Tumor-free elongation of mammalian nephrogenesis by excess fetal GDNF

ABSTRACTDue to poor regenerative capacity of adult kidneys, nephron endowment defined by the nephrogenic program during the fetal period dictates renal and related cardiovascular health throughout life. We show that the neurotropic factor GDNF, which is in clinical trials for Parkinson’s disease, is capable of prolonging the nephrogenic program beyond its normal cessation without increasing the risk of kidney tumors. Our data demonstrates that excess GDNF expands the nephrogenic program by maintaining nephron progenitors and nephrogenesis in postnatal mouse kidneys. GDNF, through its transcriptional targets excreted from the adjacent epithelium, has a major effect on nephron progenitor self-renewal and maintenance; abnormally high GDNF inhibits nephron progenitor proliferation, but lowering its level normalizes the nephrogenic program to that permissive for nephron progenitor self-renewal and differentiation. Based on our results, we propose that the lifespan of nephron progenitors is determined by mechanisms related to perception of GDNF and other signaling levels.

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Age-Dependent Changes in the Progenitor Translatome Coordinated in part by Tsc1 Increase Perception of Signaling Inputs to End Nephrogenesis

10.1101/2020.03.12.989343 ◽

2020 ◽

Author(s):

Eric Brunskill ◽

Alison Jarmas ◽

Praneet Chaturvedi ◽

Raphael Kopan

Keyword(s):

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Age Dependent ◽

Cellular Translation ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

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Progenitor translatome changes coordinated by Tsc1 increase perception of Wnt signals to end nephrogenesis

Nature Communications ◽

10.1038/s41467-021-26626-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Alison E. Jarmas ◽

Eric W. Brunskill ◽

Praneet Chaturvedi ◽

Nathan Salomonis ◽

Raphael Kopan

Keyword(s):

Progenitor Cells ◽

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that translatome analysis in Tsc1+/− nephron progenitor cells from mice with elevated nephron numbers reveals how differential translation of Wnt antagonists over agonists tips the balance between self-renewal and differentiation. Wnt agonists are poorly translated in young niches, resulting in an environment with low R-spondin and high Fgf20 promoting self-renewal. In older niches we find increased translation of Wnt agonists, including R-spondin and the signalosome-promoting Tmem59, and low Fgf20, promoting differentiation. This suggests that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and possibly clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving synchronized differentiation. As predicted by these findings, removing one Rspo3 allele in nephron progenitors delays cessation and increases nephron numbers in vivo.

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Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment

eLife ◽

10.7554/elife.40392 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 11

Author(s):

Lori L O'Brien ◽

Alexander N Combes ◽

Kieran M Short ◽

Nils O Lindström ◽

Peter H Whitney ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cell ◽

Live Imaging ◽

Rna Seq ◽

Self Renewal ◽

Mammalian Kidney ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Cell Organization ◽

Nephron Progenitor

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.

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Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance

Journal of the American Society of Nephrology ◽

10.1681/asn.2021040525 ◽

2021 ◽

Vol 32 (11) ◽

pp. 2815-2833

Author(s):

Jun Li ◽

Jinshu Xu ◽

Huihui Jiang ◽

Ting Zhang ◽

Aarthi Ramakrishnan ◽

...

Keyword(s):

Progenitor Cell ◽

Kidney Development ◽

Regulatory Elements ◽

Proximal Promoter ◽

Specific Expression ◽

Self Renewal ◽

Content Type ◽

Nephron Progenitors ◽

Genome Wide ◽

Nephron Progenitor

BackgroundEya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown.MethodsWe engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo.ResultsEya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death–inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor–specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor–specific expression in response to Six2 activity.ConclusionsOur results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

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Postnatal prolongation of mammalian nephrogenesis by excess fetal GDNF

Development ◽

10.1242/dev.197475 ◽

2021 ◽

Vol 148 (10) ◽

Author(s):

Hao Li ◽

Kristen Kurtzeborn ◽

Jussi Kupari ◽

Yujuan Gui ◽

Edward Siefker ◽

...

