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org

Regulation of Nephron Progenitor Cell Self-Renewal

by Intermediary Metabolism
Jiao Liu,* Francesca Edgington-Giordano,* Courtney Dugas,† Anna Abrams,*
Prasad Katakam,‡ Ryousuke Satou,† and Zubaida Saifudeen*
*Department of Pediatrics, Section of Nephrology, †Department of Physiology, and ‡Department of Pharmacology,
Tulane University School of Medicine, New Orleans, Louisiana

ABSTRACT
Nephron progenitor cells (NPCs) show an age-dependent capacity to balance self-renewal with differen-
tiation. Older NPCs (postnatal day 0) exit the progenitor niche at a higher rate than younger (embryonic
day 13.5) NPCs do. This behavior is reflected in the transcript profiles of young and old NPCs. Bioenergetic
pathways have emerged as important regulators of stem cell fate. Here, we investigated the mechanisms
underlying this regulation in murine NPCs. Upon isolation and culture in NPC renewal medium, younger
NPCs displayed a higher glycolysis rate than older NPCs. Inhibition of glycolysis enhanced nephrogenesis
in cultured embryonic kidneys, without increasing ureteric tree branching, and promoted mesenchymal-to-
epithelial transition in cultured isolated metanephric mesenchyme. Cotreatment with a canonical Wnt
signaling inhibitor attenuated but did not entirely block the increase in nephrogenesis observed after
glycolysis inhibition. Furthermore, inhibition of the phosphatidylinositol 3-kinase/Akt self-renewal signaling
pathway or stimulation of differentiation pathways in the NPC decreased glycolytic flux. Our findings
suggest that glycolysis is a pivotal, cell-intrinsic determinant of NPC fate, with a high glycolytic flux sup-
porting self-renewal and inhibition of glycolysis stimulating differentiation.

J Am Soc Nephrol 28: 3323–3335, 2017. doi: https://doi.org/10.1681/ASN.2016111246

Nephron abundance varies among individuals and
populations, with demonstrated influence of genet-
ics and maternal nutritional status on nephron
number in humans.1–4 Nephron progenitor cell
(NPC) availability during kidney development is a
major determinant of nephron number at birth.
Low nephron endowment results in hypertension
and CKD, both clinically significant diseases
without a cure. Self-renewal of NPCs ensures a sup-
ply of cells for nephrogenesis until the cessation of
nephrogenesis at P4 in mice and gestational age
of 35 weeks in humans. 5,6 Despite the critical im-
portance of NPC availability for renal function
across the life course, little is known about the
mechanisms controlling NPC self-renewal versus
pool, and differentiation to epithelial structures of
the nascent nephron (Figure 1).8–10 Supported by
the nephrogenic niche as the source for growth fac-
tors and nutrients, activation of signaling and met-
abolic pathways in NPCs engages renewal-specific
gene expression that sustains the maintenance of
the NPC pool for nephrogenesis. A subset of NPCs
gradually exit the self-renewing pool to the transit
compartment that undergoes differentiation.11,12
Along with a decreased proliferation rate, NPCs
Received November 26, 2016. Accepted June 9, 2017.
Published online ahead of print. Publication date available at
www.jasn.org.

differentiation. Present address: Dr. Anna Abrams, Department of Pediatrics,

NPCs reside in the nephron progenitor niche,
which includes the cap mesenchyme (CM), the ter-
minal ureteric tips and branch, and the surrounding
stroma.7 NPCs are identified by their combined ca-
pacity for self-renewal to expand the progenitor cell
Children’s National Medical Center, Washington, DC.
Correspondence: Dr. Zubaida Saifudeen, Department of Pedi-
atrics, Tulane University School of Medicine, 1430 Tulane Ave-
nue, New Orleans, LA 70112. Email: zubisaif@tulane.edu
Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 3323–3335, 2017 ISSN : 1046-6673/2811-3323 3323

