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org

Expression Profiling of Fibroblasts in Chronic and

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Acute Disease Models Reveals Novel Pathways in

Kidney Fibrosis
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Atsuko Y. Higashi,1 Bruce J. Aronow,2 and Gregory R. Dressler1

1
Department of Pathology, University of Michigan, Ann Arbor, Michigan; and 2Department of Biomedical Informatics,
Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

ABSTRACT
Background Renal interstitial fibrosis results from activation and proliferation of fibroblasts to myofibro-
blasts, secretion and accumulation of extracellular matrix, and displacement of normal renal tubules. In
contrast to chronic renal disease, acute injury may be repaired, a process that includes a decrease in the
number of myofibroblasts in the interstitium and degradation of the accumulated extracellular matrix,
leaving little evidence of prior injury.
Methods To investigate whether activated fibroblasts demonstrate changes in gene expression that cor-
respond with regression after acute injury but are not observed in chronic models of fibrosis, we used
microarrays to analyze gene expression patterns among fibroblast populations at different stages of injury
or repair. We then mined the data for signaling pathways in fibroblasts corresponding to the acute pro-
liferative, regression, and chronic phases of renal injury.
Results We identified multiple gene clusters with changes that correlate with the three phases of renal
injury, including changes in levels of receptors for the antifibrotic factor PGE2. In adult renal fibroblast
cultures, PGE2 was able to upregulate many genes that are suppressed by the profibrotic cytokine TGF-b,
whereas many PGE2-downregulated genes were activated by TGF-b. High levels of TGF-b suppressed
expression of a subset of PG receptors in fibroblast cultures, making these cells resistant to any effects of
PGE2.
Conclusions Inherent gene expression changes in activated fibroblasts accompany the transition from AKI
to repair and regeneration. In chronic models, however, activated fibroblasts are resistant to the antifi-
brotic effects of PGE2 due to suppression of a subset of PGE receptors.

J Am Soc Nephrol 30: 80–94, 2019. doi: https://doi.org/10.1681/ASN.2018060644

Fibrosis is a common pathology due to the activa- cytokines.4,5 Using definitive genetic cell lineage–
tion of interstitial fibroblasts to myofibroblasts, the tracing methods, the origins of renal interstitial
increased secretion and accumulation of extracel- fibroblasts and myofibroblasts have clearly been
lular matrix (ECM), and the loss or perturbation of established in animal models of renal fibrosis.6 The
tissue-specific epithelial, endothelial, and other cell
lineages.1,2 In CKD, this expansion and deposition
of ECM compromises renal function by displacing Received June 22, 2018. Accepted November 7, 2018.
proximal and distal tubules, by increasing glomer- Published online ahead of print. Publication date available at
ular mesangium to affect the filtration barrier, and www.jasn.org.
by affecting the hemodynamics of the renal arteri- Correspondence: Prof. Gregory R. Dressler, Department of Pa-
oles.3 Myofibroblast induction is also accompanied thology, University of Michigan, BSRB 2049, 109 Zina Pitcher
by innate immune pathways, partly through the Drive, Ann Arbor, MI 48109. Email: dressler@umich.edu

expression and secretion of immune-mediating Copyright © 2019 by the American Society of Nephrology

