Methods of Computational Analysis in Kidney Development.

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

Chapter 4 - RET Signaling in Ureteric Bud Formation and Branching

Rho kinase acts at separate steps in ureteric bud and metanephric mesenchyme morphogenesis during kidney development

Essential roles of Sall1 in kidney development

The kidney develops through mutual interactions between the metanephric mesenchyme and the ureteric bud, the former of which contains nephron progenitors that give rise to glomeruli and renal tubules. Bone morphogenetic protein (BMP) signaling and its modifiers play important roles in many steps of kidney development. BMP4 inhibits ureteric bud attraction, and the BMP antagonist Gremlin is essential for the initial stage of ureteric budding. During mid-gestation, BMP7 maintains the nephron progenitors and, at the same time, sensitizes them to the ureteric bud-derived differentiation signal. Crossveinless2 is a pro-BMP factor, and its absence leads to kidney hypoplasia. After birth, when nephron progenitors have disappeared, Dullard, a phosphatase that inactivates BMP receptors, keeps BMP signaling at an appropriate level. Deletion of Dullard results in excessive BMP signaling and apoptosis of the postnatal nephrons. In this review I discuss the similarities and differences of BMP functions in kidney development, as well as in diseases.

The significance of Itga8 and Vangl2 in kidney development: Insights from yotari mice.

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.

c-kit delineates a distinct domain of progenitors in the developing kidney

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.

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

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.

The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.

Renal epithelial cells derive from either cells of the metanephric mesenchyme or ureteric bud cells, but the origin of other renal cells is unclear. To test whether metanephric mesenchymal cells generate cells other than epithelial, we examined the developmental potential of a metanephric mesenchymal cell line (7.1.1 cells) and of primary cultures of metanephric mesenchymal cells. 7.1.1 Cells express both mesenchymal and epithelial markers and, on confluence, form well-defined monolayers expressing epithelial junctional proteins. However, 7.1.1 cells as well as primary cultures of metanephric mesenchymal cells also generate spindle-shaped cells that are positive for alpha-smooth muscle actin, indicating that they are myofibroblasts and/or smooth muscle; this differentiation pathway is inhibited by collagen IV and enhanced by fetal calf serum or transforming growth factor-beta(1). Transforming growth factor-beta(1) also induces expression of smooth muscle proteins, indicating that the cells differentiate into smooth muscle. 7.1.1 Cells as well as primary cultures of metanephric mesenchymal cells also express vascular endothelial growth factor receptor 2 and Tie-2, suggesting that the metanephric mesenchymal cells that generate epithelia may also differentiate into endothelial cells. The pluripotency of the 7.1.1 cells is self-renewing. The data suggest that the metanephric mesenchyme contains embryonic renal stem cells.

Implication of Wt1 in the Pathogenesis of Nephrogenic Failure in a Mouse Model of Retinoic Acid-Induced Caudal Regression Syndrome

The nephron is derived from the ureteric bud and metanephric mesenchyme and develops into a complex epithelial structure with a wide variety of phenotypes along its length. This segmental variation in expression of molecules provides an approach to understand the lineage of unique segments. The present study evaluated the expression of four relatively well-localized molecules--renin, Tamm-Horsfall protein (THP), oxytocin receptor (OTR), and the vasopressin type 2 receptor (V2R)--in cultured mouse-rat chimeric metanephric kidneys using reverse transcription-polymerase chain reaction (RT-PCR). Chimeric kidneys were formed by 1) separating the ureteric bud (U) from the metanephric mesenchyme (M) of mouse (m) at E11 and rat (r) at E13 days of gestation and 2) recombining the ureteric bud of one species with the metanephric mesenchyme of the other species (i.e., UrMm and UmM(r), followed by filter culture until differentiated. Species-specific restriction enzymes for all four genes were chosen to digest the PCR product from either rat or mouse. RT-PCR was performed for each mRNA species and the products digested. The V2R product from the UrMm chimera was cleaved by a restriction enzyme known to digest only rat product, suggesting the PCR product was produced predominantly by cells derived from the ureteric bud. The renin, OTR, and THP products from both chimeras were cleaved equally well by species-specific restriction enzymes, suggesting the products were made by cells originating from both the ureteric bud and the metanephric mesenchyme. These studies demonstrate that the cultured chimeric metanephric model is useful to study segment lineage. The results suggest that the lineage of at least certain portions of the nephron is heterogenous.