Diverse aspects of metanephric development.

Mammalian nephrogenesis begins by the reciprocal interaction of the ureteric bud with the undifferentiated mesenchyme. The mesenchyme differentiates into an epithelial phenotype with the development of the glomerulus and proximal and distal tubules. At the same time, the mesenchyme stimulates the branching morphogenesis of the ureteric bud that differentiates into the collecting ducts. These inductive interactions and differentiation events are modulated by a number of macromolecules, including the extracellular matrix (ECM), integrin receptors, and cell adhesion molecules. Many of these macromolecules exhibit spatiotemporal developmental regulation in the metanephros. Some are expressed in the mesenchyme, whereas others appear in the ureteric bud epithelia. The molecules expressed in the mesenchyme or at the epithelial:mesenchymal interface may serve as ligands while those in the epithelia serve as the receptors. In such a scenario the ligand and the receptor would be ideally suited for epithelial:mesenchymal paracrine/juxtacrine interactions that are also influenced by RGD sequences and Ca2+ binding domains of the ECM proteins and their receptors. This review addresses the role of such interactions in metanephric development.

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.

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.

Mutations or rearrangements in the gene encoding the receptor tyrosine kinase RET result in Hirschsprung disease, cancer and renal malformations. The standard model of renal development involves reciprocal signaling between the ureteric bud epithelium, inducing metanephric mesenchyme to differentiate into nephrons, and metanephric mesenchyme, inducing the ureteric bud to grow and branch. RET and GDNF (a RET ligand) are essential mediators of these epithelial-mesenchymal interactions. Vitamin A deficiency has been associated with widespread embryonic abnormalities, including renal malformations. The vitamin A signal is transduced by nuclear retinoic acid receptors (RARs). We previously showed that two RAR genes, Rara and Rarb2, were colocalized in stromal mesenchyme, a third renal cell type, where their deletion led to altered stromal cell patterning, impaired ureteric bud growth and downregulation of Ret in the ureteric bud. Here we demonstrate that forced expression of Ret in mice deficient for both Rara and Rarb2 (Rara(-/-)Rarb2(-/-)) genetically rescues renal development, restoring ureteric bud growth and stromal cell patterning. Our studies indicate the presence of a new reciprocal signaling loop between the ureteric bud epithelium and the stromal mesenchyme, dependent on Ret and vitamin A. In the first part of the loop, vitamin-A-dependent signals secreted by stromal cells control Ret expression in the ureteric bud. In the second part of the loop, ureteric bud signals dependent on Ret control stromal cell patterning.

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

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.

Interactions between the ureteric bud (UB) and metanephric mesenchyme are crucial for tubulogenesis during kidney development. Two immortalized cell lines derived from the day 11.5 embryonic kidney, UB cells, which appear to be epithelial (cytokeratin-positive, E-cadherin-positive, and ZO-1-positive by immunostaining) and BSN cells, which are largely mesenchymal (vimentin-positive, but negative for cytokeratin, cell surface E-cadherin, and cell surface ZO-1), were used to establish an in vitro tubulogenesis system. BSN cells expressed hepatocyte growth factor (HGF) and transforming growth factor-beta1 mRNAs, and its conditioned medium (BSN-CM) contained factors capable of activating the epidermal growth factor (EGF) receptor (EGFR). When UB cells were cultured in an extracellular matrix gel in the presence of the embryonic kidney or BSN-CM, the UB cells underwent morphogenetic changes characteristic of early in vitro branching tubulogenesis. These changes were largely inhibited by a combination of neutralizing anti-HGF antibodies and the EGFR inhibitor tyrphostin AG1478, suggesting that EGFR ligands, together with HGF, account for much of this early morphogenetic activity. Nevertheless, there was a significant fraction of tubulogenic activity that could not be inhibited, suggesting the existence of other soluble factors. Whereas HGF, EGF, transforming growth factor alpha, basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1), or a mixture of these growth factors, induced epithelial processes for up to 3 days, only IGF-1, possibly bFGF, and the mixture were able to sustain morphogenesis for longer periods, though not nearly to the same degree as BSN-CM. Moreover, only BSN-CM induced branching tubular structures with clear lumens, consistent with the existence of other soluble factors crucial for the formation and/or maintenance of branching tubular structures with lumens in vitro.

Immunohistochemical expression pattern of RIP5, FGFR1, FGFR2 and HIP2 in the normal human kidney development

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.

Mice which are homozygous for the limb deformity (ld) mutation also manifest an incompletely penetrant unilateral or bilateral renal agenesis phenotype. Intercross experiments suggest that the differences in penetrance of the renal agenesis phenotype between homozygous mice with different ld alleles are due to intrinsic differences in the strength of the mutant alleles or to one or more closely linked modifying loci, and not to generalized differences in genetic background. Analysis of ld/ld embryos between embryonic days 11-13 reveals delayed outgrowth or complete absence of the ureteric bud, the inducer of metanephric mesenchyme. Since explants of ld/ld metanephric mesenchyme differentiate in culture when apposed to embryonic spinal cord, we conclude that deficient ureteric bud outgrowth is the morphologic basis for renal agenesis in ld/ld mice. However, since ld transcripts can be detected in both metanephric mesenchyme and ureteric bud, the molecular basis for the deficiency in ureteric bud outgrowth could reside in either component.

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.

The kidney collecting system develops from the ureteric bud (UB), which undergoes multiple rounds of iterative branching. This process is controlled by growth factors and requires the interaction between the extracellular matrix and β1-containing integrin receptors. Integrin affinity for its ligands is regulated by integrin-binding proteins including kindlins, which bind well-defined motifs within the β subunit cytoplasmic tail. We show that mice expressing β1 integrins with mutations that abrogate kindlin binding in the developing UB have mild medullary hypoplasia and a moderate branching defect. Collecting duct (CD) cells expressing the same mutations in the β1 subunit have moderate tubulogenesis, spreading and adhesion defects, but show intact growth factor-dependent signaling. In contrast, mice lacking kindlins in the UB are anephric due to a complete absence of UB budding. Kindlin-knockout CD cells are unable to spread, adhere or respond to growth factors, irrespective of whether the integrins are bound to a ligand. Thus, in addition to regulating integrin function, kindlins mediate crucial growth factor signaling required for initial UB formation.

CAKUT are problems that often require surgical intervention or, in the worst case, lead to renal failure and the need for dialysis and/or renal transplantation. It is believed that these anomalies share a common genetic cause and to date there has been no good animal model with which to study these abnormalities. Although the abnormal interaction between the ureteral bud and metanephric blastema leads to renal hypodysplasia, vesicoureteral reflux, and ectopic ureters to name a few, the genetic and biochemical modulation of urinary tract development is not understood. Studies using the mouse strain mutant for angiotensin type 2 (AT2) receptors have given new insight into this mystery. The animals show defective apoptosis of undifferentiated mesenchymal cells in the area surrounding the developing kidney and urinary tract. This abnormal apoptosis may well interfere with the normal interaction between the ureteral bud and metanephric blastema resulting in CAKUT. This abnormal interaction would theoretically lead to preexisting intrinsic abnormalities of the kidney, which are programmed and take effect early in embryonic development. In the worst cases, the renal abnormalities would lead to progressive deterioration of renal function. Undoubtedly, there are more genes and biochemical modulators involved in this process other than the RAS and AT2 receptors. Our current animal model gives new and unique possibilities with which to study development of the kidney and urinary tract and ultimately seek ways of preventing an often debilitating disease process.

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