80 ISSN : 1046-6673/3001-80 J Am Soc Nephrol 30: 80–94, 2019

 www.jasn.org BASIC RESEARCH

best available data point to pericytes and perivascular fibroblastic

Significance Statement
cells, in addition to preexisting, resident interstitial fibroblasts, as
the major sources of myofibroblasts in renal fibrosis models. Activation of fibroblasts to myofibroblasts is a key factor that drives
Thus, to identify optimal therapeutic targets for CKD, it is es- renal interstitial fibrosis; regression of activated fibroblasts in the
interstitium is a key feature of repair, which can occur after acute
sential to understand both the intrinsic genomic programming
injury but not in chronic renal disease. Using models of AKI and CKD
of pericytes and interstitial, activated fibroblasts as well as their
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to study genes and signaling pathways that may mediate repair and
responses to injury and genetic abnormalities. fibrosis regression, the authors found that activated fibroblasts
Expansion of interstitial fibroblasts has been studied in AKI display changing gene expression patterns, including for PGE2
in human biopsy samples7 and in rodent models.8 AKI is the result receptors. These inherent changes accompany the transition from
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acute injury to repair, but differ from patterns observed after chronic
of renal ischemia or nephrotoxic injury and results in the death
injury. The authors also found that high levels of the profibrotic
of proximal tubule epithelial cells primarily from the S2 and S3 cytokine TGF-b in cultured adult fibroblasts may inhibit PGE2’s
segment of the nephron. However, these segments retain the abil- antifibrotic effects by suppressing a subset of PGE receptors, a
ity to regenerate and repopulate the sites of injury if the degree of finding with potential implications for therapeutic strategies.
injury is limited.9,10 Coincident with AKI is widespread activation
and proliferation of interstitial fibroblasts. As AKI is repaired, the
Michigan. Mice were housed in a specific pathogen–free fa-
activation of interstitial myofibroblasts subsides and expansion
cility with a 12-hour light/12-hour dark cycle and given free
regresses, often leaving little evidence of residual fibrosis. How-
access to food and water.
ever, repeated bouts of AKI may alter the ability of activated in-
For the UUO model, mice were anesthetized by isoflurane
terstitial myofibroblast to regress, leading to irreversible
inhalation. Through a midline abdominal incision, the left ureter
interstitial fibrosis and a chronic renal disease state.11,12 Neverthe-
was tied off at the level of the lower pole and the midureteral level
less, after an initial bout of AKI the number of activated myofi-
with fine suture materials (5–0 silk), then cut between the two
broblasts decreases as injury is repaired, suggesting that these cells
ligated points to induce a complete obstruction. Mice were al-
have the ability to reversibly deactivate or undergo apoptosis and
lowed to recover from anesthesia and were supplied food and
removal by mechanisms that remain to be determined.13
water ad libitum until they were euthanized at 14 days after the
In this report, we have begun to address inherent differences in
surgery. For the acute tubular necrosis model, mice were in-
renal interstitial fibroblasts after AKI and in chronic disease models.
jected intraperitoneally with a single dose of 250 mg/kg FA in
Using a folic acid (FA) model of AKI and the unilateral ureteral
0.15 M NaHCO3. Mice were euthanized at 3, 7, and 14 days after
obstruction model (UUO) of renal fibrosis in mice, we isolated
injury. Mice were put under constant observation for the first
interstitial fibroblasts by fluorescence-activated cell sorting (FACS)
48 hours after injection.
and determined gene expression changes with Affymetrix micro-
arrays. We compared the transcriptome of fibroblasts from unin-
jured kidneys and UUO-derived fibroblasts, as well as fibroblasts Cell Sorting
from different times after AKI. We hypothesized that there may be In brief, kidneys were minced and digested with 0.5 mg/ml Lib-
specific genes and pathways that mediate the regression of erase DH (#05401054001; Roche) and 100 U/ml DNaseI
fibroblasts after AKI that are not active in chronic models of fibro- (11284932001; Roche) in PBS solution supplemented with
sis, such as the UUO model. The data show dynamic changes in the 10% FBS at 37°C for 30 minutes with triturate by P1000 tip every
patterns of fibroblast gene expression in the acute and recovery 5 minutes, followed by centrifugation at 3003g for 5 minutes at
phases that include WNT and TGF signaling pathways, fibroblast 4°C. The resulting pellet was resuspended in ice-cold PBS with
growth factors, and genes involved in ECM deposition or turnover. 2 mM EDTA and was passed through a 70-mm cell strainer
Furthermore, we show that suppression of the PG receptor Ptger3 (Falcon), then centrifuged at 3003g for 5 minutes at 4°C. The
correlates with acutely injured fibroblasts but increases with re- pellet was incubated with 0.53 red blood cell lysis buffer
covery. Strikingly, TGF-b–cultured kidney fibroblasts suppress (0.15 M NH4Cl, 14 mM NaHCO3, and 0.1 mM Na2 EDTA)
expression of multiple PG receptors making cells recalcitrant to for 5 minutes on ice, to remove red blood cells, and centrifuged
PGE2 signaling. Given the proposed antifibrotic effects of at 3003g for 5 minutes at 4°C. The single isolated kidney cells
PGE2,14–16 these data suggest a novel mechanism for the regres- were stained with APC-conjugated anti-PDGFRa Antibodies
sion of interstitial fibroblasts that is deregulated in a high–TGF-b (Ab) (#A18382; Life Technologies) and PE-Cyanine7–conju-
environment. gated anti-CD11b Ab (#25–0112–81; eBioscience) and subjected
to cell sorting using FACS Aria II cell sorter (BD Biosciences).
FACS Aria II was also used for surface pattern analysis. The
METHODS antibodies used for surface pattern are anti-PDGFRa, APC-Cy-
anine7–conjugated anti-CD45 Ab (#103115; Biolegend), PE-
Animals Cyanine7–conjugated anti-CD105 Ab (#120409; Biolegend),
Mice were kept according to National Institutes of Health PE-conjugated anti-CD44 Ab (#103007; Biolegend), PE-con-
guidelines and all procedures were approved by the University jugated anti-PDGFrb Ab (#136005; Biolegend), and PE-con-
Committee on Use and Care of Animals at the University of jugated anti-CD31 Ab (#102507; Biolegend).

J Am Soc Nephrol 30: 80–94, 2019 Gene Expression Changes in Activated Kidney Fibroblasts 81
 BASIC RESEARCH www.jasn.org

No injury FA3 FA7 FA14 U14

HE staining
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PDGFR / Nuclei
SMA / Nuclei
PDGFR / Nuclei
Collagen1 / Nuclei
Fibronectin / Nuclei

Figure 1. Interstitial fibroblasts expand and regress after acute kidney injury (FA3–FA14) but not in the chronic obstruction model
(U14). Representative sections of kidneys taken at the indicated times post-FA or -UUO or from uninjured controls are shown. Sections
were stained with hematoxylin/eosin (HE) or with the indicated antibodies in color. Note the increase in PDGFra/b- and aSMA-
positive cells, and the matrix proteins Col1 and fibronectin at FA3, FA7, and U14. Note the decreases in matrix and fibroblasts
by FA14.

82 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
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Microarray Expression Analysis tissue-Tek O.C.T. compound (Sakura Finetek) and sectioned
Total RNA was extracted from FACS-sorted cells using TRIzol using a cryostat. Frozen sections (6 mm) were treated with
reagent (Life Technologies) and purified by RNeasy Mini Kit 100% methanol for 20 minutes at 24°C and subsequently blocked
(Qiagen). Microarray expression analysis was performed us- with 5% normal goat serum in PBS/0.05% Tween for 1 hour at
ing three independent mice and carried out by the University room temperature. The following primary antibodies were used:
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of Michigan Comprehensive Cancer Center Affymetrix and anti-PDGFRa Ab (#14–1401–82; eBioscience), anti-PDGFRb Ab
Microarray Core Facility using Mouse 430 2.0 Affymetrix Gene (#14–1402; eBioscience), anti-fibronectin Ab (sc-9068; Santa
Chip 3 expression arrays (Affymetrix), as described.17 Expres- Cruz), anti-laminin Ab (#L9393; Sigma-Aldrich), anti-collagen I
sion values for each gene were calculated using the robust mul- Ab (#ab34710; Abcam), anti-CD31 Ab (#ab28364; abcam), Fluo-
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tiarray average method and fitted to weighted linear models in rescein-labeled Lotus Tetragonolobus Lectin (#FL-1321; Vector
R using the Affymetrix package of Bioconductor.17,18 The Af- Labs), Cyanine3-conjugated anti–a-Smooth Muscle Actin Ab
fymetrix datasets have been submitted to the Gene Expression (#C6198; Sigma-Aldrich), and anti-Gja1 Ab (sc-9059; Santa
Omnibus (https://www.ncbi.nlm.nih.gov/geo/), accession Cruz). All images were taken on an Olympus IX71 epifluorescent
number GSE121190. microscope. Morphometric analysis was carried out using
ImageJ software.
RNA Reverse Transcription and Quantitative Real-Time
PCR Kidney Primary Fibroblast Culture
Total RNA samples of FACS-sorted cells were reverse transcribed Tissue culture dishes were coated with 1% gelatin solution
into cDNA using SuperScript III Reverse Transcription (Life before tissue explanting. Sections of kidney cortex were diced
Technologies). cDNA templates were amplified with iTaq Uni- and digested in 0.05% trypsin/EDTA (Gibco) for 10 minutes at
versal SYBR Green Supermix (Bio-Rad) on an Applied Biosys- 37°C. The kidney pieces were placed in gelatin-coated dishes
tems 7500 Real-Time PCR System. The primers (59 to 39) used: and incubated in DMEM/F12 (Gibco) supplemented with
mPDGFRa: forward, cattgaccctgttccagaggag, reverse, ggtggaac- 20% FBS, 13 ITS-X (Gibco), 13 Glutamax (Gibco), 13
tactggaacctgtc; mPtger3 (EP3): forward, gaaccagatcttggatccctgg, sodium pyruvate (Gibco), and 5 mM Y-27632 (Cayman
reverse, cagggaaacaggtactgcaatg; mFGF9: forward, tcatttaga- Chemical) for 3 days. After 3 days, kidney pieces were gently
gatcttccccaacg, reverse, gttcatgccgaggtagagtcc; mGja1: forward, removed and medium was changed. Culture medium was
gtgccggcttcactttcattaag, reverse, gaaaatgaagagcaccgacagc; renewed twice weekly until the monolayer had covered ap-
mGapdh: forward, ccagaacatcatccctgcatc, reverse, cctgcttcac- proximately 75% of the dish surface. For passage, cells were
caccttcttga. The relative standard method was used to obtain dissociated with 0.05% trypsin/EDTA. Y-27632 was not added
relative expression for each grouping comparison, where the to the medium after passage 5. Cells could be continuously
amount of target was normalized to endogenous GAPDH. cultured for .36 passages.
For expression analyses, cells were cultured in six-well plates
Histology and Immunohistochemistry to confluency then shifted to a low-serum (0.05%) media for
The kidneys were fixed in Carnoy solution, embedded in paraffin, 24 hours before addition of 10 ng/ml TGF-b or 1 mM PGE2 or
and sections (6-mm thick) were stained with Hematoxylin and both factors. RNA was harvested with TRIzol after 48 hours
Eosin for routine histologic examination. For immunofluo- in culture and analyzed by Affymetrix microarray gene chips
rescent stainings, tissues from adult animals were frozen in as above.

