryonically different structures, the collecting duct originating from ureteric bud (UB), and the nephron derived from the cap mesenchyme. However, the cellular detail at the initial connection is limited. The present study demonstrated that the initial connection was composed of cells which were closely associated with the renal vesicle (RV), the initial nephron, and connected with the basal epithelium of the terminal UB tip at discrete points. The identification of the RV and UB tip was based on tracing of tubules on serial epoxy sections at mouse embryonic day 17.5. The cells at the initial connection were characterized by 1) irregularly-shaped nuclei and cells with cytoplasmic processes, 2) electron dense nuclei, 3) abundant intercellular spaces, 4) extensive cell-cell contacts with cell junctions, often zonulae adherences and occasionally focal fusion of opposing plasma membranes, and 5) numerous mitochondria, densely packed rosette-like polyribosomes, and widespread rER in the cytoplasm. Moreover, the tracing revealed that a terminal UB tip frequently connected to two nephrons at different developing stages. The UB tips, the initial connections, and the distal tubules of the S-shaped bodies did not express Na + -Cl - cotransporter, H + -ATPase, or aquaporin 2, while they were expressed in immature CNT of the capillary-loop stage nephrons throughout the kidney development. Consequently, the cells at the initial connection exhibit the morphological features suggestive of energy demanding, protein producing, and intercellular communicating. The cell morphology together with transporter development indicates that these cells serve several functions during the development of the initial connection, and that these functions are different from the cells' final functions as transportation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The ability to explant and then maintain embryonic tissues in organ culture makes it feasible to study the growth and differentiation of whole organs, or parts or combinations of them, in three dimensions. Moreover, the possible effects of biochemical manipulations or mutations can be explored by visualizing a growing organ. The mammalian renal tract comprises the kidney, ureter, and urinary bladder, and the focus of this chapter is organ culture of the embryonic mouse ureter in serum-free defined medium. Over the culture period, rudiments grow in length, smooth muscle differentiates, and the ureters then undergo peristalsis in a proximal to distal direction.

Ureteric bud branching and nephrogenesis are performed through large-scale proliferation and apoptosis events during renal development. Reactive oxygen species (ROS), produced by NADPH oxidase, may contribute to cell behaviors, including proliferation and apoptosis. We investigated the role of NADPH oxidase expression and ROS production in developing kidneys. Immunohistochemistry revealed that NADPH oxidase componentswere expressed on epithelial cells in ureteric bud branches, as well as on immature glomerular cells and epithelial cells in nephrogenic zones. ROS production, detected by dihydroethidium assay, was strongly observed in ureteric bud branches and nephrogenic zones, corresponding with NADPH oxidase localization. Organ culture of E14 kidneys revealed that the inhibition of NADPH oxidase significantly reduced the number of ureteric bud branches and tips, consistent with reduced ROS production. This was associated with reduced expression of phosphorylated ERK1/2 and increased expression of cleaved caspase-3. Organ culture of E18 kidneys showed that the inhibition of NADPH oxidase reduced nephrogenic zone size, accompanied by reduced ROS production, fewer proliferating cell nuclear antigen-positive cells, lower p-ERK1/2 expression, and increased expression of cleaved caspase-3. These results demonstrate that ROS produced by NADPH oxidase might play an important role in ureteric bud branching and nephrogenesis by regulating proliferation and apoptosis. J.Med. Invest. 66 :93-98, February, 2019.

Background: The kidney develops from an intimate interaction between the ureteric bud and the metanephric mass. We attempted to differentially stain the derivatives of the ureteric bud and the metanephric mass in ovine fetuses., Methods: After appropriate approval, 47 fetal lambs' kidneys at 50 (4), 60 (6), 70 (5), 80 (4), 100 (10), 110 (8), 145 (10) days' gestation (term is 140-145 days) were obtained. After confirming the pregnancy, the sheep were anesthetized, and the fetuses sacrificed. The fetal kidneys were prepared for histological examination, using immunostaining for β-catenin, Laminin, CK34βE12, CK7, E-cadherin, and EMA., Results: In the nephrogenic zone, positive staining was only seen for β-catenin and Laminin. Areas with linear β-catenin expression increased with increasing gestational age, whereas cytoplasmic granular expression in the nephrogenic zone diminished. At 50 days, Laminin-positive cells appeared in the ureteric bud epithelial cells, but not in the proximal tubule epithelium. They were found only in the immature collecting duct at 60 days., Conclusion: We have shown that the distribution of β-catenin and Laminin positive-stained cells initially appearing in the ureteric bud changes with gestational age. Further studies may help inform the optimal timing of fetal shunt insertion in obstructive uropathy., (Copyright © 2018. Published by Elsevier Inc.)

