Regulation of Renal Differentiation by Trophic Factors

Classically, trophic factors are considered as proteins which support neurons in their growth, survival, and differentiation. However, most neurotrophic factors also have important functions outside of the nervous system. Especially essential renal growth and differentiation regulators are glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). Here we discuss how trophic factor-induced signaling contributes to the control of ureteric bud (UB) branching morphogenesis and to maintenance and differentiation of nephrogenic mesenchyme in embryonic kidney. The review includes recent advances in trophic factor functions during the guidance of branching morphogenesis and self-renewal versus differentiation decisions, both of which dictate the control of kidney size and nephron number. Creative utilization of current information may help better recapitulate renal differentiation in vitro, but it is obvious that significantly more basic knowledge is needed for development of regeneration-based renal therapies.Peer reviewe

Regulation of Renal Differentiation by Trophic Factors

Classically, trophic factors are considered as proteins which support neurons in their growth, survival, and differentiation. However, most neurotrophic factors also have important functions outside of the nervous system. Especially essential renal growth and differentiation regulators are glial cell line-derived neurotrophic factor (GDNF), bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs). Here we discuss how trophic factor-induced signaling contributes to the control of ureteric bud (UB) branching morphogenesis and to maintenance and differentiation of nephrogenic mesenchyme in embryonic kidney. The review includes recent advances in trophic factor functions during the guidance of branching morphogenesis and self-renewal versus differentiation decisions, both of which dictate the control of kidney size and nephron number. Creative utilization of current information may help better recapitulate renal differentiation in vitro, but it is obvious that significantly more basic knowledge is needed for development of regeneration-based renal therapies

MAPK/ERK Signaling in Regulation of Renal Differentiation

Congenital anomalies of the kidney and urinary tract (CAKUT) are common birth defects derived from abnormalities in renal differentiation during embryogenesis. CAKUT is the major cause of end-stage renal disease and chronic kidney diseases in children, but its genetic causes remain largely unresolved. Here we discuss advances in the understanding of how mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity contributes to the regulation of ureteric bud branching morphogenesis, which dictates the final size, shape, and nephron number of the kidney. Recent studies also demonstrate that the MAPK/ERK pathway is directly involved in nephrogenesis, regulating both the maintenance and differentiation of the nephrogenic mesenchyme. Interestingly, aberrant MAPK/ERK signaling is linked to many cancers, and recent studies suggest it also plays a role in the most common pediatric renal cancer, Wilms’ tumor

ShapeMetrics: a userfriendly pipeline for 3D cell segmentation and spatial tissue analysis

The demand for single-cell level data is constantly increasing within life sciences. In order to meet this demand, robust cell segmentation methods that can tackle challenging in vivo tissues with complex morphology are required. However, currently available cell segmentation and volumetric analysis methods perform poorly on 3D images. Here, we generated ShapeMetrics, a MATLAB-based script that segments cells in 3D and, by performing unbiased clustering using a heatmap, separates the cells into subgroups according to their volumetric and morphological differences. The cells can be accurately segregated according to different biologically meaningful features such as cell ellipticity, longest axis, cell elongation, or the ratio between cell volume and surface area. Our machine learning based script enables dissection of a large amount of novel data from microscope images in addition to the traditional information based on fluorescent biomarkers. Furthermore, the cells in different subgroups can be spatially mapped back to their original locations in the tissue image to help elucidate their roles in their respective morphological contexts. In order to facilitate the transition from bulk analysis to single-cell level accuracy, we emphasize the user-friendliness of our method by providing detailed step-by-step instructions through the pipeline hence aiming to reach users with less experience in computational biology.Peer reviewe

Omics profiling identifies the regulatory functions of the MAPK/ERK pathway in nephron progenitor metabolism

Publisher Copyright: © 2022. Published by The Company of Biologists Ltd.Nephron endowment is defined by fetal kidney growth and crucially dictates renal health in adults. Defects in the molecular regulation of nephron progenitors contribute to only a fraction of reduced nephron mass cases, suggesting alternative causative mechanisms. The importance of MAPK/ERK activation in nephron progenitor maintenance has been previously demonstrated, and here, we characterized the metabolic consequences of MAPK/ERK deficiency. Liquid chromatography/mass spectrometry-based metabolomics profiling identified 42 reduced metabolites, of which 26 were supported by in vivo transcriptional changes in MAPK/ERK-deficient nephron progenitors. Among these, mitochondria, ribosome and amino acid metabolism, together with diminished pyruvate and proline metabolism, were the most affected pathways. In vitro cultures of mouse kidneys demonstrated a dosage-specific function for pyruvate in controlling the shape of the ureteric bud tip, a regulatory niche for nephron progenitors. In vivo disruption of proline metabolism caused premature nephron progenitor exhaustion through their accelerated differentiation in pyrroline-5-carboxylate reductases 1 (Pycr1) and 2 (Pycr2) double-knockout kidneys. Pycr1/Pycr2-deficient progenitors showed normal cell survival, indicating no changes in cellular stress. Our results suggest that MAPK/ERK-dependent metabolism functionally participates in nephron progenitor maintenance by monitoring pyruvate and proline biogenesis in developing kidneys.Peer reviewe