40 60
35

30
30 50
PDGFr (%area)
SMA (%area)

Col1 (%area)

25

40 20
20
15
30 10
10
5
20
0 0
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14

Figure 2. Quantitative morphometry of interstitial area shows regression after AKI. Multiple random images were analyzed by calculating the
total positive area of micrographs stained with the indicated antibodies. Scatter plots show data points with the mean and 1 SD indicated. Note
the significant decrease in interstitial fibroblast area by FA14 treatment, whereas U14 remains high. SMA, Smooth Muscle Actin; UN, Uninjured.

J Am Soc Nephrol 30: 80–94, 2019 Gene Expression Changes in Activated Kidney Fibroblasts 83
 BASIC RESEARCH www.jasn.org

A living (PI - ) CD11b (-) cells

(X 1,000)
(X 1,000)
250 250
FA3 FA3

200 200

SSC-A
SSC-A
SSC-A
150 150
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100 100

50 50
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2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10
660(20 (633)-A 660(20 (633)-A

APC PDGFR-APC
(X 1,000)

(X 1,000)
250 250

200 200
SSC-A

SSC-A
150 150

100 100

50 50

2 3 4 5 2 3 4 5
10 10 10 10 10 10 10 10

PDGFR PDGFR

B living (PI - ) cells C 90

In situ surface pattern
35
80
30
70
%PDGFR + cells

%expression

25 60
20 50
15 40
30
10
20
5 10
0 0
UN FA3 FA7 FA14 U14 CD45 CD31 CD44 PDGFR CD105

D SMA Fibronectin Laminin

PDGFR / Nuclei

CD31 LTA E
60
p=0.0016
PDGFR / GAPDH

50
PDGFR / Nuclei

40
30
20
10
0
input FACS

84 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
 www.jasn.org BASIC RESEARCH

RESULTS using FACS (Figure 3A). Uninjured control kidneys were par-
ticularly challenging because very few PDGFra cells are ob-
Expansion of PDGFra-Positive Cells in the Interstitium served in tissue sections. However, sufficient numbers of cells
of Acute and Chronically Injured Kidneys could be isolated by FACS by pooling multiple uninjured adult
In order to examine renal interstitial fibroblasts from both kidney preparations. We also tried PDGFrb antibodies for
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acutely injured and chronically injured kidneys, we used the separating cells, but the affinity was lower and gating could
established FA nephrotoxicity model and the UUO model of not separate the positive cells as cleanly as anti-PDGFra. The
fibrosis. To induce AKI, mice were injected with 250 mg/kg FA PDGFra-positive cells separated by FACS represent a popula-
and kidneys were isolated at 3 days (FA3), 7 days (FA7), and tion ranging from approximately 2% in uninjured to .20% in
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14 days (FA14) postinjection. The chronic injury was examined U14 kidneys (Figure 3B). The PDGFra-sorted population was
in UUO kidneys 14 days after ligation of a single ureter (U14). also costained for endothelial cell markers (CD31), hemato-
We initially confirmed kidney injury and expansion of fibro- poietic cells markers (CD45), and pericyte/mesenchymal stem
blasts and myofibroblasts by histology and immunostaining cell markers (CD44, PDGFrb, and CD105) to ensure a specific
for PDGFra, PDGFrb, smooth muscle actin (aSMA), colla- population (Figure 3C). These data indicate that our PDGFra+
gen1, and fibronectin (Figure 1). Representative images show population contains few endothelial and hematopoietic cells
dilated renal tubules and significant expansion of fibroblasts but does overlap with pericytes. Immunostaining of kidney
and ECM in FA-treated kidneys (FA3 and FA7). However, by sections, with the same antibody as used for FACS, also dem-
FA14, the acute injury has resolved because the injured renal onstrates that the vast majority of PDGFra+ cells were local-
tubules have regenerated and the interstitial expansion of ized within the interstitium and associated with ECM, as
fibroblasts and myofibroblasts has regressed. Both PDGFra- marked by aSMA, fibronectin, and laminin (Figure 3D).
positive cells and aSMA-positive cells are greatly reduced in However, PDGFra+ cells were distinct from CD31+ endothe-
number at FA14, compared with FA3 or FA7. Staining for the lial cells or LTA+ proximal tubule epithelium (Figure 3D).
ECM proteins collagen1 and fibronectin is also reduced by Total RNA was isolated from sorted cells and assayed for ex-
FA14, although it remains stronger than in uninjured controls. pression of the PDGFra mRNA and compared with whole-
In the U14 kidney sections, staining for aSMA, collagen1, and kidney RNA to assess the degree of enrichment for PDGFra
PDGFra was more widespread than even in the FA3 or FA7 fibroblasts (Figure 3E). These data show a 50–100-fold in-
kidneys. The immunostained micrographs were subject to crease for PDGFra expression, again indicating that a signifi-
quantitative morphometry, whereby the stained area was calcu- cantly enriched population was obtained.
lated on multiple independent images from different animals
(Figure 2). These data confirm that there is a rapid expansion Gene Signatures of PDGFra-Positive Cells from Acute
of interstitial fibroblasts and myofibroblasts in the acute in- and Chronically Injured Kidneys
jury phase at 3 and 7 days post–FA injection that resolves by Total RNAs obtained from PDGFra-positive cells from FA3,
14 days upon recovery from injury. However, the chronic FA7, FA14, and U14 kidneys and from uninjured kidneys were
UUO model continues to exhibit interstitial expansion of used to assess gene expression differences between any and all
PDGFra- and aSMA-positive cells with no evidence for of the five samples to determine what unique signatures might
regression. be expressed in the different populations. For each time point,
samples were done in triplicate. The entire Affymetrix expres-
Enrichment of PDGFra-Positive Cells by FACS sion dataset is available as an Excel file (Supplemental Table 1)
In order to determine what inherent differences among the or can be accessed at https://www.ncbi.nlm.nih.gov/geo/ (ac-
fibroblast populations could account for the regression of cells cession number GSE121190). A total of 2460 probe sets, rep-
after acute injury, we isolated populations of PDGFra-positive resenting 1885 unique transcription units, showed statistically
cells from the kidney cortex of both acute and chronic models significant differences in expression in at least one of the