Keyword(s):

Cardiovascular Health ◽

Postnatal Period ◽

Activity Level ◽

Ureteric Bud ◽

Negative Effects ◽

Multi Stage ◽

Nephron Progenitors ◽

Mechanistic Analysis ◽

Nephron Progenitor ◽

Progenitor Maintenance

ABSTRACT 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.

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Nephron progenitor commitment is a stochastic process influenced by cell migration

eLife ◽

10.7554/elife.41156 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Kynan T Lawlor ◽

Luke Zappia ◽

James Lefevre ◽

Joo-Seop Park ◽

Nicholas A Hamilton ◽

...

Keyword(s):

Cell Migration ◽

Progenitor Cell ◽

Computational Modelling ◽

Rna Seq ◽

Self Renewal ◽

Tissue Microenvironment ◽

Nephron Progenitors ◽

Nephron Progenitor ◽

Robust Regulation ◽

Over Time

Progenitor self-renewal and differentiation is often regulated by spatially restricted cues within a tissue microenvironment. Here, we examine how progenitor cell migration impacts regionally induced commitment within the nephrogenic niche in mice. We identify a subset of cells that express Wnt4, an early marker of nephron commitment, but migrate back into the progenitor population where they accumulate over time. Single cell RNA-seq and computational modelling of returning cells reveals that nephron progenitors can traverse the transcriptional hierarchy between self-renewal and commitment in either direction. This plasticity may enable robust regulation of nephrogenesis as niches remodel and grow during organogenesis.

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Abstract 9: Histone Deacetylases 1 And 2 Regulate Six2 Function To Maintain Nephron Progenitor Cells During Nephrogenesis

Hypertension ◽

10.1161/hyp.76.suppl_1.9 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Hongbing Liu ◽

Mahitha M Koduri ◽

Andrea Dragon ◽

Chao Hui Chen ◽

Samir S El-Dahr

Keyword(s):

Progenitor Cells ◽

Histone Deacetylases ◽

Target Genes ◽

Fusion Gene ◽

Gene Expression Pattern ◽

Proximity Ligation Assay ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitor

Low nephron endowment is strongly associated with cardiovascular disease, especially hypertension. Sine oculis homeobox 2 (Six2) is the master transcriptional regulator in balancing self-renewal and differentiation of nephron progenitor cells (NPCs) for appropriate nephron endowment. Loss of Six2 in mice causes early-onset loss of self-renewal and premature differentiation of NPCs. However, it is unclear how Six2 is functionally regulated during nephrogenesis. In vivo interaction of histone deacetylase1 and 2 (Hdac1/2) to Six2 was detected in developing kidney by co-immunoprecipitation and proximity ligation assay. Chromatin immunoprecipitation and DNA sequencing experiments in isolated E16.5 NPCs revealed 1,180 (84.58%) of the Six2 peaks overlapped with Hdac2 peaks, implying the involvement of Hdac1/2 in Six2 DNA binding and its function in NPCs. To test whether Hdac1/2 are required for Six2 function to regulate nephron formation, we employed Six2 GC mouse line, in which the eGFP and Cre fusion gene (GC) replaces and fully recapitulates the endogenous Six2 gene expression pattern. Analysis of kidneys at embryonic day (E) 19.5 and newborn (P0) showed that Six2 heterozygous (Six2 GC ) together with three alleles knockout of Hdac1/2 resulted in severely hypoplastic kidneys, while three alleles knockout of Hdac1/2 by transgenic Six2-Cre only led to very subtle phenotypes. Immunostaining at E 19.5 and P0 revealed about 50% reduction of Six2 level in the kidney of Six2 GC only and Six2 GC together with three alleles knockout of Hdac1/2 mice. In the kidneys of Six2 heterozygous mice, no change was observed for most of the NPC identity makers for self-renewal, including the three Six2 targets, Pax2, Sall1 and WT1. However, sequential removal of three alleles of Hdac1/2 of Six2 GC mice did not change Six2 protein level but significantly decreased the expression of Pax2, Sall1 and WT1, suggesting the requirement of Hdac1/2 for Six2’s function to transcriptionally activate the expression of its target genes. We also observed the premature differentiation and decreased nephron formation in mutant kidneys. Therefore, we conclude that Hdac1/2 are required for Six2’s function to promote NPC self-renewal and repress premature differentiation during nephrogenesis.