Dynamic MAPK/ERK Activity Sustains Nephron Progenitors through Niche Regulation and Primes Precursors for Differentiation

The in vivo niche and basic cellular properties of nephron progenitors are poorly described. Here we studied the cellular organization and function of the MAPK/ERK pathway in nephron progenitors. Live-imaging of ERK activity by a Forster resonance energy transfer biosensor revealed a dynamic activation pattern in progenitors, whereas differentiating precursors exhibited sustained activity. Genetic experiments demonstrate that MAPK/ERK activity controls the thickness, coherence, and integrity of the nephron progenitor niche. Molecularly, MAPK/ERK activity regulates niche organization and communication with extracellular matrix through PAX2 and ITGA8, and is needed for CITED1 expression denoting undifferentiated status. MAPK/ERK activation in nephron precursors propels differentiation by priming cells for distal and proximal fates induced by the Wnt and Notch pathways. Thus, our results demonstrate a mechanism through which MAPK/ERK activity controls both progenitor maintenance and differentiation by regulating a distinct set of targets, which maintain the biomechanical milieu of tissue-residing progenitors and prime precursors for nephrogenesis.Peer reviewe

Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF

Nephron endowment, defined during the fetal period, dictates renal and related cardiovascular health throughout life. We show here that, despite its negative effects on kidney growth, genetic increase of GDNF prolongs the nephrogenic program beyond its normal cessation. Multi-stage mechanistic analysis revealed that excess GDNF maintains nephron progenitors and nephrogenesis through increased expression of its secreted targets and augmented WNT signaling, leading to a two-part effect on nephron progenitor maintenance. Abnormally high GDNF in embryonic kidneys upregulates its known targets but also Wnt9b and Axin2, with concomitant deceleration of nephron progenitor proliferation. Decline of GDNF levels in postnatal kidneys normalizes the ureteric bud and creates a permissive environment for continuation of the nephrogenic program, as demonstrated by morphologically and molecularly normal postnatal nephron progenitor self-renewal and differentiation. These results establish that excess GDNF has a bi-phasic effect on nephron progenitors in mice, which can faithfully respond to GDNF dosage manipulation during the fetal and postnatal period. Our results suggest that sensing the signaling activity level is an important mechanism through which GDNF and other molecules contribute to nephron progenitor lifespan specification.Peer reviewe

Comparative whole-genome transcriptome analysis in renal cell populations reveals high tissue specificity of MAPK/ERK targets in embryonic kidney

Background: MAPK/ERK signaling is a well-known mediator of extracellular stimuli controlling intracellular responses to growth factors and mechanical cues. The critical requirement of MAPK/ERK signaling for embryonic stem cell maintenance is demonstrated, but specific functions in progenitor regulation during embryonic development, and in particular kidney development remain largely unexplored. We previously demonstrated MAPK/ERK signaling as a key regulator of kidney growth through branching morphogenesis and normal nephrogenesis where it also regulates progenitor expansion. Here, we performed RNA sequencing-based whole-genome expression analysis to identify transcriptional MAPK/ERK targets in two distinct renal populations: the ureteric bud epithelium and the nephron progenitors. Results: Our analysis revealed a large number (5053) of differentially expressed genes (DEGs) in nephron progenitors and significantly less (1004) in ureteric bud epithelium, reflecting likely heterogenicity of cell types. The data analysis identified high tissue-specificity, as only a fraction (362) of MAPK/ERK targets are shared between the two tissues. Tissue-specific MAPK/ERK targets participate in the regulation of mitochondrial energy metabolism in nephron progenitors, which fail to maintain normal mitochondria numbers in the MAPK/ERK-deficient tissue. In the ureteric bud epithelium, a dramatic decline in progenitor-specific gene expression was detected with a simultaneous increase in differentiation-associated genes, which was not observed in nephron progenitors. Our experiments in the genetic model of MAPK/ERK deficiency provide evidence that MAPK/ERK signaling in the ureteric bud maintains epithelial cells in an undifferentiated state. Interestingly, the transcriptional targets shared between the two tissues studied are over-represented by histone genes, suggesting that MAPK/ERK signaling regulates cell cycle progression and stem cell maintenance through chromosome condensation and nucleosome assembly. Conclusions: Using tissue-specific MAPK/ERK inactivation and RNA sequencing in combination with experimentation in embryonic kidneys, we demonstrate here that MAPK/ERK signaling maintains ureteric bud tip cells, suggesting a regulatory role in collecting duct progenitors. We additionally deliver new mechanistic information on how MAPK/ERK signaling regulates progenitor maintenance through its effects on chromatin accessibility and energy metabolism.Peer reviewe

Cellular and molecular regulation of renal progenitor populations : The roles of receptor tyrosine kinase signaling in nephron and collecting duct progenitor maintenance and differentiation