Figure 3. PDGFra-positive cells can be sorted with purity and specificity from adult kidneys. (A) Representative flow cytometry plot for
sorting PDGFRa-positive cells from whole adult kidney 3 days post-FA, using either a control (left panel) or anti-PDGFRa mAb (APA5,
right panel). Cells in gated area were selected. (B) The percentage of PDGFRa-positive cells from whole kidneys sorted from each
disease model and uninjured control kidneys (no injury, n=7; FA3, n=9, FA7, n=5; FA14, n=5; U14, n=4). (C) Flow cytometry analysis
shows that PDGFRa-sorted cells include pericyte/MSC marker–positive cells (CD44, PDGFRb, and CD105), but not endothelial (CD31)
and hematopoietic (CD45) cell populations (n=3). (D) Immunostaining demonstrates that anti-PDGFRa mAb (APA5) recognizes cells
specifically confined to the tubulointerstitial compartment. In FA3 kidney, PDGFRa-positive (red) cells stain positive for aSMA, overlap
with ECM (fibronectin and laminin), but do not costain with endothelial marker (CD31) and proximal tubular marker (LTL-lectin).
(E) Quantitative RT-PCR analyses of PDGFRa mRNA from whole FA3-treated kidneys and PDGFRa-sorted cells show approximately
50-fold enrichment (n=3). SSC-A, Side scatter; UN, Uninjured.

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Color Key cluster1: Translation

6
5
–5 5
4
3
Value 1
2
1
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0
FA3 FA7 FA14 UUO UN

cluster 2: Ribosomes, ER,

6
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2 5
4
3
2
1
0
FA3 FA7 FA14 UUO UN


cluster 3: ECM, Tgf-
14
12
3 10
8
6
4
2
0
FA3 FA7 FA14 UUO UN

cluster 4: ECM, development

7
6
4
5
4
3
2
1
5
0
FA3 FA7 FA14 UUO UN

cluster 5: Immunity, inflammation

8

2
6 0
FA3 FA7 FA14 UUO UN

cluster 6: Metabolism, transporters

1.2
1.0
0.8
0.6
0.4
0.2
0
FA3 FA7 FA14 UUO UN

cluster 7: Respiration, mitochondria

1.2
7
1.0
0.8
0.6
0.4
0.2
FA3 FA7 UUO FA14 UN 0
FA3 FA7 FA14 UUO UN

Figure 4. Gene expression changes in isolated fibroblasts after renal injury exhibit distinct clustering. The normalized relative ex-
pression values for 2640 Affymetrix probe sets that show statistically significant changes in at least one of the renal disease models
were clustered. The graphs on the left show mean expression values for individual clusters (dots) with the 25th and 75th percentiles
represented by the vertical lines. The complete dataset with gene names is available as a supplemental Excel file (Supplemental Table 2).
ER, Endoplasmic reticulum; UN, Uninjured.

86 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
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Table 1. Pathway analyses of clustered gene sets clustered to group genes depending on their
Cluster Pathways P Value # Genes Sourcea level of expression under the different condi-
1 Translation, ribosomal proteins tions (Figure 4). Gene expression changes
RNA binding 9.6E26 29 of 1632 T showed seven distinct clusters as follows:
Translation initiation 2.4E28 12 of 194 T cluster 1 genes were highest at FA3 and lowest
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rRNA processing 5.9E27 12 of 260 T in uninjured, cluster 2 genes were high in all
Translation initiation 8.1E29 12 of 191 G injured kidneys, cluster 3 genes were high at
rRNA processing 1.6E26 11 of 257 G FA3 and lower at FA7 and FA14 and high in
Protein metabolism 1.1E24 32 of 2061 G the U14 kidneys, cluster 4 genes were highest
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2 Ribosomes, translation, ER targeting in U14, cluster 5 genes were increased at FA7

