Human ureteric bud organoids recapitulate branching morphogenesis and differentiate into functional collecting duct cell types.

Causes of congenital anomalies of the kidney and urinary tract (CAKUT) remain unclear. The authors investigated whether and how inactivation of Ash2l -which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide histone H3 lysine K4 (H3K4) methylation-might contribute to CAKUT. In a mouse model, inactivation of Ash2l in the ureteric bud (UB) lineage led to CAKUT-like phenotypes. Removal of ASH2L led to deficient H3K4 trimethylation, which slowed cell proliferation at the UB tip, delaying budding and impairing branching morphogenesis. The absence of ASH2L also downregulated the expression of Ret , Gfra1 , and Wnt11 genes involved in RET/GFRA1 signaling. These findings identify ASH2L-mediated H3K4 methylation as an upstream epigenetic regulator of signaling crucial for UB morphogenesis and indicate that deficiency or dysregulation of these processes may lead to CAKUT. Ureteric bud (UB) induction and branching morphogenesis are fundamental to the establishment of the renal architecture and are key determinants of nephron number. Defective UB morphogenesis could give rise to a spectrum of malformations associated with congenital anomalies of the kidney and urinary tract (CAKUT). Signaling involving glial cell line-derived neurotrophic factor and its receptor rearranged during transfection (RET) and coreceptor GFRA1 seems to be particularly important in UB development. Recent epigenome profiling studies have uncovered dynamic changes of histone H3 lysine K4 (H3K4) methylation during metanephros development, and dysregulated H3K4 methylation has been associated with a syndromic human CAKUT. To investigate whether and how inactivation of Ash2l , which encodes a subunit of the COMPASS methyltransferase responsible for genome-wide H3K4 methylation, might contribute to CAKUT, we inactivated Ash2l specifically from the UB lineage in C57BL/6 mice and examined the effects on genome-wide H3K4 methylation and metanephros development. Genes and epigenome changes potentially involved in these effects were screened using RNA-seq combined with Cleavage Under Targets and Tagmentation sequencing. UB-specific inactivation of Ash2l caused CAKUT-like phenotypes mainly involving renal dysplasia at birth, which were associated with deficient H3K4 trimethylation. Ash2l inactivation slowed proliferation of cells at the UB tip, delaying budding and impairing UB branching morphogenesis. These effects were associated with downregulation of Ret , Gfra1 , and Wnt11 , which participate in RET/GFRA1 signaling. These experiments identify ASH2L-dependent H3K4 methylation in the UB lineage as an upstream epigenetic regulator of RET/GFRA1 signaling in UB morphogenesis, which, if deficient, may lead to CAKUT.

Ca2+ signaling in metanephric mesenchymal (MM) cells contributes to key pattern forming events including branching morphogenesis in the embryonic rat kidney (Fontana et al., FASEB J, 2019). We previously reported that basolateral exposure of tubular structures isolated from MM-derived kidney organoids to the PIEZO1 channel agonist Yoda1 elicited a maturation stage-dependent elevation of intracellular Ca2+ concentration, [Ca2+]i (Carrisoza-Gaytán et al., AJP Cell Physiol, 2023). The aim of our current study is to explore whether tubular cells in human iPSC-derived UB and CD organoids exhibit similar developmental increases in PIEZO1 function.To investigate this, tubular structures from UB and CD organoids cultured for 34-35 or 61-65 d (4 tubules/group; n=38-40 cells/group) are loaded with the Ca2+-sensitive fluorophore Fura2-AM to measure [Ca2+]i before and after basolateral exposure to Yoda1 (5 µM). Baseline [Ca2+]i was 108.5±3.6 nM across all tubular cells studied, with no differences detected between UBs and CDs or days in culture. Subsequent exposure to Yoda1 induced an increase in [Ca2+]i and appearance of Ca2+ oscillations. The Yoda1-induced [Ca2+]i signals of individually identified cells are then subject to spectral analysis using the multitaper method along with a harmonic F-test to identify predominant frequencies in the signal. We found that with advancing days in culture from 34-35 to 61-65 d: (i) Yoda1 induced an increase in [Ca2+]i in UBs from 45.3±18.5 to 275.6±43.7 nM (p≤0.001) and in CDs from 61.7±16.1 to 254.3±56.2 nM (p≤0.001), (ii) the proportion of cells exhibiting Ca2+ oscillations increased in UBs from 70 to 83% and in CDs from 75 to 84%, and (iii) the predominant oscillatory frequency fell by 50% in UBs and 30% in CDs from 15 mHz. These observations suggest that kidney organoids undergo a developmental increase in PIEZO1 abundance/activity and maturation of Ca2+ signal transduction pathways, especially those involved in Ca2+ oscillatory activity. Decoding Ca2+ oscillations may unveil molecular mechanisms underlying maturation/differentiation. This work was supported by the NIH Re(Building) a Kidney Consortium (NIH UC2DK126023) and the Welcome LEAP Human Organs, Physiology and Engineering (HOPE) program. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Human Pluripotent Stem Cell-Derived Kidney Organoids with Improved Collecting Duct Maturation and Injury Modeling