 BASIC RESEARCH www.jasn.org
Figure 1. Experimental scheme of NPC isolation for metabolic profiling and ddPCR.
Cited1+/Six2+ NPCs from different ages were isolated by MACS (see Concise Methods) by
negative depletion. Isolated NPCs were expanded and used for extracellular flux analysis
or ddPCR. Also see Supplemental Figure 1, A and B. CS, comma-shaped body; ECAR,
extracellular acidification rate; OCR, oxygen consumption rate; PA, pretubular aggregate;
RV, renal vesicle; UB, ureteric bud.
have increased exit rates from the renewing niche as develop-
ment progresses.13 This shift in behavior is largely cell-intrinsic,
demonstrated by heterochronic transplantation of young and
old NPCs into young niches.13 The young and old NPCs also
exhibit differential growth factor signaling and distinct transcrip-
tional profiles.13 Therefore, the young NPCs from early (E13.5)
CM are predominantly self-renewing, whereas old NPCs from
late CM (E19.5 and P0) are poised to differentiate en masse.
The self-renewing Cited1+/Six2+ cells are refractory to differ-
entiation signals, whereas the transit Cited12/Six2+ population
is induced by cell-autonomous and nonautonomous differenti-
ation signals to undergo differentiation to Six2low/Wnt4+ cells.
These further differentiate to the Wnt4/Lef1+ pretubular aggre-
gates (Figure 1), which undergo epithelialization (to the renal
vesicle) and progressive maturation.14 Six2 expression is essen-
tial to maintain the progenitor state.15 FGF/PI3K-Akt, Bmp7/
MAPK/JNK/Jun-ATF2/Smad, and Wnt9b/b-catenin signaling
pathways are required for NPC self-renewal and differentiation,
demonstrated by pharmacologic or genetic inactivation of dif-
ferent steps of these pathways, resulting in NPC renewal and
differentiation defects.10,14,16,17
Bioenergetic pathways have emerged as important regula-
tors of stem cell fate. Besides supporting the rapidly changing
energy needs of the developing embryo, cellular metabolism
provides macromolecules required to support self-renewal,
proliferation, differentiation, or quiescence. Moreover, several
metabolites that are intermediates of cellular metabolism path-
ways are substrates or cofactors for chromatin modifying en-
zymes (e.g., acetyl CoA, folate and methyl group donors, or
a-ketoglutarate in histone demethylation).18,19 Thus, perturbations
in cellular energy pathways and consequently metabolite avail-
ability directly influence the epigenome and can have
3324 Journal of the American Society of Nephrology
drastic consequences on the ability of the
stem cell to self-renew and differentiate,
suggesting that the metabolic status of a
stem cell may actively dictate cell fate rather
than just be a marker of differentiation.20–23
In this study we demonstrate that NPC
age and fate are intimately linked to interme-
diary metabolism pathways. Young NPCs
demonstrate a high glycolysis and high en-
ergy state compared with old NPCs. Inhibi-
tion of the PI3K/Akt self-renewal signaling
pathway or stimulation of differentiation in
the young NPCs decreased their glycolysis
flux. Ex vivo inhibition of glycolysis aug-
mented nephrogenesis, with premature
NPC differentiation and depletion indepen-
dent of ureteric bud branching in E12.5
kidneys. Our data demonstrate an intrinsic
difference between young and old NPCs and
suggest intermediary metabolism as a
balancing mechanism for NPC fate.
RESULTS
Distinct Energy Metabolism Profiles of Young and
Old NPCs
Glycolysis
To unravel the underlying mechanism of the recently docu-
mented differential cellular behavior of young (E13.5) versus
old NPCs (P0) in the nephrogenic niche,13 we hypothesized on
the basis of stem cell literature that young NPCs preferentially
utilize glycolysis, an established driver of self-renewal in em-
bryonic stem cells, to maintain their self-renewing state in
contrast to old NPCs that are predominantly exiting the self-
renewing niche. We therefore undertook an assessment of the
metabolic profiles of young versus old NPCs. Using magnetic-
activated cell sorting (MACS) as described by Brown et al.,11,14
NPCs were isolated to near 100% purity for extracellular flux
measurements (Supplemental Figure 1). Isolated cells were
cultured in the defined NPC expansion medium (NPEM11),
which simulates nephron progenitor niche conditions for
maintaining undifferentiated progenitor proliferation and
self-renewal. Comparable levels of Cited1 and Six2 expression
(Supplemental Figure 1B) validate the successful isolation and
expansion of the same subpopulations of undifferentiated
NPCs from E13.5 and P0 kidneys. Moreover, young and old
NPCs demonstrate similar cell growth metrics (Supplemental
Table 1).
As old time points, we chose E19.5 and P0 as time of harvest
to evaluate whether prenatal and postnatal NPCs exhibit dif-
ferences in extracellular metabolic flux. Glycolysis is signifi-
cantly higher in E13.5 NPCs than E19.5 and P0 NPCs
(P,0.05), shown by increased extracellular acidification rate
(ECAR) after the addition of a saturating dose (10 mM) of
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glucose-dependent ECAR increase (Figure

2, A and B), to specifically allow measure-
ment of glycolysis-mediated acidification of
the media. To ascertain that the glycolysis
flux difference between young and old
NPCs is not an artifact of cell expansion,
we measured glycolysis in primary E13.5
and E19.5 NPCs that were not passaged af-
ter isolation. Glycolysis is significantly
higher in E13.5 than in E19.5 NPCs (Figure
2F), as with expanded cells. Therefore, high
glycolysis appears to be a distinguishing
metabolic feature of young NPCs.