The organized array of smooth muscle cells (SMCs) and fibroblasts in the walls of visceral tubular organs arises by patterning and differentiation of mesenchymal progenitors surrounding the epithelial lumen. Here, we show that the TBX2 and TBX3 transcription factors have novel and required roles in regulating these processes in the murine ureter. Co-expression of TBX2 and TBX3 in the inner mesenchymal region of the developing ureter requires canonical WNT signaling. Loss of TBX2/TBX3 in this region disrupts activity of two crucial drivers of the SMC program, Foxf1 and BMP4 signaling, resulting in decreased SMC differentiation and increased extracellular matrix. Transcriptional profiling and chromatin immunoprecipitation experiments revealed that TBX2/TBX3 directly repress expression of the WNT antagonists Dkk2 and Shisa2 , the BMP antagonist Bmper and the chemokine Cxcl12 These findings suggest that TBX2/TBX3 are effectors of canonical WNT signaling in the ureteric mesenchyme that promote SMC differentiation by maintaining BMP4 and WNT signaling in the inner region, while restricting CXCL12 signaling to the outer layer of fibroblast-fated mesenchyme., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Branching morphogenesis of the ureteric bud is integral to kidney development; establishing the collecting ducts of the adult organ and driving organ expansion via peripheral interactions with nephron progenitor cells. A recent study suggested that termination of tip branching within the developing kidney involved stochastic exhaustion in response to nephron formation, with such a termination event representing a unifying developmental process evident in many organs. To examine this possibility, we have profiled the impact of nephron formation and maturation on elaboration of the ureteric bud during mouse kidney development. We find a distinct absence of random branch termination events within the kidney or evidence that nephrogenesis impacts the branching program or cell proliferation in either tip or progenitor cell niches. Instead, organogenesis proceeds in a manner indifferent to the development of these structures. Hence, stochastic cessation of branching is not a unifying developmental feature in all branching organs., Competing Interests: KS, AC, VL, JL, LJ, NH, IS No competing interests declared, ML has consulted for and received research funding from Organovo Inc., (© 2018, Short et al.)

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

Background: Vesicle-associated membrane protein 5 (VAMP5) is a member of the SNARE protein family, which regulates the docking and fusion of membrane vesicles within cells. Previously, we reported ubiquitous expression of VAMP5 proteins in various organs except the brain and small intestine. However, the precise roles of VAMP5 in each organ remain unclear. To explore the roles of VAMP5 in vivo, we generated VAMP5 knockout (KO) mice., Results: VAMP5 KO mice showed low birth rate and low body weight. KO embryos grew normally in the uterus, and tended to die around birth. Anatomical analysis revealed that viable KO mice often exhibited duplication of the ureter, and dead KO mice showed insufficient expansion of the lung. VAMP5 was localized in the epithelial cells of the ureter and terminal bronchiole., Conclusions: VAMP5 KO mice showed a low birth rate and abnormalities of the urinary and respiratory systems. VAMP5 KO mice died around birth, possibly due to defects in vesicoureteral flow and breathing. The results presented could provide a basis for future studies to understand the roles of VAMP5 protein. Developmental Dynamics 247:754-762, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Nonobstructive hydronephrosis, defined as dilatation of the renal pelvis with or without dilatation of the ureter, is the most common antenatal abnormality detected by fetal ultrasound. Yet, the etiology of nonobstructive hydronephrosis is poorly defined. We previously demonstrated that defective development of urinary tract pacemaker cells (utPMCs) expressing hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) and the stem cell marker cKIT causes abnormal ureteric peristalsis and nonobstructive hydronephrosis. However, further investigation of utPMC development and function is limited by lack of knowledge regarding the embryonic derivation, development, and molecular apparatus of these cells. Here, we used lineage tracing in mice to identify cells that give rise to utPMCs. Neural crest cells (NCCs) indelibly labeled with tdTomato expressed HCN3 and cKIT. Furthermore, purified HCN3+ and cKIT+ utPMCs were enriched in Sox10 and Tfap-2α , markers of NCCs. Sequencing of purified RNA from HCN3+ cells revealed enrichment of a small subset of RNAs, including RNA encoding protein kinase 2 β (PTK2 β ), a Ca 2+ -dependent tyrosine kinase that regulates ion channel activity in neurons. Immunofluorescence analysis in situ revealed PTK2 β expression in NCCs as early as embryonic day 12.5 and in HCN3+ and cKIT+ utPMCs as early as embryonic day 15.5, with sustained expression in HCN3+ utPMCs until postnatal week 8. Pharmacologic inhibition of PTK2 β in murine pyeloureteral tissue explants inhibited contraction frequency. Together, these results demonstrate that utPMCs are derived from NCCs, identify new markers of utPMCs, and demonstrate a functional contribution of PTK2 β to utPMC function., (Copyright © 2018 by the American Society of Nephrology.)