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney

Ret signaling promotes branching morphogenesis during kidney development, but the underlying cellular mechanisms remain unclear. While Ret-expressing progenitor cells proliferate at the ureteric bud tips, some of these cells exit the tips to generate the elongating collecting ducts, and in the process turn off Ret. Genetic ablation of Ret in tip cells promotes their exit, suggesting that Ret is required for cell rearrangements that maintain the tip compartments. Here, we examine the behaviors of ureteric bud cells that are genetically forced to maintain Ret expression. These cells move to the nascent tips, and remain there during many cycles of branching; this tip-seeking behavior may require positional signals from the mesenchyme, as it occurs in whole kidneys but not in epithelial ureteric bud organoids. In organoids, cells forced to express Ret display a striking self-organizing behavior, attracting each other to form dense clusters within the epithelium, which then evaginate to form new buds. The ability of forced Ret expression to promote these events suggests that similar Ret-dependent cell behaviors play an important role in normal branching morphogenesis.

Angiotensinogen-, angiotensin-converting enzyme-, and angiotensin II (Ang II) type 1 receptor (AT(1)R)-deficient mice exhibit a dilated renal pelvis (hydronephrosis) and a small papilla. These abnormalities have been attributed to impaired development of the ureteral and pelvic smooth muscle. Defects in the growth and branching of the ureteric bud (UB), which gives rise to the collecting system, have not been examined carefully. This study tested the hypothesis that Ang II stimulates UB growth and branching in the intact metanephros. Immunohistochemistry demonstrated that embryonic mouse kidneys express AT(1)R in the UB and its branches. Embryonic day 11.5 metanephroi were microdissected from Hoxb7-green fluorescence protein mice and grown for 48 h in serum-free medium in the presence or absence of Ang II. The number of green fluorescence protein-positive UB branch points (BP) and tips was monitored in each explant at 24 and 48 h. Ang II increased the number of UB tips and BP at 24 h (tips: 24.3 +/- 1.1 versus 18.3 +/- 0.7, P < 0.01; BP: 14.4 +/- 0.6 versus 11.7 +/- 0.6, P < 0.01) and 48 h (tips: 30.2 +/- 1.3 versus 22.9 +/- 0.8, P < 0.01; BP: 21.3 +/- 0.9 versus 15.7 +/- 0.6, P < 0.01) compared with control. In contrast, treatment of metanephroi with the AT(1)R antagonist candesartan inhibited UB branching, decreasing the number of UB tips and BP. Similarly, inhibition of EGF receptor (EGFR) tyrosine kinase activity abrogated Ang II-stimulated UB branching. A cross-talk between the renin-angiotensin system and EGFR signaling was elicited at the cellular level by the ability of Ang II to induce tyrosine phosphorylation of EGFR in UB cells and through abrogation of Ang II-induced UB cell branching using an EGFR tyrosine kinase inhibitor. These data demonstrate that Ang II, acting via the AT(1)R, stimulates UB branching morphogenesis. This process depends on tyrosine phosphorylation of the EGFR. Cooperation of AT(1)R and EGFR signaling therefore is important in the development of the renal collecting system.

Overexpression of Robo2 causes defects in the recruitment of metanephric mesenchymal cells and ureteric bud branching morphogenesis

The collecting duct network and the urothelium of the ureter of the metanephric kidney are derived from the ureteric bud epithelium, initially an outgrowth from the caudal end of the Wolffian duct at the onset of the metanephric kidney development. The tips of the ureteric bud epithelium undergo reiterative branching morphogenesis, which generates more tips and trunks, whereas the ureteric trunks grow and differentiate into principal cells and intercalated cells of the collecting ducts that regulate body water and acid-base homeostasis. microRNAs (miRNAs) are a family of small non-coding RNAs that regulate a diversity of biological processes including organogenesis, mostly by negatively regulating their target gene expression. In this review, I will summarize the current knowledge on the critical roles of miRNAs expressed in the ureteric bud epithelium in ureteric bud morphogenesis and differentiation, including ureteric bud branching morphogenesis, collecting duct terminal differentiation, cystogenesis of the collecting ducts, and ureter development.