Oxidative Phosphorylation
Next, we evaluated oxidative phosphoryla-
tion (OxPhos) in E13.5, E19.5 and P0 NPCs
by measuring their oxygen consumption
rate (OCR) and mitochondrial function
by employing the mitochondrial stress
test (Figure 3). Addition of specific inhibi-
tors of mitochondrial function during the
test revealed basal and maximal mitochon-
drial respiration, the spare respiratory ca-
pacity, ATP-linked and -unlinked (proton
leak) respiration, and nonmitochondrial
respiration (Figure 3, A and B). Younger
NPCs registered significantly higher base-
line OCR than older NPCs (Figure 3C).
Maximal OxPhos capacity was also signif-
icantly higher in younger NPCs (Figure
Figure 2. Young (E13.5) NPCs have a significantly higher glycolytic rate and capacity than 3D). Higher ECAR and OCR are suggestive
old (E19.5 and P0) NPCs. (A) Graph to explain basal, maximal, and reserve glycolysis of higher ATP production in E13.5 NPCs.
capacity measurements. (B–E) Measurement of ECARs in real time in E13.5, E19.5, and P0 Accordingly, 30% higher ATP levels were
NPCs. Each data point is a biologic replicate. (B) Graph plot of a representative ECAR recorded in younger NPCs (Figure 3E).
measurement from E13.5, E19.5, and P0 NPCs. (C) Basal glycolysis rate, measured after However, there was a significantly higher
addition of glucose, is significantly lower in P0 NPCs. *P,0.05 (D) Maximal glycolytic OCR/ECAR ratio in E19.5 NPCs, showing
capacity, measured after inhibiting mitochondrial oxygen consumption by oligomycin that old NPCs are more dependent on OxPhos
addition, is significantly lower in old than young NPCs. *P,0.05; ***P,0.001. (E) than glycolysis (Figure 3F).
Glycolysis reserve (excess glycolytic capacity) is significantly higher in E13.5 young NPCs
than old NPCs. **P,0.01. (F) Glycolysis in primary E13.5 and E19.5 NPCs that were not
Inhibition of Glycolysis Promotes NPC
passaged after isolation. Glycolysis is significantly higher in E13.5 than in E19.5 NPCs.
n=4 biologic replicate experiments. Paired t test, *P,0.05. Plotted values were obtained
Differentiation in Embryonic Kidneys
from three to five biologic replicate experiments. Error bars indicate SEM. P values were Older NPCs (P0) demonstrate an increased
obtained by performing t test (C) or one-way ANOVA (D and E). predilection to exit self-renewal and differ-
entiate than younger (E13.5) NPCs.13 Be-
cause young NPCs demonstrate increased
glucose (Figure 2, B and C). Pharmacologic modulation of glycolysis compared with old NPCs (Figure 2), we hypothe-
glycolysis allowed calculation of the glycolytic flux and glyco- sized that decreasing the glycolysis flux would switch E13.5
lytic capacity. As expected, addition of oligomycin to inhibit NPC behavior from young to old, i.e., from predominantly
mitochondrial respiration further augments the glycolytic flux self-renewing toward differentiation. To test this idea, we
as the cells try to maintain cellular ATP homeostasis, revealing added glycolysis inhibitor YN1 (Figure 4A) to culture media.
the cellular glycolytic capacity (Figure 2D). In line with the Dose-dependent reduction in glycolysis rate was achieved,
higher flux, the glycolytic capacity and reserve of younger cells monitored as a decrease in ECAR measurements (data points
is also higher than that of older cells (Figure 2, D and E). Ad- after glucose addition, Supplemental Figure 2A) after 18 hours
dition of glucose analog 2-deoxyglucose completely inhibits of treatment with YN1. Addition of YN1 to organ culture

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Supplemental Figure 2C). The induced

nephrons continue to mature, demonstrated
by the significant increase of proximal tubule
marker lotus tetragonolobus lectin staining
72 hours after YN1 treatment (Figure 4, E
and F). The CM in YN1-treated kidneys
appeared diffused and diminished, and ex-
hibited ectopic Lhx1 staining (Figure 5A).
Markers for cell proliferation (phospho-
Histone 3) and apoptosis (PARP) were
unchanged between control kidneys and
those with 24 hours of YN1 treatment (Fig-
ure 5, B and C). Lack of change in prolifera-
tion rate after 24 hours of YN1 treatment was
confirmed by flow cytometry–based cell-cycle
analysis of Six2GFP+ NPCs from E12.5 kid-
neys (Supplemental Figure 3). Quantification
showed a decrease in Six2+ cell number that is
statistically significant after 48 hours but not
24 hours of YN1 treatment (Figure 5, D and
E). As neither proliferation nor survival of
NPCs was altered after 24 hours of YN1 treat-
ment, the gradual depletion of the CM is
likely from increased differentiation (exit) of
NPCs from the renewing pool.
Wnt/b-catenin signaling induces the
Cited12/Six2+ NPCs to undergo nephro-
genesis.24 Increased foci of Wnt4 mRNA
expression in E12.5 kidneys accompanied
the increase of Lhx1+ nascent nephrons
after YN1 treatment, suggesting that the
Figure 3. Young (E13.5) NPCs have a significantly higher basal and maximal respiratory increased differentiation is b-catenin–
capacity than old (E19.5 and P0) NPCs. (A) Measurement of OCR in real time in young and dependent (Figure 6B). To test this impli-
old NPC, under basal conditions (before oligomycin) and in response to the indicated
cation, we cotreated embryonic kidney
mitochondrial inhibitors. (B) Sequential addition of indicated mitochondrial inhibitors
cultures with glycolysis and canonical
reveal different aspects of mitochondrial respiration. Addition of the protonophore FCCP
reveals the maximal respiratory capacity of the cell. (C and D) Basal respiration decreases
Wnt signaling inhibitors, YN1 and IWR1,
significantly during NPC aging (C), as does the maximal respiratory capacity (D). *P,0.05. respectively. Addition of IWR1 reduced
(E) Young NPCs have significantly higher ATP levels than old NPCs. Luminescence was YN1-induced nephrogenesis (Figure 6, C
recorded as counts per second (CPS). ****P,0.001. (F) OCR/ECAR ratio is significantly and D), suggesting Wnt/b-catenin signal-
higher in E19.5 NPCs. *P,0.05. OCR measurements are from two to five biologic rep- ing is a likely mediator of increased neph-
licates. ATP measurements are from three biologic replicates with three to six technical rogenesis. However, the significant increase
replicates per assay. Error bars indicate SEM. P values were obtained by performing one- in Lhx1+ nascent nephrons on addition of
way ANOVA (C) or paired t test (D–F). YN1 to IWR1-treated kidneys in compari-
son to IWR1-only treatment suggests dif-
media for 24–72 hours enhanced nephrogenesis (Figure 4, B, C ferentiation may occur via a b-catenin–independent pathway
and E, Supplemental Figure 2C). The increase in nephrogenesis as well.
was observed with up to 25 mM YN1, whereas high-dose To determine whether the decrease in glycolysis is sufficient
(100 mM) YN1 treatment proved to be lethal (Supplemental to stimulate differentiation gene expression in isolated NPCs,
Figure 2B). As our aim was to achieve inhibition of glycolysis we assessed expression of renewal and differentiation genes by
and not complete repression, with the expectation that this droplet digital PCR (ddPCR)(Supplemental Figure 4). Although
would result in lethality, we used 5–25 mM YN1 to achieve ap- addition of YN1 to E13.5 NPCs growing in NPEM decreased
proximately 30% reduction in glycolysis (see below, and Supple- glycolysis, differentiation gene expression was not induced (data
mental Figure 2A). Note that the increase in nascent nephrons not shown). Media supporting renewal and inhibiting differenti-
(Lhx1+) is not accompanied by an increase in ureteric bud tip ation is not conducive to induction of differentiation genes. There-
number after 24 hours of YN1 treatment (Figure 4, C and D, fore, we added YN1 to DMEM/F12 without (DMEM–CHIR)