Structural constituent of ribosome 6.4E236 46 of 216 T
and FA14 but low at FA3 and in U14 kidneys,
Translation 3.5E250 54 of 165 T
cluster 6 genes were highest at FA14 and in
Translation termination 4.8E250 45 of 97 T
Translation elongation 8.6E250 45 of 98 T
uninjured kidneys, and cluster 7 genes were
Translation 1.7E240 35 of 91 G low in all injured kidneys. We utilized both
ER targeting, membrane targeting 1.6E239 35 of 96 G ToppGene19 and Genomatix (www.genoma-
Translation initiation 5.4E233 39 of 191 G tix.de) for gene ontology and pathway analyses
Ribosome 1.8E236 35 of 119 G to determine specific themes for each cluster
3 ECM, collagens, integrins (Table 1). Clusters 1 and 2 are enriched for
ECM organization 6.5E238 56 of 354 T genes involved in protein synthesis or mem-
TGF-b 6.7E210 44 of 824 G brane targeting. Clusters 3 and 4 are enriched
MMPs 2.1E26 21 of 331 G in genes for matrix synthesis, deposition, and
Integrin signaling 6.6E213 15 of 70 G
signaling. Cluster 5 highlights the inflamma-
ECM organization 5.5E239 53 of 331 G
tory response, whereas clusters 6 and 7 are
Smad2 2.9E212 31 of 436 G
4 ECM, development
enriched in genes for metabolisms, cellular
Regulation of development 1.7E214 60 of 1893 T respiration, and transporters.
ECM 2.0E218 33 of 444 T We examined candidate genes associated
ECM 3.4E215 29 of 530 G with signaling pathways, ECM secretion, or
Smad signaling 1.3E27 20 of 491 G transcription, or previously implicated in
TGF-b 8.2E27 25 of 824 G fibrosis (Figure 5). Receptors for the profi-
5 Immunity, inflammatory response brotic cytokine TGF-b were up slightly at
Antigen processing and presentation 2.1E211 10 of 94 T FA3 and U14, but lower at FA14. Most
Defense response 3.8E211 30 of 1651 T strikingly, the TGF-b–induced gene was
Immune, inflammatory response 1.9E222 34 of 549 T
up 15-fold at FA3 and continued to increase
Innate immune system 8.8E25 18 of 1459 G
despite a reduction of PDGFra-positive
Immune response 7.1E29 25 of 2042 G
6 Metabolism, transporters, kidney disease
cells by FA14. For WNT ligands, Wnt5a in-
Transmembrane transporters 1.4E219 81 of 1014 T creased seven-fold by FA3 and declined by
Organic acid metabolic process 3.0E230 104 of 1131 T FA14, whereas Wnt4 peaked at FA7 in the
Acidosis 2.5E212 50 of 475 T acute model. WNT inhibitors Dkk3 and
Metabolic pathways 1.1E223 109 of 1272 T Frzb also peaked at FA3 in the acute model
Kidney disease 1.3E215 53 of 569 T with even higher levels in the chronic model
Metabolism 6.6E220 126 of 2100 G at U14, whereas the inhibitor Dkk2 was
TCA cycle 1.1E213 27 of 169 G down 3–4-fold at FA3. These data suggest
Transmembrane transporters 2.6E221 68 of 1034 G that Wnt4 may be more specific to the res-
Renal tissue 2.7E249 122 of 1247 G
olution phase after AKI and that dynamic
Metabolic disease 3.6E214 71 of 903 G
changes in WNT inhibitors could modulate
7 Respiration, mitochondria
Cellular respiration 1.1E27 16 of 178 T
signaling. Other signaling effectors that in-
Response to hypoxia 2.5E27 14 of 143 T creased during the acute phase include
TCA cycle 6.4E26 14 of 171 T Bmp1 and Vegfd, whereas Bmp7 was re-
Cellular respiration 3.2E27 14 of 168 G duced at FA3 and FA7. The tumor suppressor
ER, Endoplasmic reticulum; MMPs, Matrix metalloproteinase; rRNA, Ribosomal RNA; TCA, Pten, a phosphatidylinositol phosphatase,
tricarboxylic acid.
a
also increases four-fold at FA3 and declines
T, ToppGene; G, Genomatix.
by FA14, but remains high in the U14 kidneys.
As expected, almost all collagen chains
samples. The differential gene expression dataset is available showed differential expression, which increased at FA3 and
as a single Excel file (Supplemental Table 2). These genes were declined slowly thereafter, but remained high in the U14

J Am Soc Nephrol 30: 80–94, 2019 Gene Expression Changes in Activated Kidney Fibroblasts 87
 BASIC RESEARCH www.jasn.org

WNT Signaling TGF- Signaling Other Signaling

32 32.00 8.00
16
16.00 4.00
8
4 8.00
2.00
2
4.00
1 1.00
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0.5 2.00
0.25 0.50
1.00
0.125
0.50 0.25
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14
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Wnt5a Wnt4 Dkk3 Tgfbi Tgfb1i1 Tgfbr1 Vegfd Pten Bmpr1a

Frzb Dkk2 Sfrp1 Tgfb2 Tgfbr2 Tgfbr3 Bmp7 Bmp1 Pdgfra

Collagens MMPs TIMPs

128.00 16 64.00
64.00 32.00
8
32.00 16.00
16.00 4 8.00
8.00 4.00
4.00 2 2.00
2.00 1.00
1
1.00 0.50
0.50 0.5 0.25
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14
Col4a2 Col6a1 Col1a2 Mmp3 Mmp19 Mmp14 Timp1 Timp2 Timp3
Col6a2 Col1a1 Col5a1 Mmp11 Mmp2 Mmp12
Col4a1 Col3a1

Integrins Transcription Factors Fibrosis Related

16 32.00 16
8 16.00 8
4
4 8.00
2
2 4.00 1
1 2.00 0.5
0.25
0.5 1.00
0.125
0.25 0.50 0.0625
0.125 0.25
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14
Itgb5 Itga9 Itgb2 Tcf12 Tfap2b Runx2 Fgf1 Fgf2 Ptger3
Itga6 Itga4 Itga8 Stat3 Runx1 Hoxa10 Fgf9 Kl
Tcf21 Hoxd11

Figure 5. Expression profiles of selected genes and pathways exhibit dynamic changes. Normalized RNA expression levels from
PDGFra-selected fibroblasts isolated from uninjured (UN) kidneys; or AKI kidneys at 3, 7, or 14 days post-FA; or kidneys 4 days post-
UUO. RNAs were assayed with Affymetrix microarrays in triplicate from three independent sorted samples, with data points rep-
resenting the mean. Expression levels are relative to UN and graphed on a log2 scale. Signaling pathways include TGF-b–related,
WNT-related, and other ligands or receptors. ECM genes include the collagen family, the matrix metalloproteinase (MMPs), and the
tissue inhibitors of MMPs (TIMPs).