The epithelial Wolffian duct (WD) inserts into the cloaca (primitive bladder) before metanephric kidney development, thereby establishing the initial plumbing for eventual joining of the ureters and bladder. Defects in this process cause common anomalies in the spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). However, developmental, cellular, and molecular mechanisms of WD-cloaca fusion are poorly understood. Through systematic analysis of early WD tip development in mice, we discovered that a novel process of spatiotemporally regulated apoptosis in WD and cloaca was necessary for WD-cloaca fusion. Aberrant RET tyrosine kinase signaling through tyrosine (Y) 1062, to which PI3K- or ERK-activating proteins dock, or Y1015, to which PLC γ docks, has been shown to cause CAKUT-like defects. Cloacal apoptosis did not occur in RetY1062F mutants, in which WDs did not reach the cloaca, or in RetY1015F mutants, in which WD tips reached the cloaca but did not fuse. Moreover, inhibition of ERK or apoptosis prevented WD-cloaca fusion in cultures, and WD-specific genetic deletion of YAP attenuated cloacal apoptosis and WD-cloacal fusion in vivo Thus, cloacal apoptosis requires direct contact and signals from the WD tip and is necessary for WD-cloacal fusion. These findings may explain the mechanisms of many CAKUT., (Copyright © 2018 by the American Society of Nephrology.)

Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species., (Copyright © 2018 by the American Society of Nephrology.)

Intrinsic ureteropelvic junction obstruction is the most common cause of congenital hydronephrosis, yet the underlying pathogenesis is undefined. Hedgehog proteins control morphogenesis by promoting GLI-dependent transcriptional activation and inhibiting the formation of the GLI3 transcriptional repressor. Hedgehog regulates differentiation and proliferation of ureteric smooth muscle progenitor cells during murine kidney-ureter development. Histopathologic findings of smooth muscle cell hypertrophy and stroma-like cells, consistently observed in obstructing tissue at the time of surgical correction, suggest that Hedgehog signaling is abnormally regulated during the genesis of congenital intrinsic ureteropelvic junction obstruction. Here, we demonstrate that constitutively active Hedgehog signaling in murine intermediate mesoderm-derived renal progenitors results in hydronephrosis and failure to develop a patent pelvic-ureteric junction. Tissue obstructing the ureteropelvic junction was marked as early as E13.5 by an ectopic population of cells expressing Ptch2 , a Hedgehog signaling target. Constitutive expression of GLI3 repressor in Ptch1- deficient mice rescued ectopic Ptch2 expression and obstructive hydronephrosis. Whole transcriptome analysis of isolated Ptch2 + cells revealed coexpression of genes characteristic of stromal progenitor cells. Genetic lineage tracing indicated that stromal cells blocking the ureteropelvic junction were derived from intermediate mesoderm-derived renal progenitors and were distinct from the smooth muscle or epithelial lineages. Analysis of obstructive ureteric tissue resected from children with congenital intrinsic ureteropelvic junction obstruction revealed a molecular signature similar to that observed in Ptch1 -deficient mice. Together, these results demonstrate a Hedgehog-dependent mechanism underlying mammalian intrinsic ureteropelvic junction obstruction., (Copyright © 2018 by the American Society of Nephrology.)