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Post-translational modifications by SIRT3 de-2-hydroxyisobutyrylase activity regulate glycolysis and enable nephrogenesis

Scientific Reports ◽

10.1038/s41598-021-03039-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Luca Perico ◽

Marina Morigi ◽

Anna Pezzotta ◽

Daniela Corna ◽

Valerio Brizi ◽

...

Keyword(s):

Kidney Development ◽

Renal Diseases ◽

Nephron Number ◽

Self Renewal ◽

Post Translational Modifications ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Lysine Residues ◽

Epigenetic Processes

AbstractAbnormal kidney development leads to lower nephron number, predisposing to renal diseases in adulthood. In embryonic kidneys, nephron endowment is dictated by the availability of nephron progenitors, whose self-renewal and differentiation require a relatively repressed chromatin state. More recently, NAD+-dependent deacetylase sirtuins (SIRTs) have emerged as possible regulators that link epigenetic processes to the metabolism. Here, we discovered a novel role for the NAD+-dependent deacylase SIRT3 in kidney development. In the embryonic kidney, SIRT3 was highly expressed only as a short isoform, with nuclear and extra-nuclear localisation. The nuclear SIRT3 did not act as deacetylase but exerted de-2-hydroxyisobutyrylase activity on lysine residues of histone proteins. Extra-nuclear SIRT3 regulated lysine 2-hydroxyisobutyrylation (Khib) levels of phosphofructokinase (PFK) and Sirt3 deficiency increased PFK Khib levels, inducing a glycolysis boost. This altered Khib landscape in Sirt3−/− metanephroi was associated with decreased nephron progenitors, impaired nephrogenesis and a reduced number of nephrons. These data describe an unprecedented role of SIRT3 in controlling early renal development through the regulation of epigenetics and metabolic processes.

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Loss of miR-17~92 results in dysregulation of Cftr in nephron progenitors

AJP Renal Physiology ◽

10.1152/ajprenal.00450.2018 ◽

2019 ◽

Vol 316 (5) ◽

pp. F993-F1005 ◽

Cited By ~ 6

Author(s):

Yu Leng Phua ◽

Kevin Hong Chen ◽

Shelby L. Hemker ◽

April K. Marrone ◽

Andrew J. Bodnar ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Kidney Disease ◽

Kidney Development ◽

The Self ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Increased Risk ◽

Renal Hypodysplasia

We have previously demonstrated that loss of miR-17~92 in nephron progenitors in a mouse model results in renal hypodysplasia and chronic kidney disease. Clinically, decreased congenital nephron endowment because of renal hypodysplasia is associated with an increased risk of hypertension and chronic kidney disease, and this is at least partly dependent on the self-renewal of nephron progenitors. Here, we present evidence for a novel molecular mechanism regulating the self-renewal of nephron progenitors and congenital nephron endowment by the highly conserved miR-17~92 cluster. Whole transcriptome sequencing revealed that nephron progenitors lacking this cluster demonstrated increased Cftr expression. We showed that one member of the cluster, miR-19b, is sufficient to repress Cftr expression in vitro and that perturbation of Cftr activity in nephron progenitors results in impaired proliferation. Together, these data suggest that miR-19b regulates Cftr expression in nephron progenitors, with this interaction playing a role in appropriate nephron progenitor self-renewal during kidney development to generate normal nephron endowment.

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