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or with CHIR (DMEM+CHIR). Cited1 and

Six2 expression did not show an additional
decrease with YN1 than that observed with
DMEM–CHIR (Figure 7A). However,
b-catenin target gene expression (Wnt4
and Lef1) is stimulated comparably to that
with low-dose 0.1 mM CHIR (Figure 7B).
Simultaneous addition of 10 mM YN1 and
low-dose CHIR (0.1 mM) further upregulat-
ed Wnt4, Lef1, Lhx1, and Jag1 expression
than with either treatment alone, indicating
an additive effect of YN1 and low-dose
CHIR on differentiation gene expression in
isolated NPCs. Maximal expression of the
four differentiation marker genes was ob-
tained with low-dose 0.1 mM CHIR with
10 mM YN1, and is even greater than expres-
sion with 0.5 mM CHIR treatment.
To evaluate whether YN1 treatment re-
duces the requirement for CHIR for epithe-
lial induction, E11.5 mesenchyme cultures
were treated with YN1 and/or CHIR. Lhx1
expression was used as a marker of induc-
tion because Lhx1 expression precedes
epithelialization. Mesenchyme cultures
treated with YN1 alone demonstrated Lhx1
immunostaining after 48 hours of YN1 treat-
ment, which was enhanced in cultures co-
treated with YN1 and CHIR (low dose,
0.01 and 0.1 mM) (Figure 7C). Addition of
low-dose CHIR alone did not induce Lhx1
expression, whereas high-dose CHIR (1 mM)
induced Lhx1 expression (data not shown).
Therefore, YN1 treatment facilitates differ-
entiation by reducing the requirement for
CHIR for epithelial induction of the meta-
nephric mesenchyme.

Inhibition of PI3K/Akt Signaling or

Activation of Differentiation
Programs Reduces the Glycolytic Flux
in NPCs
Figure 4. Inhibition of glycolysis promotes nephrogenesis in embryonic kidneys. (A) FGF/PI3K and BMP/MAPK pathways
YN1 inhibits PFKFB3, the enzyme required for generation of F-2, 6-bisP, the potent
maintain NPCs in a self-renewing state,
stimulator of glycolysis via PFK1.45 (B) Addition of YN1 to organ culture media for 24
whereas decreased PI3K or activation of
hours enhanced nephrogenesis, visualized by Lhx1 immunostaining. Number of Lhx1+
structures are indicated. (C) Significant increase in Lhx1(+) nascent nephrons after 24
Bmp/Smad signaling promotes NPC differ-
hours of YN1 treatment; ***P,0.001. (D) Ureteric bud tip number did not show a entiation, 11,14,25,26 similar to our treat-
significant change after 24 hours of YN1 treatment. (E) The induced nephrons continue ments with glycolysis inhibitor YN1. We
to mature, demonstrated by increased staining for proximal tubule marker lotus tet- therefore hypothesized that self-renewing
ragonolobus lectin (LTL) detected after 72 hours of YN1 treatment. (F) Increased LTL signaling pathways stimulate glycolysis for
staining in (E) is statistically significant. *P,0.05. Error bars indicate SEM. P values NPC maintenance; thus, inhibition of these
were obtained by performing paired t test. 43 magnification. Also see Supplemental self-renewal signals in NPCs should result
Figure 2. in decreased glycolysis. Indeed, addition of
PI3K inhibitor LY294002 to NPCs signifi-
cantly decreased glycolysis, similar to the

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of LY-treated NPCs was confirmed by in-

duction of Lhx1 expression (Supplemental
Figure 5E), as demonstrated by Lindstrom
et al.26 In contrast to the inhibition, activa-
tion of PI3K/Akt pathway by addition of
phosphatase and tensin homolog (PTEN)
inhibitor bpV(Phen) to NPEM increased
ECAR (Figure 8, A and B). PTEN inhibits
the PI3K signaling pathway via its lipid
phosphatase activity, which prevents the
phosphorylation and activation of Akt by
PIP3 and PDK1.27 Thus, the analogous ef-
fect of PI3K inhibition or activation on gly-
colysis levels suggests that the glycolytic
flux in NPCs is downstream to PI3K/Akt
signaling.
We next examined whether NPC growth
in differentiation inducing DMEM/F12 plus
3 mM CHIR14 media altered glycolysis. We
confirmed decrease in Cited1 and Six2 ex-
pression and increase of Wnt4 expression
(Supplemental Figure 6). In addition to de-
creasing the basal glycolysis flux (Figure 8B),
induction of differentiation by DMEM–
CHIR or PI3K inhibition also decreased the
maximal glycolysis capacity of NPCs (Figure
8C). Note that old NPCs (E19.5 and P0)
demonstrate a lower glycolytic capacity
than younger NPCs (E13.5) (Figure 2D).