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Metanephric kidney development is orchestrated by the iterative branching morphogenesis of the ureteric bud. We describe an underlying patterning associated with the ramification of this structure and show that this pattern is conserved between developing kidneys, in different parts of the organ and across developmental time. This regularity is associated with a highly reproducible branching asymmetry that is consistent with locally operative growth mechanisms. We then develop a class of tip state models to represent elaboration of the ureteric tree and describe rules for 'half-delay' branching morphogenesis that describe almost perfectly the patterning of this structure. Spatial analysis suggests that the observed asymmetry may arise from mutual suppression of bifurcation, but not extension, between the growing ureteric tips, and demonstrates that disruption of patterning occurs in mouse mutants in which the distribution of tips on the surface of the kidney is altered. These findings demonstrate that kidney development occurs by way of a highly conserved reiterative pattern of asymmetric bifurcation that is governed by intrinsic and locally operative mechanisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe conditionally deleted Frs2α in peri-Wolffian duct ST with a Tbx18cre mouse line (Frs2α ST-/- ). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions by three-dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2α ST-/- embryos had many displaced ureteric bud induction sites when compared with controls. E11.0 Frs2α ST-/- embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2α ST-/- mice had higher VUR rates and grades vs., Controls: Mutant refluxing ureters that inserted improperly into the bladder had shortened intravesicular tunnels (IVTs) when compared with controlsConclusionFrs2α ST-/- embryos have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths, and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits; crucial integrins in the kidney collecting system express the β1 subunit. The β1 cytoplasmic tail has two NPxY motifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the β1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the β1 integrin subunit NPxY motif., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Competing Interests: Competing interests: None declared.

Odd-skipped related 1 (Osr1) is a transcriptional repressor that plays critical roles in maintaining the mesenchymal stem cell population within the developing kidney. Here, we report that newborn pups with a heterozygous null mutation in Osr1 exhibit a 21% incidence of vesicoureteric reflux and have hydronephrosis and urinary tract duplications. Newborn pups have a short intravesical ureter, resulting in a less competent ureterovesical junction which arises from a delay in urinary tract development. We describe a new domain of Osr1 expression in the ureteral mesenchyme and within the developing bladder in the mouse. OSR1 was sequenced in 186 children with primary vesicoureteric reflux, and 17 have single nucleotide polymorphisms. Fifteen children have a common synonymous variant, rs12329305, one child has a rare nonsynonymous variant, rs3440471, and one child has a rare 5'-UTR variant, rs45535040 The impact of these SNPs is not clear; therefore, the role of OSR1 in human disease remains to be elucidated. Osr1 is a candidate gene implicated in the pathogenesis of vesicoureteric reflux and congenital abnormalities of the kidney and urinary tract in mice., (Copyright © 2017 the American Physiological Society.)

BackgroundWe tested the hypothesis that Foxd1, a transcription factor essential for normal kidney development, is an upstream regulator of the renin-angiotensin system (RAS) during ureteric bud (UB)-branching morphogenesis.MethodsUB branching, RAS gene, and protein expression were studied in embryonic mouse kidneys. RAS mRNA expression was studied in mesenchymal MK4 cells.ResultsThe number of UB tips was reduced in Foxd1 -/- compared with that in Foxd1 +/+ metanephroi on embryonic day E12.5 (14±2.1 vs. 28±1.3, P<0.05). Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that renin, angiotensin I-converting enzyme (ACE), and angiotensin (Ang) II receptor type 1 (AT 1 R) mRNA levels were decreased in Foxd1 -/- compared with those in Foxd1 +/+ E14.5 metanephroi. Western blot analysis and immunohistochemistry showed decreased expression of AGT and renin proteins in Foxd1 -/- metanephroi compared with that in Foxd1 +/+ metanephroi. Foxd1 overexpression in mesenchymal MK4 cells in vitro increased renin, AGT, ACE, and AT 1 R mRNA levels. Exogenous Ang II stimulated UB branching equally in whole intact E12.5 Foxd1 -/- and Foxd1 +/+ metanephroi grown ex vivo (+364±21% vs. +336±18%, P=0.42).ConclusionWe conclude that Foxd1 is an upstream positive regulator of RAS during early metanephric development and propose that the cross-talk between Foxd1 and RAS is essential in UB-branching morphogenesis.