samples. The matrix metalloproteases that are involved in (HoxD11 and HoxA10) are also upregulated 2–3-fold in the
matrix turnover showed different patterns, with Mmp3 in- acute phase and decline back to uninjured levels by FA14.
creasing rapidly at FA3 and then declining, but with Mmp12 Of genes associated with fibrosis, the Fgf2 but not Fgf1
showing maximum levels at FA14. Again, this suggests that expression increased strongly at FA3, whereas Fgf1 and Fgf9
Mmp12 may be involved primarily in the degradation of ma- declined in the AKI model, suggesting that Fgf2 is a primary
trix that accumulates as a result of injury. Integrins also show driver of fibroblast proliferation. Among the objectives of the
dynamic changes, with Itgb5 and Itga9 strongly induced at screen were to examine the expression levels of potential anti-
FA3, FA7, and U14, whereas Itgb2 peaks at FA14 and is low at fibrotic genes during the recovery phase after acute injury.
U14. Again, these data suggest that Itgb2 may be associated with PGs are lipid compounds with hormone-like activity, of
the regression of fibroblasts. Of transcription factors, the Tcf21 which PGE2 has been implicated in tissue repair and ECM
gene is the most highly upregulated in AKI and declines in the degradation.15,16 Thus, PGE2 has been implicated as an anti-
resolution phase at FA14. Similarly, kidney-specific Hox genes fibrotic factor.5,14 We noted that one of its receptors, Ptger3,

88 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
 www.jasn.org BASIC RESEARCH

A Ptger3/ Gapdh B Fgf9/ Gapdh the levels of the profibrotic factor TGF-b1 in
p=0.0029 p=0.0024 whole-tissue RNA from kidneys isolated
before and after injury, which increased
p=0.0031 p=0.010
at FA3 and FA7, decreased by FA14, but
1.4 1.4 was .20-fold higher in U14 compared
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1.2 with uninjured (Figure 6D).

1.2
1.0 1.0
Effects of TGF-b and PGE2 on
0.8 0.8 Cultured Kidney Fibroblasts
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0.6 0.6 To examine and compare the effects of pro-

0.4 0.4
fibrotic and potential antifibrotic agents,
we cultured immortalized adult kidney fi-
0.2 0.2
broblasts with PGE2, TGF-b, or both com-
pounds for 48 hours and measured gene
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 expression profiles with Affymetrix micro-
arrays (Supplemental Tables 3–5). PGE2
C Gja1/ Gapdh D treatment resulted in .1000 genes with
p=0.017
TGF1/Gapdh statistically significant altered expression
30 values; approximately 514 genes were up-
p=0.031
40
regulated and 508 genes were downregula-
ted two-fold or more (Figure 7). TGF-b
35
20 also greatly altered gene expression pat-
30
terns in adult fibroblast cultures with
25 .2500 genes affected (Figure 7). Analyses
20 of the TGF-b–regulated gene set show great
15 10 similarity (P,3.8E243) to the response
10 seen in mouse embryo fibroblasts (MEFs)
5 in Gene Set Enrichment Analysis M2445
0
(PLASRI_TGFB_TARGETS_10HR_UP),
UN FA3 FA7 FA14 U14 UN FA3 FA7 FA14 U14 as reported by Plasari et al.20 (Table 2). Sig-
nificant overlap is also seen with genes
Figure 6. Quantitative RT-PCR for selected genes confirms expression changes. (A–C) downregulated by TGF-b in kidney fibro-
Total RNA samples from PDGFRa-sorted cells were analyzed by quantitative RT-PCR for
blasts and MEFs. Conversely, the gene set
the indicated genes to confirm Affymetrix array expression differences. Note decline of
Ptger3 in the early acute phase (FA3) and its increase during the recovery phase (FA7,
upregulated by PGE2 overlaps with genes
FA14). The gap junction protein Gja1, expressed in fibroblasts, shows an expression suppressed by TGF-b in MEFs, whereas the
pattern opposite that of Ptger or Fgf9, with maximum levels in the chronic UUO model gene set downregulated by PGE2 is most
(U14). (D) Total RNA was isolated from whole kidneys at the indicated times postinjury similar to genes upregulated by TGF-b.
and assayed for TGF-b1 by quantitative RT-PCR. The y axis is relative RNA levels in Within our datasets, of the 1139 genes
arbitrary units. downregulated with TGF-b, approxi-
mately 166 were upregulated by PGE2, a
highly significant number of overlapping
was down more than three-fold at FA3 and began to increase genes some of which are associated with TGF-b signaling
by FA7 and FA14, whereas the U14 samples remained low. The (Smad), Wnt signaling (b-catenin), and extracellular matrix
Klotho gene has also been implicated in renal disease20,21 and remodeling. Similarly, 96 genes that are upregulated by TGF-b
its expression was also strongly reduced at FA3 and U14 but were also downregulated by PGE2 and correlated with matrix
approached normal levels by FA14. We confirmed by quanti- metalloproteinase, Hif1a, as well as TGF-b pathways. The list
tative RT-PCR that expression levels of the PGE2 receptor of overlapping genes can be found in Supplemental Table 6.
Ptger3 were low in acutely injured fibroblasts at FA3, but be- Given that PGE2 and TGF-b may have opposite effects on
gan to increase at FA7 and FA14, while remaining low in the gene expression in fibroblasts and the progression of fibrosis,
UUO model (Figure 6). Similarly, we independently con- we also examined whether PGE2 could counter the effects of
firmed the reduction of Fgf9 and the marked increase in TGF-b by comparing cells cultured with TGF-b to those cul-
Gja1, a gap junction protein highly expressed in U14 kidneys tured with TGF-b and PGE2 together. Surprisingly, there were
(Figure 6, B and C). These data suggest that the antifibrotic few changes in gene expression between these two sets after
activities of PGE2 may be limited in the UUO model due to a 48 hours in culture (Figure 7). For both genes that were up-
specific decrease in Ptger3 receptor expression. We also examined regulated by PGE2 (Figure 7D) and genes downregulated by

J Am Soc Nephrol 30: 80–94, 2019 Gene Expression Changes in Activated Kidney Fibroblasts 89
 BASIC RESEARCH www.jasn.org

A D F
TGF vs. Control (>2x) - 1245 up, 1139 down 450
PGE2 vs. Control (>2x) - 514 up, 508 down Ptger3

RNA expression (rel. units)

400
TGF/PGE2 vs. TGF (>2x) - 2 up, 11 down 160

RNA expression (rel. units)

