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

Abnormal 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.

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

Mammalian 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.

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

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.

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.

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

Mammalian 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.

In vivo measurements of kidney glomerular number and size in healthy and Os/+ mice using MRI

The development of chronic kidney disease (CKD) is associated with the loss of functional nephrons. However, there are no methods to directly measure nephron number in living subjects. Thus, there are no methods to track the early stages of progressive CKD before changes in total renal function. In this work, we used cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) to enable measurements of glomerular number ( Nglom) and apparent glomerular volume (aVglom) in vivo in healthy wild-type (WT) mice ( n = 4) and mice with oligosyndactylism (Os/+; n = 4), a model of congenital renal hypoplasia leading to nephron reduction. We validated in vivo measurements of Nglom and aVglom by high-resolution ex vivo MRI. CFE-MRI measured a mean Nglom of 12,220 ± 2,028 and 6,848 ± 1,676 (means ± SD) for WT and Os/+ mouse kidneys in vivo, respectively. Nglom measured in all mice in vivo using CFE-MRI varied by an average 15% from Nglom measured ex vivo in the same kidney (α = 0.05, P = 0.67). To confirm that CFE-MRI can also be used to track nephron endowment longitudinally, a WT mouse was imaged three times by CFE-MRI over 2 wk. Values of Nglom measured in vivo in the same kidney varied within ~3%. Values of aVglom calculated from CFE-MRI in vivo were significantly different (~15% on average, P < 0.01) from those measured ex vivo, warranting further investigation. This is the first report of direct measurements of Nglom and aVglom in healthy and diseased mice in vivo.

State of the Art on Kidney Development: How Nephron Endowment at Birth Can Shape Our Susceptibility to Renal Dysfunction Later in Life

In the present article, we discuss the following topics: (1) the fetal programming of adult kidney diseases and (2) the role of neonatologists in the regenerative renal medicine, based on the activation of resident renal SC. Here, we report the most important steps of our collaboration between neonatologists, nephrologists, and pathologists. Nephrologists should be more interested in clinical data regarding the first month of life in the womb of their adult patients, being particularly focused on birth weight and on the weeks of gestation at birth, without forgetting data regarding maternal status during gestation and neonatal asphyxia. Neonatologists should be aware that any preterm or low birthweight infant should be considered as a subject with fewer glomeruli, probably predicted to develop renal disease later in life.

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

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.