The role of the prorenin receptor (PRR) in the regulation of ureteric bud (UB) branching morphogenesis is unknown. Here, we investigated whether PRR acts specifically in the UB to regulate UB branching, kidney development and function. We demonstrate that embryonic (E) day E13.5 mouse metanephroi, isolated intact E11.5 UBs and cultured UB cells express PRR mRNA. To study its role in UB development, we conditionally ablated PRR in the developing UB (PRR UB−/−) using Hoxb7 Cre mice. On E12.5, PRR UB−/− mice had decreased UB branching and increased UB cell apoptosis. These defects were associated with decreased expression of Ret, Wnt11, Etv4/Etv5, and reduced phosphorylation of Erk1/2 in the UB. On E18.5, mutants had marked kidney hypoplasia, widespread apoptosis of medullary collecting duct cells and decreased expression of Foxi1, AE1 and H+-ATPase α4 mRNA. Ultimately, they developed occasional small cysts in medullary collecting ducts and had decreased nephron number. To test the functional consequences of these alterations, we determined the ability of PRR UB−/− mice to acidify and concentrate the urine on postnatal (P) day P30. PRR UB−/− mice were polyuric, had lower urine osmolality and a higher urine pH following 48 hours of acidic loading with NH4Cl. Taken together, these data show that PRR present in the UB epithelia performs essential functions during UB branching morphogenesis and collecting duct development via control of Ret/Wnt11 pathway gene expression, UB cell survival, activation of Erk1/2, terminal differentiation and function of collecting duct cells needed for maintaining adequate water and acid-base homeostasis. We propose that mutations in PRR could possibly cause renal hypodysplasia and renal tubular acidosis in humans.

Our studies have established that ureteric bud (UB) prorenin receptor (PRR/ATP6AP2), an accessory subunit of the vacuolar H+-ATPase (V-ATPase), is critical for normal UB branching. Here, we tested the hypothesis that V-ATPase activity, acidosis, and UB cell intracellular pH (pHi) regulate UB branching morphogenesis during kidney development. The effect of specific V-ATPase inhibitor Bafilomycin, hypercapnic (high CO2), and metabolic (low [Formula: see text]) acidosis on UB branching was determined in whole intact E12.5 Hoxb7GFP+ mouse kidneys grown ex vivo by time-lapse photomicroscopy. The effect of Bafilomycin on UB cell migration in vitro was examined using a transwell migration assay (n = 3 wells/treatment group). The presence of V-ATPase and Na+-H+ exchanger (NHE) activity in UB cells was investigated by measurements of intracellular pH (pHi). The ability of UB cells to regulate cell pHi in vitro was determined by measurements of Na-dependent and Na-independent pHi recovery from acid loads. The mean number of UB cells that migrated through the membrane after 24-h culture was reduced with Bafilomycin compared with control. Treatment with Bafilomycin, hypercapnic acidosis (induced by high CO2), or metabolic acidosis (induced by low [Formula: see text] concentration) in the culture media caused a marked reduction in the number of UB tips compared with control. We conclude that intact V-ATPase activity is essential for normal UB branching during kidney development. V-ATPase-dependent reduction in UB cell pHi is likely a cause of decreasing UB branching by inhibiting directional movements of UB cells.NEW & NOTEWORTHY Disruption of normal kidney development results in a spectrum of congenital anomalies of the kidney and urinary tract (CAKUT), the major cause of end-stage kidney disease in children. We demonstrate that acidosis, kidney cell intracellular pH, and activity of V-ATPase pump are essential for normal kidney development.

BackgroundThe 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.MethodsMutant Hoxb7Cre+/PRRflox/flox (PRRUB-/-) and control PRRUB+/+, 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.ResultsDOT1L 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).ConclusionTargeted 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.ImpactThe 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.

The mammalian kidney, the metanephros, is a mesodermal organ classically regarded as arising from the intermediate mesoderm (IM). Indeed, both the ureteric bud (UB), which gives rise to the ureter and the collecting ducts, and the metanephric mesenchyme (MM), which forms the rest of the kidney, derive from the IM. Based on an understanding of the signalling molecules crucial for IM patterning and kidney morphogenesis, several studies have now generated UB or MM, or both, in vitro via the directed differentiation of human pluripotent stem cells. Although these results support the IM origin of the UB and the MM, they challenge the simplistic view of a common progenitor for these two populations, prompting a reanalysis of early patterning events within the IM. Here, we review our understanding of the origin of the UB and the MM in mouse, and discuss how this impacts on kidney regeneration strategies and furthers our understanding of human development.

Chapter 4 - RET Signaling in Ureteric Bud Formation and Branching

Generation of esophageal organoids and organotypic raft cultures from human pluripotent stem cells.

The metanephric, or definitive, kidney forms as a result of inductive processes between tissues of two distinct embryologic origins, the metanephric mesenchymal blastema and the ureteric bud. After inductive signalling between these primordial tissues, mesenchymal cells aggregate next to the branching ureteric bud tip, convert to epithelial cells and differentiate into the diverse cell populations of the nephron. The ureteric bud enters branching morphogenesis and gives rise to the collecting duct system. Nephrogenesis has become a target system by which to study developmental processes including embryonic inductive interactions, mesenchyme-to-epithelium conversion, cell lineage pathways, epithelial cell polarization, branching morphogenesis and spatio-temporal expression of transcription factors. This review summarizes data on cellular and extracellular events during epithelial metanephrogenesis.