DISCUSSION

NPCs have a limited life span. The mecha-

Figure 5. Increased nephrogenesis depletes the CM in YN1-treated kidneys. (A) The nistic aspects of what determines NPC ag-
CM (Six2+, green) exhibits ectopic Lhx1 (white arrow) staining after 24 hours of YN1 ing are unknown. The dissimilar behavior
treatment of E12.5 kidneys. 603 magnification. (B and C) No significant difference in patterns of young and old NPCs, namely
proliferation or apoptosis rate was observed in CM of YN1-treated kidneys. (B) Pro- decreased proliferation and increased exit
liferation was quantified by counting phosphohistone 3(+) immunostained cells in
rate from the progenitor niche of the old
Six2+ CM. (C) Apoptosis was quantified by counting PARP(+) cells in Six2(+) CM. Each
NPC, is reflected in the differential tran-
data point shows number of positive cells per 1000 Six2+ cells, from n=3–4 biologic
replicates. (D and E) Quantification by flow cytometry of Six2GFP+ cells from kidneys script profiles and utilization of different
treated with YN1 for 24 hours (n=9 kidney pairs) or 48 hours (n=6 kidney pairs). A signaling pathways.13 Here, we report that
significant decrease in Six2+ cell number 48 hours after YN1 treatment, but not at 24 native (freshly harvested) young and old
hours. Each data point represents GFP+ cells per kidney. Error bars indicate SEM. NPCs have distinct metabolic profiles,
P values were obtained by performing paired t test. *P,0.05. with young NPCs displaying a higher en-
ergy state as a result of significantly higher
glycolysis and mitochondrial respiration
rate decrease observed with addition of glycolysis inhibitor rates. Higher glycolysis rate in young NPCs appears to be a
YN1 (Figure 8). Decreased P-Akt immunostaining confirmed cell-intrinsic function as it persists when both young and old
inhibition of PI3K activity after LY294002 addition to culture NPCs were cultured in the same NPEM. Moreover, manipu-
media (Supplemental Figure 5, A and B). The ability of LY- lating intermediary metabolism shifts the balance between
treated NPCs to mount a compensatory response to metabolic stem cell self-renewal and differentiation. Interestingly, com-
stress and basal OCR comparable to YN1- or Wnt9b-treated parison of transcriptomes of embryonic and postnatal kidneys
NPCs confirmed the viability of NPCs treated with 10 mM by microarray analysis revealed decreased expression of several
LY294002 (Supplemental Figure 5, C and D). Differentiation glycolytic pathway genes between P0 and P2 mouse kidneys, but

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Figure 6. Inhibition of glycolysis stimulates b-catenin–dependent differentiation of NPCs. (A and B) Increased Wnt4 mRNA expression
accompanies the increased nephrogenesis observed after 24 hours of YN1 treatment in E12.5 kidneys, shown by whole mount in situ
hybridization. (C and D) Cotreatment of E12.5 kidneys with YN1 and b-catenin inhibitor IWR1 for 24 or 48 hours. Glycolysis inhibitor
YN1-mediated nephron induction is abrogated by addition of 2 mM IWR1. Lhx1+ nascent nephrons were counted after indicated treatments
and mean number is shown in the bar graph. Data from at least two independent experiments, with at least n=3 kidneys per treatment. Error
bars indicate SEM. P values were obtained by performing the paired t test. *P,0.05; **P,0.01. 103 magnification.

not at earlier ages.28 It is possible that the decrease in glycolysis
we observe in functional assays between young and old embry-
onic age NPCs is from post-translational regulation, leaving the
pathway open to modulation by niche signals. However, post-
natally toward the cessation of nephrogenesis as the progeni-
tors differentiate the reduction in enzyme expression is likely
permanent and achieved transcriptionally.
Metabolism has emerged as an active regulator of stem cell
fate, and niche-directed (nutrients, oxygen tension, growth
factors) changes in metabolic flux and redox homeostasis di-
rect cell fate decisions from renewal to differentiation. Methods
promoting a metabolic shift toward glycolysis facilitate stem-
ness maintenance and are a critical step in reprogramming
somatic cells to induced pluripotent stem cells.20,21,23 In

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Figure 7. Glycolysis inhibition reduces CHIR requirement to induce differentiation gene expression. (A and B) Quantitation of mRNA by
ddPCR on RNA isolated from NPCs cultured in differentiation medium. YN1 was added to media without (DMEM–CHIR) or with in-
dicated amount of CHIR (DMEM+CHIR). Data show mean6SD from technical replicates from pooled kidneys. Also see Supplemental
Figure 3. (C, a–f) Isolated E11.5 mesenchyme cultured for 48 hours as indicated. Immunostaining for Six2 (red) and Lhx1 (green).
103 magnification.