The differentiated cell types of the mature ureter arise from the distal ureteric bud epithelium and its surrounding mesenchyme. Uncommitted epithelial cells first become intermediate cells from which both basal and superficial cells develop. Mesenchymal progenitors give rise to separated layers of adventitial fibrocytes, smooth muscle cells and lamina propria fibrocytes. How progenitor expansion and differentiation are balanced is poorly understood. Here, we addressed the role of retinoic acid (RA) signaling in these programs. Using expression analysis of components and target genes, we show that pathway activity is restricted to the mesenchymal and epithelial progenitor pools. Inhibition of RA signaling in ureter explant cultures resulted in tissue hypoplasia with a relative expansion of smooth muscle cells at the expense of lamina propria fibroblasts in the mesenchyme, and of superficial cells at the expense of intermediate cells in the ureteric epithelium. Administration of RA led to a slight reduction of smooth muscle cells, and almost completely prevented differentiation of intermediate cells into basal and superficial cells. We identified cellular programs and transcriptional targets of RA signaling that may account for this activity. We conclude that RA signaling is required and sufficient to maintain mesenchymal and epithelial progenitors in early ureter development.

The elimination of unwanted cells by apoptosis is necessary for tissue morphogenesis. However, the cellular control of morphogenetic apoptosis is poorly understood, notably the modulation of cell sensitivity to apoptotic stimuli. Ureter maturation, the process by which the ureter is displaced to the bladder wall, represents an exquisite example of morphogenetic apoptosis, requiring the receptor protein tyrosine phosphatases (RPTPs): LAR and RPTPσ. Here we show that LAR-RPTPs act through cellular inhibitor of apoptosis protein 1 (cIAP1) to modulate caspase 3,7-mediated ureter maturation. Pharmacologic or genetic inactivation of cIAP1 reverts the apoptotic deficit of LAR-RPTP-deficient embryos. Moreover, Birc2 (cIAP1) inactivation generates excessive apoptosis leading to vesicoureteral reflux in newborns, which underscores the importance of apoptotic modulation during urinary tract morphogenesis. We finally demonstrate that LAR-RPTP deficiency increases cIAP1 stability during apoptotic cell death. Together these results identify a mode of cIAP1 regulation playing a critical role in the cellular response to apoptotic pathway activation in the embryo., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Heterozygous loss of Bmp4 results both in humans and mice in severe malformation of the urinary tract. These defects have at least partially been attributed to loss of expression of Bmp4 in the ureteric mesenchyme, yet the cellular and molecular function of this signal as well as its effector pathways in this tissue have remained incompletely resolved. Here, we show that mice with a conditional deletion of Bmp4 in the ureteric mesenchyme exhibited hydroureter and hydronephrosis at newborn stages due to functional and physical ureter obstruction. Proliferation in both the mesenchymal and epithelial progenitor pools was severely reduced and smooth muscle cell and urothelial differentiation programs were not activated. Epithelial expression of P-ERK1/2, P-AKT and P-P38, and mesenchymal expression of P-SMAD1/5/9, P-P38 and P-AKT were abrogated. Pharmacological inhibition and activation experiments in ureter cultures defined AKT as the most relevant downstream effector for epithelial and mesenchymal proliferation as well as for epithelial differentiation. Epithelial proliferation and differentiation were also influenced by P-38 and ERK1/2, while SMAD signaling, together with AKT and P-38, were required for smooth muscle cell differentiation. Our analysis suggests that BMP4 is the signal that couples the proliferation and differentiation programs in the epithelial and mesenchymal tissue compartments of the developing ureter by different downstream effectors, most importantly AKT and SMAD., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The purpose of the study was to identify the characteristics of apoptosis in the kidneys, ureters and bladder of fetuses and newborns in the modeling of chronic intrauterine hypoxia, acute postnatal hypoxia and mixed hypoxia. An experiment was conducted on WAG rats for modeling high altitude hypoxia. Experimental animals were divided into four groups: I - control - fetuses and newborns from healthy rats; II - modeling of chronic intrauterine hypoxia; III - modeling of acute postnatal hypoxia; IV - modeling of mixed hypoxia. The material of the study was the tissue of the kidneys, ureters and bladder of fetuses and newborns. In group I in the kidneys of fetuses the mean value of the number of p53-positive cells was 7.83±0.31, newborns - 5.40±0.28; in the ureters and bladder of fetuses - 5.77±0.29 and 6.97±0.32, newborns - 3.58±0.21 and 5.36±0.28. In the kidneys in group II the mean value of the number of p53-expressing cells in fetuses was 1.43±0.50, in newborns - 21.72±0.58; in group III in newborns - 15.03±0.63; in group IV in newborns - 33.33±0.72. The mean value of the number of p53-expressing cells in the ureters and bladder in group II in fetuses was 13.17±0.49 and 11.83±0.43, in newborns - 16.24±0.37 and 15.38±0.37; in group III in newborns - 7.25±0.27 and 8.68±0.32; in group IV in newborns - 19.63±0.31and 21.03±0.40. As the result of the study it was found that experimental hypoxia induced apoptotic processes in the kidneys, ureters and bladder of fetuses and newborns, the severity of which was moderate in the modeling of acute postnatal hypoxia, expressed in the modeling of chronic intrauterine hypoxia and strongly expressed in the modeling of mixed hypoxia. Under the influence of acute postnatal hypoxia, chronic intrauterine hypoxia and mixed hypoxia in the ureters and bladder of fetuses and newborns p53-positive cells were located evenly in all layers of the wall of these organs, whereas in the kidneys p53-positive cells prevailed in the tubular component. In the modeling of chronic intrauterine hypoxia apoptotic processes in the kidneys, ureters and bladder increased in newborns in comparison with fetuses.