350
140
300
120
B
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250
100
TGF PGE2 200 80
down 973 166 348 up 150 60
100 40
Common elements - 166 (p < 1E-18)
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50 20
Pathway Analyses #Genes P value 0 0
Con. PGE2 TGF T+P Con. PGE2 TGF T+P
Smad 11 2E-3
B-catenin 13 3E-3
WNT signaing 8 4E-4
Pi3K/AKT 4 3E-3
E G
ECM organization 7 4E-3 600 Ptger4
350
RNA expression (rel. units)

RNA expression (rel. units)

500
300
C
400 250
TGF PGE2
1149 96 412 300 200
up down
150
200
Common elements - 96 (P < 1E-17) 100
100 50
Pathway Analyses #Genes P value
MMPs 11 5E-7 0 0
Hif1a 8 2E-5 Con. PGE2 TGF T+P Con. PGE2 TGF T+P
TGF-b 10 6E-3
Smad 8 2E-3

Figure 7. TGF-b suppresses the effects of PGE2 on adult renal fibroblast gene expression. (A) The number of genes up- or
downregulated by TGF-b or PGE2 is compared with untreated cells after 48 hours in culture. However, TGF-b treatment alone
abrogates almost all of the effects of PGE2 treatment. The complete datasets are in Supplemental Tables 3–5. (B) Pathway analyses
of the 166 genes that are downregulated by TGF-b and upregulated by PGE2. (C) Pathway analyses of 96 genes that are upre-
gulated by TGF-b and downregulated by PGE2 in adult kidney fibroblasts. (D) Median expression values of genes upregulated
two-fold by PGE2 (2) with vertical lines representing the 75th and 25th percentiles. Note that TGF-b treatment suppresses any
increase by PGE2 on average gene expression values (T+P). (E) Median expression values of genes downregulated two-fold by
PGE2 (2) with vertical lines representing the 75th and 25th percentiles. Note that TGF-b treatment suppresses any decrease by
PGE2 (T+P) on average gene expression values. (F) Relative RNA expression levels of the PGE2 receptor Ptger3 in adult kidney
fibroblasts after treatment with PGE2, TGF-b, or both factors (T+P) compared with controls. (G) Similar analyses as in (F) but for the
PGE2 receptor Ptger4.

PGE2 (Figure 7E), TGFb inhibited the effects of added PGE2, more lamellipodia, and more compact, dense monolayers
suggesting that PGE2 had little effect on gene expression when (Figure 8, A and B), suggesting that more cells can be com-
present in a high–TGF-b environment, as seen in the U14 pacted into the interstitial space. This increased density is also
kidneys. Given that TGF-b appeared to suppress almost all observed in vivo when kidney sections were stained with anti-
of the effects of PGE2, we examined the level of PG receptor PDGFra (Figure 8, C and D). We next examined adult kidney
expression in fibroblasts before and after treatment with fac- fibroblasts (AKF) from the U14 kidneys and from uninjured
tors (Figure 7, D and E). Strikingly, TGF-b suppressed the kidneys to determine whether the ability to respond to PGE2
expression of PG receptors Ptger3 and Ptger4, although there was a stable trait in cultured cells. When treated with TGF-b,
was no effect on Ptger1 or Ptger2. These data suggest that AKF cells suppressed both Ptger3 and Ptger4, whereas the U14
TGF-b suppression of a subset of PG receptors and presum- fibroblasts had virtually no Ptger3 expression and significantly
ably subsequent downstream signaling contribute to drive the less Ptger4, compared with AKFs (Figure 8E). We then asked
profibrotic phenotype of activated fibroblasts in chronically whether the U14 fibroblasts could respond to PGE2 by assay-
injured kidneys and inhibit the antifibrotic effects of PG. ing for two responsive genes identified in the AKFs (see Sup-
To test whether fibroblasts isolated from healthy or diseased plemental Table 4). Sox9 is significantly upregulated, whereas
kidneys showed differences in their morphology and ability to Ccl2 is suppressed by PGE2 in AKFs (Figure 8F). In the U14
respond to PGE2, we examined primary fibroblasts in culture fibroblasts, baseline expression of Sox9 is approximately 60%
(Figure 8). Interestingly, cells isolated from U14 kidneys of AKF levels and there is no response to PGE2. Ccl2 expres-
showed different morphology in culture, with less cytoplasm, sion is downregulated by PGE2 in AKFs and is also suppressed

90 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
 www.jasn.org BASIC RESEARCH

Table 2. Pathway analyses of gene expression in response to TGFb or PGE2

Gene Cluster Pathways P Value # Genes Sourcea
TGF-b: Genes upregulated
Genes upregulated in MEFs by TGFb 3.8E243 71 of 199 T
Hif1a transcription 2.1E214 25 of 65 T
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Glucose metabolism 2.9E210 23 of 81 T

Hif1a 1.6E211 21 of 67 G
Carbohydrate metabolism 1.9E28 39 of 276 G
TGF-b: Genes downregulated
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Genes downregulated in MEFs by TGFb 1.4E243 77 of 244 T

Cell adhesion 2.1E221 176 of 1530 T
Cell migration 2,6E220 155 of 1300 T
ECM and associated proteins 7.2E214 119 of 1028 T
ECM organization 5.7E27 37 of 298 G
Cell invasion 6.0E29 76 of 845 G
PGE2: Genes upregulated
Genes downregulated in MEFs by TGFb 4.3E237 51 of 244 T
Proteinaceous ECM 1.2E27 28 of 379 T
Smad-related signaling 2.1E24 25 of 491 G
ECM components 3.4E26 12 of 120 G
PGE2: Genes downregulated
Genes upregulated in MEFs by TGFb 1.8E269 71 of 199 T
Regulation of cell death 4.6E218 100 of 1650 T
Smad signaling 2.9E26 31 of 491 G
Matrix metalloproteases 1.3E25 23 of 331 G
Fibrosis 6.4E211 25 of 268 G
a
T, ToppGene; G, Genomatix.