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glycolysis-driven acetyl CoA production and

histone acetylation in human and mouse em-
bryonic stem cells.30 Inhibition of glycolysis
leads to histone deacetylation and differenti-
ation, whereas decrease in NAD+, a cofactor
for the sirtuin class of HDACs, inhibits his-
tone deacetylation. 19,30,31 O-linked N-
acetylglucosamine protein modification
directly reflects changes in ambient glucose
levels; occurrence of this modification on his-
tones is another link to nutrient availability–
directed chromatin modification by
intermediary metabolism. 32 Hence, the
metabolite milieu directly influences epi-
genetic modifications and associated
changes in transcription and cell fate.
The documented heterogeneity of the
NPC pool and changes in signaling path-
ways may well result from focal variations
in microniche biochemistry. In line with
the Barker hypothesis on the in utero origin
of adult disease,33–35 changes in gestational
metabolism that accompany maternal nutri-
tion habits are recognized to affect the met-
abolic status of stem cell populations in the
developing fetus.
NPC self-renewal is dependent on PI3K
activity.26 The PI3K/Akt pathway positively
regulates glycolysis at multiple points in the
pathway: it facilitates glucose uptake36,37;
Figure 8. Inhibition of PI3K/Akt signaling or activation of differentiation programs stimulates hexokinase activity38; stimulates
decreases glycolysis in NPCs. (A) Pharmacologic inhibition or activation of the PI3K/ PFK1, the rate-determining step of glycol-
Akt pathway by LY294002 or bpV(Phen), respectively. (B) Addition of 10 mM LY294002 ysis38; and activates expression and activity
to NPEM for 20 hours decreased glycolysis (ECAR) significantly in E13.5 NPCs. In of rate-regulating pyruvate kinase isoform
contrast, addition of 30–60 mM bpV(Phen) (Phen, PTEN inhibitor) significantly in-
M2 (PKM2), which is expressed exclusively
creased ECAR. Growth in differentiation media (DMEM+CHIR) also significantly re-
in embryonic, proliferating, and cancer cells
duced NPC glycolysis. YN1 was added to NPEM as positive control for monitoring
decrease in ECAR measurement. Error bars indicate SEM. *P,0.05; **P,0.005, t test.
via the mTOR/Hif1a/cMyc pathway.39 On
(C) Maximal glycolysis capacity of NPCs decreased after culture in NPEM with PI3K the basis of our data, we propose a model
inhibitor LY294002 (10 mM) or YN1 (5 or 20 mM), whereas addition of PTEN inhibitor whereby a high glycolysis flux is an essential
bpV(Phen) significantly increased the glycolytic capacity. Replacement of NPEM by intermediary of growth factor PI3K signaling–
DMEM+CHIR differentiation medium showed a comparable decrease in glycolytic mediated NPC self-renewal (Figure 9A).
capacity as with LY or YN1. Error bars indicate SEM. All changes relative to ECAR PI3K and glycolysis downregulation
measured in NPCs cultured in NPEM. *P,0.05; **P,0.01 by t test. Also see Sup- switches the NPC to a Wnt/b-catenin–
plemental Figure 4. dependent (26 and this study) or –independent
differentiation program in embryonic kid-
contrast, promoting mitochondrial biogenesis and OxPhos in neys. As PI3K inhibition results in decreased glycolysis, we
embryonic and somatic stem cells accelerates their commit- posit that the PI3K pathway potentiates NPC renewal by pro-
ment to a more differentiated state.23 Low oxygen concentra- moting glycolysis (Figure 9, A and B). Single-cell transcrip-
tions (1%–5%) are conducive to stem cell proliferation and tomics shows changes in mTOR, growth factor signaling, and
maintenance of pluripotency, whereas higher levels promote dif- extracellular matrix pathway genes, as well as in chromatin
ferentiation.29 Nutrient availability in the niche also influences modifiers between young and old NPCs.13 As mTOR activ-
cellular metabolic pathway usage. How does intermediary me- ity may also support mitochondrial respiration,40 an increase
tabolism direct cell fate? In addition to ATP, metabolism pro- in mTOR pathway gene expression in P0 (old) NPCs relative
duces metabolites that are critical substrates or cofactors for to young (E13.5) NPCs may restrict the old cells to a lower
essential processes. A direct link was demonstrated between glycolytic flux.

J Am Soc Nephrol 28: 3323–3335, 2017 Glycolysis Promotes Nephron Progenitor Cell Self-Renewal 3331
 BASIC RESEARCH www.jasn.org