The formation of the proper number of nephrons requires a tightly regulated balance between renal progenitor cell self-renewal and differentiation. The molecular pathways that regulate the transition from renal progenitor to renal vesicle are not well understood. Here, we show that Sall1interacts with the nucleosome remodeling and deacetylase complex (NuRD) to inhibit premature differentiation of nephron progenitor cells. Disruption of Sall1-NuRD in vivo in knock-in mice (Δ SRM ) resulted in accelerated differentiation of nephron progenitors and bilateral renal hypoplasia. Transcriptional profiling of mutant kidneys revealed a striking pattern in which genes of the glomerular and proximal tubule lineages were either unchanged or upregulated, and those in the loop of Henle and distal tubule lineages were downregulated. These global changes in gene expression were accompanied by a significant decrease in THP-, NKCC2- and AQP1-positive loop of Henle nephron segments in mutant Δ SRM kidneys. These findings highlight an important function of Sall1-NuRD interaction in the regulation of Six2-positive multipotent renal progenitor cells and formation of the loop of Henle., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Branching morphogenesis creates arborized epithelial networks. In the mammalian kidney, an epithelial progenitor pool at ureteric branch tips (UBTs) creates the urine-transporting collecting system. Using region-specific mouse reporter strains, we performed an RNA-seq screen, identifying tip- and stalk-enriched gene sets in the developing collecting duct system. Detailed in situ hybridization studies of tip-enriched predictions identified UBT-enriched gene sets conserved between the mouse and human kidney. Comparative spatial analysis of their UBT niche expression highlighted distinct patterns of gene expression revealing novel molecular heterogeneity within the UBT progenitor population. To identify kidney-specific and shared programs of branching morphogenesis, comparative expression studies on the developing mouse lung were combined with in silico analysis of the developing mouse salivary gland. These studies highlight a shared gene set with multi-organ tip enrichment and a gene set specific to UBTs. This comprehensive analysis extends our current understanding of the ureteric branch tip niche., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The differentiated cell types of the epithelial and mesenchymal tissue compartments of the mature ureter of the mouse arise in a precise temporal and spatial sequence from uncommitted precursor cells of the distal ureteric bud epithelium and its surrounding mesenchyme. Previous genetic efforts identified a member of the Hedgehog (HH) family of secreted proteins, Sonic hedgehog (SHH) as a crucial epithelial signal for growth and differentiation of the ureteric mesenchyme. Here, we used conditional loss- and gain-of-function experiments of the unique HH signal transducer Smoothened (SMO) to further characterize the cellular functions and unravel the effector genes of HH signaling in ureter development. We showed that HH signaling is not only required for proliferation and SMC differentiation of cells of the inner mesenchymal region but also for survival of cells of the outer mesenchymal region, and for epithelial proliferation and differentiation. We identified the Forkhead transcription factor gene Foxf1 as a target of HH signaling in the ureteric mesenchyme. Expression of a repressor version of FOXF1 in this tissue completely recapitulated the mesenchymal and epithelial proliferation and differentiation defects associated with loss of HH signaling while re-expression of a wildtype version of FOXF1 in the inner mesenchymal layer restored these cellular programs when HH signaling was inhibited. We further showed that expression of Bmp4 in the ureteric mesenchyme depends on HH signaling and Foxf1, and that exogenous BMP4 rescued cell proliferation and epithelial differentiation in ureters with abrogated HH signaling or FOXF1 function. We conclude that SHH uses a FOXF1-BMP4 module to coordinate the cellular programs for ureter elongation and differentiation, and suggest that deregulation of this signaling axis occurs in human congenital anomalies of the kidney and urinary tract (CAKUT).