Disruptions in MAPK/ERK signaling have been implicated in the development of various renal diseases including renal cancer, nephrotic syndrome, and Wilms’ tumor. Our lab has shown that MAPK/ERK is a crucial regulator of normal kidney development, where its functions are required both for ureteric bud branching morphogenesis as well as for nephrogenesis and progenitor expansion. Thus, understanding the role of MAPK/ERK signaling in kidney development is crucial for advancing our understanding of the mechanisms underlying renal diseases and for the development of new therapeutic strategies. Kidney development begins when signals originating from the metanephric mesenchyme, including GDNF, induce the initial ureteric bud outgrowth from the Wolffian duct and drive subsequent branching morphogenesis. The ureteric bud is comprised of the collecting duct progenitors, and their maintenance depends on the metanephric mesenchyme-derived signals. In turn, signals from the ureteric bud induce the metanephric mesenchyme, comprised of the nephron progenitor cells, to differentiate and begin the process of nephron formation. Due to the reciprocal interactions, the extent of ureteric bud branching dictates not only the size and shape of the organ but also the final nephron number. Nephron number additionally depends on the molecular regulation and maintenance of the nephron progenitors. As the kidney is a non-regenerating organ, nephron endowment is ultimately defined during the fetal period, and low nephron number predisposes renal and related cardiovascular issues throughout life. This thesis work found that MAPK/ERK maintains ureteric bud cells as undifferentiated and regulates renal progenitor cell maintenance through effects on chromatin accessibility and energy metabolism. MAPK/ERK-dependent nephron progenitor metabolism functionally contributes to progenitor preservation by controlling pyruvate availability and proline metabolism in developing kidneys. Furthermore, my data shows that monitoring the degree of signaling activity is a crucial way by which GDNF and possibly other molecules contribute to the specification of the lifespan of nephron progenitors. These studies provide some previously missing information about the regulation of renal development, which could contribute to the improvement of diagnostic and/or therapeutic strategies for renal diseases.Munuaisten kehitys alkaa, kun metanefrisestä mesenkyymistä, peräisin olevat signaalit, pääosin GDNF, indusoivat primaarisen virtsajohtimen silmun muodostumisen Wolffian tiehyestä. Samat signaalit ohjaavat myös silmun myöhempää haarautumismorfogeneesiä. Induktiivisien kudosvuorovaikutusten takia virtsajohtimen silmun haarautumisaste määrää munuaisen koon ja muodon lisäksi myös sen, kuinka monta toiminnallista nefroniyksikköä siihen muodostuu. Koska munuainen ei muodostumisensa jälkeen enää kykene uusiutumaan, sikiönkehityksen aikana muodostunut yksilöllinen nefronivara on äärimmäisen tärkeä tekijä munuaisterveyden kannalta, sillä alhainen nefronimäärä altistaa munuais- ja sydän- ja verisuoniongelmille myöhemmin elämässä. Virtsanjohtimen silmu sisältää kokoojaputkiston esisoluja, joiden ylläpito riippuu metanefrisestä mesenkyymistä tulevista signaaleista. Virtsajohtimen silmun signaalit puolestaan indusoivat metanefrisen mesenkyymin sisältämien nefronin esisolujen erilaistumisen ja nefronin muodostumisprosessin aloituksen. Nefronien lukumäärä riippuu lisäksi nefronien esiastesolujen molekyylisäätelystä ja ylläpidosta. MAPK/ERK-signalointi välittää useiden kasvutekijöiden kuten GDNF:n vaikutuksia solun sisään. MAPK/ERK-signaloinnin häiriöt ovat osallisena erilaisten sairauksien kuten munuaissyöpä, nefroottinen oireyhtymä ja Wilmsin kasvain, muodostumisessa. Tutkimuksemme on osoittanut, että MAPK/ERK on tärkeä munuaisen kehityksen säätelijä, joka se tarvitaan sekä virtsaputken silmun normaaliin haaroittumiseen että nefronien erilaistumiseen. Väitöskirjassani havaitsin, että MAPK/ERK signalointia tarvitaan pitämään virtsajohtimen silmun esiastesolut erilaistumattomina. Tutkimukseni osoittaa, että MAPK/ERK säätelee munuaisen esisolujen ylläpitoa vaikuttamalla kromatiinin rakenteeseen ja energia-aineenvaihduntaan. MAPK/ERK-signaloinnista riippuva metabolia kehittyvissä munuaisissa edistää nefronin esiastesolujen säilymistä erilaistumattomina säätelemällä proliinimetaboliaa sekä rajoittamalla pyruvaatin saatavuutta. Lisäksi tutkimukseni osoittavat, että GDNF transkription kvantitatiivinen nousu eli korkeat pitoisuudet pystyvät pidentämään esiastesolujen elinikää. Tutkimukseni tulokset tuottivat aiemmin tuntematonta tietoa munuaisten kehityksen säätelystä, mitä voidaan tulevaisuudessa käyttää munuaissairauksien diagnostisten ja/tai terapeuttisten strategioita suunnittelussa. MAPK/ERK-signaloinnin roolin ymmärtäminen munuaisten kehityksessä on ratkaisevan tärkeää, sillä se edistää munuaissairauksien taustalla olevien mekanismien ymmärrystä, joka puolestaan mahdollistaa uusien hoitostrategioiden kehittämisen