pathways in the NPC (for example, Bmp7/

MAPK, YAP/TAZ, or Wnt17,41) and extend
these analyses to human induced pluripo-
tent stem cells and NPCs. Strategies to ex-
pand NPCs in vivo or in vitro will benefit
greatly from understanding how cellular
metabolic processes influence the balance
between NPC renewal and differentiation.
For example, glycolysis downmodulation
may be beneficial in the treatment of
Wilms’ tumor, where stimulation of cellu-
lar differentiation may be a desirable out-
come. In contrast, glycolysis activators or
inhibitors of OxPhos may favor NPC ex-
pansion.
While this article was under revision two
articles were published that address the role
of a glycolysis gradient in body axis elonga-
tion42 and presomitic mesoderm differen-
tiation.43 In the former publication, the
authors demonstrate a gradient of glycoly-
sis enzymes regulated by FGF signaling in
the mouse and chick embryo tail bud, and
that inhibition of glycolysis reduces cell
motility, Wnt signaling, and changes cell
fate decisions. The focus to date on genetic
signals has allowed mapping growth factor
signaling and transcriptional networks that
regulate NPC renewal and differentiation.
This study adds another dimension that
needs to be considered when describing
Figure 9. Role of glycolysis in NPC self-renewal. (A) Schematic illustration of NPC fate the self-renewing versus differentiating po-
specification by glycolysis. Per our model, we propose a high glycolysis flux is an tential of NPCs.
essential mediator of PI3K signaling–driven NPC self-renewal. Stimulation of PI3K/Akt
signaling increases glycolysis (green box arrow). High glycolysis flux in self-renewing
NPCs may inhibit Wnt-induced differentiation that occurs on glycolysis inhibition. CONCISE METHODS
MAPK, cMyc, and mTOR signaling are also known stimulators of glycolysis and may
contribute to the glycolysis flux (not tested in this study), denoted by blue arrows. (B)
Both PI3K and glycolysis downregulation switches the NPC to a Wnt-dependent dif-
MACS and Expansion of NPCs for
ferentiation program in embryonic kidneys. As PI3K inhibition results in decreased Extracellular Flux Measurements and
glycolysis, we posit that the PI3K pathway potentiates NPC renewal by promoting RNA Isolation
glycolysis. E13.5, E19.5, or P0 CD1 mouse kidneys were dis-
sected into HBSS (Invitrogen) and treated with 1%
pancreatin/0.25% collagenase mixture to collect the
A dissociated response in expression of renewal and differ- suspension of nephrogenic zone cells as described.11 NPCs were enriched
entiation genes was observed with both CHIR and YN1 treat- by negative bead depletion with MACS–Phycoerythrin-conjugated
ment of NPCs in that the mRNA of renewal genes did not antibodies against endothelial progenitors (anti-CD105), RBCs and
decrease although differentiation gene expression (Wnt4, erythroid cells (anti-TER119), cortical interstitium (anti-CD140a),
Lef1, Lhx1, and Jag1) increased (Figure 7, A and B). Whereas and renal epithelial cells (anti-CD326) following the manufacturer’s
in vivo more subtle gradations of signaling and metabolic (Miltenyi Biotec) and published protocol.11 Isolated cells were cul-
pathways influence the temporal and spatial pattern of gene tured on Matrigel-coated plates in a monolayer in the APEL-based
expression, the manipulations here are tools to demonstrate a (Stem Cell Technologies) NPEM exactly as described.11 The NPCs
proof of principle and do not necessarily reflect the physio- were expanded for two to three passages for all experiments, and split
logic situation. It would be of interest to compare the meta- at 60%–70% confluence. Animal protocols utilized in this study were
bolic profiles of NPCs in different expansion or differentiation approved by and in strict adherence to guidelines established by the
media, and in response to other critical regulatory signaling Tulane University Institutional Animal Care and Use Committee.