The mammalian ureter consists of a mesenchymal wall composed of smooth muscle cells and surrounding fibrocytes of the tunica adventitia and the lamina propria and an inner epithelial lining composed of layers of basal, intermediate, and superficial cells. How these cell types arise from multipotent progenitors is poorly understood. Here, we performed marker analysis, cell proliferation assays, and genetic lineage tracing to define the lineage relations and restrictions of the mesenchymal and epithelial cell types in the developing and mature mouse ureter. At embryonic day (E) 12.5, the mesenchymal precursor pool began to subdivide into an inner and outer compartment that began to express markers of smooth muscle precursors and adventitial fibrocytes, respectively, by E13.5. Smooth muscle precursors further diversified into lamina propria cells directly adjacent to the ureteric epithelium and differentiated smooth muscle cells from E16.5 onwards. Uncommitted epithelial progenitors of the ureter differentiated into intermediate cells at E14.5. After stratification into two layers at E15.5 and three cell layers at E18.5, intermediate cells differentiated into basal cells and superficial cells. In homeostasis, proliferation of all epithelial and mesenchymal cell types remained low but intermediate cells still gave rise to basal cells, whereas basal cells divided only into basal cells. These studies provide a framework to further determine the molecular mechanisms of cell differentiation in the tissues of the developing ureter., (Copyright © 2017 by the American Society of Nephrology.)

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H + -ATPase. Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is essential for mammalian kidney development. Previously, we demonstrated that conditional ablation of the PRR in the UB in PRR UB-/- mice causes severe defects in UB branching, resulting in marked kidney hypoplasia at birth. Here, we investigated the UB transcriptome using whole genome-based analysis of gene expression in UB cells, FACS-isolated from PRR UB-/- , and control kidneys at birth (P0) to determine the primary role of the PRR in terminal differentiation and growth of UB-derived collecting ducts. Three genes with expression in UB cells that previously shown to regulate UB branching morphogenesis, including Wnt9b , β-catenin, and Fgfr2 , were upregulated, whereas the expression of Wnt11 , Bmp7 , Etv4 , and Gfrα1 was downregulated. We next demonstrated that infection of immortalized UB cells with shPRR in vitro or deletion of the UB PRR in double-transgenic PRR UB-/- / BatGal + mice, a reporter strain for β-catenin transcriptional activity, in vivo increases β-catenin activity in the UB epithelia. In addition to UB morphogenetic genes, the functional groups of differentially expressed genes within the downregulated gene set included genes involved in molecular transport, metabolic disease, amino acid metabolism, and energy production. Together, these data demonstrate that UB PRR performs essential functions during UB branching and collecting duct morphogenesis via control of a hierarchy of genes that control UB branching and terminal differentiation of the collecting duct cells., (Copyright © 2017 the American Physiological Society.)