by TGF-b alone, whereas in U14 fibroblasts, PGE2 again has populations of interstitial fibroblasts from uninjured kidneys
no effect on Ccl2 expression. These data suggest that the in- with fibroblasts isolated from acutely injured and chronically
ability to respond to PGE2 is a direct result of low levels of injured kidneys. Given that expression values were derived
Ptger3 and/or Ptger4 and that this is an innate property of from relatively pure populations of PDFGra-positive cells
fibrotic PDGFra+ cells. and were normalized, these differences in gene expression
do not reflect differences in cell number; rather, they must
represent inherent differences in gene expression levels per
DISCUSSION cell.
These data allow clustering of gene sets that correlate with
Wound healing is a natural response after injury and inflam- critical steps in the progression or resolution of fibrosis.
mation such that damaged cells and tissues can be repaired. Among these are genes in cluster 3, which are high in the early
Essential for repair is the activation of fibroblasts to myofibro- acute phase of AKI, but decline to near normal levels after 14
blasts, driven in part by profibrotic cytokines such as TGF-b. days, and yet remain high in the UUO model of fibrosis. Many
This results in increased secretion of ECM proteins, which cluster 3 genes are associated with ECM organization and
promote rigidity and form a scaffold for tissue repair.5 In synthesis or signaling from the ECM to cells through integrins.
the kidney, much of the current literature addressing the tissue Thus, reduced ECM expression and integrin signaling within
repair and regeneration after AKI has focused on the renal fibroblasts correlate with the resolution phase of the fibrotic
epithelium. However, dramatic changes in the numbers of in- response after AKI. Similarly, genes associated with the in-
terstitial activated fibroblasts, myofibroblasts, and immune flammatory response in cluster 5 are upregulated in the res-
cells are observed after AKI, even at sites far removed from olution phase, suggesting that PDGFra-positive fibroblasts
localized injury. Yet, by 2–4 weeks post-AKI in mouse models, can regulate the immune response that may be necessary for
this expansion of interstitial cells is no longer observed be- clearing activated fibroblasts and help to degrade the associ-
cause the numbers of aSMA- and PDGFra-positive fibroblasts ated ECM.
have regressed nearly to uninjured levels. We proposed that Among critical signaling pathways, dynamic changes in
there are inherent gene expression differences in fibroblasts WNT components can be seen in the acute injury phase and
that correlate with active proliferation during the acute injury during recovery. WNT5a and Wnt4 are increased at 3 and
phase when compared with fibroblasts that are regressing dur- 7 days post-AKI and decline by 14 days, whereas the WNT
ing the repair process. In this report, we compared inhibitor Dkk2 is down more than five-fold 3 days post-AKI.

J Am Soc Nephrol 30: 80–94, 2019 Gene Expression Changes in Activated Kidney Fibroblasts 91
 BASIC RESEARCH www.jasn.org

A B C D
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PDGFR/Hoechst

E F
AKF U14

AKF 8
1.2

mRNA levels (Rel. units)

mRNA levels (Rel. units)

AKF+T 7 Sox9 Sox9

1.2
1.0 U14 Ccl2 Ccl2
U14+T 6 1.0
0.8 5
0.8
0.6 4
0.6
3
0.4 0.4
2
0.2 1 0.2
0 0 0
Ptger3 Ptger4 cont. PGE2 , T+P cont. PGE2

Figure 8. Activated fibroblasts from fibrotic kidneys exhibit alterations in PGE2 responses. Fibroblast from control adult kidneys (AKF)
or from fibrotic kidneys were isolated by FACS and cultured as primary cell lines. Morphologies of PDGRa+ fibroblasts in primary
cultures from (A) control AKFs or (B) U14 fibroblasts show higher densities of cells with less cytoplasm and more lamellipodia in U14 cell
cultures. (C) Densities of PDGFRa+ cells from (C) control and (D) FA14 kidney sections are shown. Insets in (C and D) are higher
magnifications. (E) Quantitative RT-PCR for PG receptors Ptger3 and Ptger4 in AKFs and U14-isolated fibroblasts cultured with or
without TGF-b (T). (F) Sensitivity to PGE2 (1 mM) in control AKFs and U14 fibroblasts as measured by two PGE2-responsive genes, Sox9
and Ccl2. Cont., control media; Rel., relative; T+P, TGF-b and PGE2.

Consistent with our data, genetic fibroblast-specific ablation fibrosis, low levels of PGE2 correlate with enhanced fibro-
of b-catenin, the canonical WNT signaling effector, results in sis.31,32 However, in our adult fibroblast cultures, these anti-
less injury and inflammation after renal ischemia, suggesting fibrotic effects of PGE2 are diminished, likely because the
that increased WNT signaling in activated fibroblasts inhibits receptors Ptger3 and Ptger4 are significantly downregulated
tubular repair and promotes immune cell infiltration. 23 by TGF-b. In the presence of TGF-b, PGE2 has little effect on
Many of these pathways have previously been identified in the transcriptional program of adult kidney fibroblasts in cell
animal models of renal injury and fibrosis using other meth- culture. In the absence of TGF-b, PGE2 activates many genes
ods, such as sophisticated genetic labeling of different cell that are suppressed by TGF-b alone, whereas many genes
populations, 24,25 demonstrating that FACS for PDGFra+ downregulated by PGE2 are activated by TGF-b. These recip-
cells is a viable alternative to genetic lineage markers for rocal effects on potential target genes could explain in part the
fibroblasts. antifibrotic effects of PGE2. However, those effects are likely
A novel finding among fibroblasts isolated from the acute diminished, because a high concentration of TGF-b sup-
phase 3 days after AKI was the reduction of PG receptor Ptger3. presses expression of PGE2 receptors in fibroblasts. In cul-
During the recovery phase, Ptger3 levels increased but re- tured fibroblasts isolated from UUO kidneys after 14 days,
mained low in the chronic UUO model at 14 days. In lung the expression of Ptger3 and 4 remains low, which strongly
fibrosis models, PGE2 is thought to promote repair in part by attenuates any response to PGE2. This would suggest that
inhibition of fibroblast proliferation,26 migration,27 and/or there may be stable silencing mechanisms in activated fibro-
differentiation into myofibroblasts.28 The suppression of fi- blasts derived from high–TGF-b environments.
broblast activation by PGE2 is thought to occur primarily In summary, our data point to inherent differences in gene
through the cAMP and protein kinase A pathways.26,28–30 expression patterns among PDGFra-positive fibroblasts iso-
In samples isolated from patients with idiopathic pulmonary lated from different phases of renal injury. These changes

92 Journal of the American Society of Nephrology J Am Soc Nephrol 30: 80–94, 2019
 www.jasn.org BASIC RESEARCH

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