3332 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 3323–3335, 2017

Extracellular Flux Measurements
Kidneys were pooled from multiple litters for E13.5 NPC isolation.
Cells were seeded 18–20 hours before the assay, at 150,000 cells per
well, on Matrigel-coated Seahorse microplates for flux measurements
on the XFe24 Extracellular Flux Analyzer (Agilent Seahorse Technol-
ogies). For flux measurements after media manipulations or glycol-
ysis or signaling pathway inhibition, cells were plated 40 hours before
the assay in NPEM, and media changed 18–20 hours before the assay
with the indicated treatments. Optimal cell density was determined
empirically, on the basis of attaining flux measurements in the linear
range. To probe glycolysis and OxPhos beyond basal measurements,
glycolysis and mitochondrial stress tests, respectively, were done. The
stress tests require sequential addition of specific pathway inhibitors
to query various aspects of these two metabolic pathways. All meta-
bolic inhibitors were titrated to obtain maximal response in the ab-
sence of toxicity. The XF sensor cartridges were hydrated overnight at
37°C without carbon dioxide in the Seahorse XF calibrant solution, as
recommended in the manufacturer’s protocol. For glycolysis rate
measurements, NPEM was replaced with the XF assay medium (un-
buffered DMEM [pH 7.4] with 2 mM glutamine) and the microplate
with cells was placed in a 37°C incubator without carbon dioxide.
Glucose (10 mM, final), oligomycin (2 mg/ml), and 2-deoxyglucose
(0.1 M final) were added to the ports at concentrations to achieve
appropriate final dilutions. For OCR measurements, the assay
medium was Seahorse XF base medium (unbuffered DMEM) supple-
mented with 3–5 mM pyruvate, 5 mM D-glucose, and 2 mM
L-glutamine (pH 7.4). Oligomycin (1 mg/ml), FCCP (1.5 mM), rotenone
(0.1 mM), and antimycin (1 mM) were added to the ports. Both ECAR
and OCR measurements were performed at 8-minute intervals (2 min-
utes mixing, 2 minutes recovery, and 4 minutes measuring) for 3 hours,
using the Seahorse XF24 analyzer. Measurements were recorded from
three to five biologic replicates with three technical replicates per exper-
iment from young and old NPCs. All data are normalized to cell number.
Embryonic Kidneys and Metanephric Mesenchyme
Culture and Immunofluorescence Staining
E12.5 kidneys were collected from CD1 mice for culture in DMEM/
F12 complete media as previously described.44 Glycolysis inhibitor
YN1 (Sigma) was added at 5–20 mM for the times indicated. YN1 was
developed using a structure-based approach and has high specificity
to PFKFB3 over the other isoenzymes, specifically targeted toward the
fructose 6-phosphate–binding site.45,46 This dose was determined
after titration of 1–100 mM YN1, added to media for 24 hours culture.
IWR1 [4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-
isoindole-2-yl)-N-8-quinolinyl-benzamide], LY294002, and potas-
sium bisperoxo(1,10-phenanthroline) oxovanadate [bpV(Phen)] were
all purchased from Sigma and added as indicated. Kidneys were fixed in
4% paraformaldehyde and processed for immunofluorescence staining
in Tris-Saponin buffer. For mesenchyme cultures, E11.5 mesenchymes
were manually dissected from whole kidneys after gentle trypsinization
and neutralization in complete media.14,26 Per experiment, pooled mes-
enchymes in dissection media from 12 to 15 kidney pairs were equally
divided for individual treatments and placed on transwell filters. Cul-
tures were incubated in NPEM with or without YN1 as indicated for
0–24 hours, and then in DMEM/F12 with additions for 24–48 hours as
J Am Soc Nephrol 28: 3323–3335, 2017
www.jasn.org BASIC RESEARCH
noted in Figures 4–7. At 48 hours, cultures were fixed per whole-
mount kidney protocol for immunofluorescence staining. For kidney
sections, YN1-treated kidneys were fixed in 10% formalin and pro-
cessed for paraffin embedding and sectioning as previously de-
scribed.47 Antibodies against the following proteins were used: Lhx1
(4F2; Developmental Studies Hybridoma Bank), Pax2 (Invitrogen),
Six2 (Proteintech), and phosphohistone 3 (Ser10) (Cell Signaling).
RNA Isolation and ddPCR
E13.5 NPCs were isolated and cultured in NPEM. After 24 hours of
YN1 treatment, total RNA was isolated using a commercially available
RNA isolation kit (Qiagen). RNA concentration was quantified using
Nanodrop 2000 (Thermo Scientific). In addition, b-actin levels were
detected by ddPCR before analysis of target genes to adjust for minor
differences in sample RNA concentrations. ddPCR was performed
on a Bio-Rad ddPCR system to determine Cited1, Six2, Osr1,
Wnt4, Lef1, Lhx1, and Jag1 gene expression. Primers, probes, and
reagents for the One-step RT-ddPCR system were purchased from
Bio-Rad to generate cDNA and quantify gene expression. After drop-
let generation and PCR amplification, droplets were analyzed on the
QX200 droplet reader and target cDNA concentration was deter-
mined using the QuantaSoft analysis software (Bio-Rad). For each
target gene, the amount of total RNA in a PCR reaction was deter-
mined by a pilot ddPCR, using serially diluted total RNA, in which the
determined amount is in a linear range. Primer sequences (Supple-
mental Table 2) and a representative standard curve (Supplemental
Figure 4) are provided in the Supplemental Material. Data are ex-
pressed as copy numbers of target gene in 1 ml PCR reaction. Exper-
imental and biologic replicates were applied.
Measurement of ATP Levels
ATPlite (Perkin Elmer) luminescence assay was performed as per the
manufacturer’s instructions as previously described.47 This assay is
on the basis of light produced by conversion of luciferin by luciferase
to oxyluciferin in an ATP-dependent reaction. The emitted light is
proportional to ATP concentration in the cell lysate. NPCs (2.53104
per well) were seeded onto an ATP-free microplate. At 18–20 hours
after seeding, cells were lysed in the well, and the luminescence was
measured after the addition of substrate. Values were normalized to
cell number. Measurements were recorded from three biologic rep-
licates (n=3), with three to six technical replicates per experiment
from young and old NPCs.
Statistical Analyses
For extracellular flux analysis (ECAR and OCR), paired two-tailed t
test was used to calculate P values. Values of P,0.05 are considered
significant. Error bars represent 6SEM for biologic replicates from
pooled kidneys from three to five mouse litters per age. Each data
point represents a biologic replicate with NPCs from distinct isola-
tions and cultures. For Lhx1+ nascent nephron counting in organ
culture experiments, 19 paired kidneys from four litters were used
for 24-hour treatment and control. Paired two-tailed paired t test was
used to calculate P values, with P,0.05 considered significant. For
ddPCR analysis, error bars represent 6SD for technical replicates.
RNA was isolated from pooled kidneys (10–12 pairs) from two litters,
Glycolysis Promotes Nephron Progenitor Cell Self-Renewal 3333
 BASIC RESEARCH www.jasn.org
with two biologic replicates. Cell quantification by flow cytometry or
manual counts was done on multiple kidney pairs, as indicated in
Figure 5 legend. Data points represent experimental replicates, and
error bars denote 6SEM unless noted otherwise. All experiments
utilized kidneys harvested from two or more different litters.
ACKNOWLEDGMENTS
We are extremely grateful to Samir El-Dahr for helpful discussions
over the course of the project and critical review of the manuscript. We
thank the Tulane Hypertension and Renal Centers of Excellence
Molecular Core Service. This work was partially funded by grants
from National Institute of Diabetes and Digestive and Kidney Dis-
eases of the National Institute of Health (R56DK104779) and the
Diabetic Complications Consortium (DiaComp) (DK076169) to Z.S.
DISCLOSURES
None.
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See related editorial, “New Insights into Fuel Choices of Nephron Progenitor
Cells,” on pages 3133–3135.
This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2016111246/-/DCSupplemental.
Glycolysis Promotes Nephron Progenitor Cell Self-Renewal 3335