The renal vasculature is integral to the physiologic function of the kidneys in regulating hemodynamics of the body and maintaining organ health. The close inter-relationship of capillaries and the renal epithelium is key to renal physiology, but how renal tubules regulate capillary development remains unclear. Our previous work showed that Wnt7b is expressed in the ureteric trunk epithelium and activates canonical Wnt signaling in the surrounding medullary interstitium, where the capillaries reside. In this study, we showed by immunofluorescence that the target interstitial cells of Wnt7b/canonical Wnt signaling are mural cells of periureteric bud capillaries in the nascent renal medulla of embryonic mice. Genetic ablation of Wnt7b enhanced the proliferation of Wnt7b target mural cells, an effect that associated with decreased expression of PDGFRβ and p57kip2, a cyclin-dependent kinase inhibitor, in these cells. Furthermore, Wnt7b regulated lumen formation of the capillary endothelium in the renal medulla. In the absence of Wnt7b signaling, the periureteric bud medullary capillaries displayed narrower lumens lined with less flattened endothelial cells and a significantly increased presence of luminal endothelial cell-cell junctions, a transient configuration in the forming blood vessels in the controls. Moreover, the absence of Wnt7b led to greatly diminished levels of vascular endothelial (VE)-cadherin at the cell surface in these blood vessels. VE-cadherin is essential for blood vessel lumen formation; thus, Wnt7b may regulate lumen formation through modulation of VE-cadherin localization. Overall, these results indicate a novel role of Wnt7b signaling and the ureteric bud epithelium in renal medullary capillary development., (Copyright © 2016 by the American Society of Nephrology.)

The mammalian kidney develops from a simple epithelial bud to an arborized network of tubules, which are fated to form the ureter, renal pelvis and collecting ducts. This process of ductal elaboration is achieved through an ancient developmental mechanism known as branching morphogenesis that is widely employed in glandular organs, the vasculature and lungs. It breaks up large solid tissues facilitating secretion, excretion and gas exchange, depending on the tissue. In the kidney, growth of the ureteric bud is driven by interactions between progenitor cells in the tips of the epithelial tree and their mesenchymal 'caps'. The cells of the cap mesenchyme give rise to nephrons; therefore, the interaction between these two cell populations is likely to be a critical driver of nephron number, which is determined during gestation. These cellular interactions are potentially affected by genetic mutations (congenital kidney diseases) and by changes in the fetal environment. Understanding the aetiology of congenital and acquired kidney diseases therefore requires a full appreciation of the processes involved in establishing the cellular architecture of the kidney and of the factors that affect the commitment of progenitor cells to form nephrons.

Morphogenesis of the mammalian kidney requires reciprocal interactions between two cellular domains at the periphery of the developing organ: the tips of the epithelial ureteric tree and adjacent regions of cap mesenchyme. While the presence of the cap mesenchyme is essential for ureteric branching, how it is specifically maintained at the tips is unclear. Using ex vivo timelapse imaging we show that cells of the cap mesenchyme are highly motile. Individual cap mesenchyme cells move within and between cap domains. They also attach and detach from the ureteric tip across time. Timelapse tracks collected for >800 cells showed evidence that this movement was largely stochastic, with cell autonomous migration influenced by opposing attractive, repulsive and cell adhesion cues. The resulting swarming behaviour maintains a distinct cap mesenchyme domain while facilitating dynamic remodelling in response to underlying changes in the tip., (Copyright © 2016 Elsevier Inc. All rights reserved.)