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Impairment of PPARa and the Fatty Acid Oxidation

Pathway Aggravates Renal Fibrosis during Aging
Ki Wung Chung,1,2 Eun Kyeong Lee,1,2,3 Mi Kyung Lee,4 Goo Taeg Oh,5 Byung Pal Yu,6
and Hae Young Chung1,2
1
Molecular Inflammation Research Center for Aging Intervention and 2Department of Pharmacy, College of Pharmacy,
Pusan National University, Busan, Republic of Korea; 3Korea Institute of Toxicology, Yuseong-gu, Daejeon, Republic
of Korea; 4Department of Pathology, Ilsin Christian Hospital, Busan, Republic of Korea; 5Department of Life Sciences,
Ewha Womans University, Seoul, Republic of Korea; and 6Department of Physiology, The University of Texas Health
Science Center at San Antonio, San Antonio, Texas

ABSTRACT
Defects in the renal fatty acid oxidation (FAO) pathway have been implicated in the development of renal fibrosis.
Although, compared with young kidneys, aged kidneys show significantly increased fibrosis with impaired kidney
function, the mechanisms underlying the effects of aging on renal fibrosis have not been investigated. In this study,
we investigated peroxisome proliferator–activated receptor a (PPARa) and the FAO pathway as regulators of age-
associated renal fibrosis. The expression of PPARa and the FAO pathway–associated proteins significantly de-
creased with the accumulation of lipids in the renal tubular epithelial region during aging in rats. In particular,
decreased PPARa protein expression associated with increased expression of PPARa-targeting microRNAs. Among
the microRNAs with increased expression during aging, miR-21 efficiently decreased PPARa expression and im-
paired FAO when ectopically expressed in renal epithelial cells. In cells pretreated with oleic acid to induce lipid
stress, miR-21 treatment further enhanced lipid accumulation. Furthermore, treatment with miR-21 significantly
exacerbated the TGF-b–induced fibroblast phenotype of epithelial cells. We verified the physiologic importance
of our findings in a calorie restriction model. Calorie restriction rescued the impaired FAO pathway during aging and
slowed fibrosis development. Finally, compared with kidneys of aged littermate controls, kidneys of aged PPARa2/2
mice showed exaggerated lipid accumulation, with decreased activity of the FAO pathway and a severe fibrosis
phenotype. Our results suggest that impaired renal PPARa signaling during aging aggravates renal fibrosis devel-
opment, and targeting PPARa is useful for preventing age-associated CKD.

J Am Soc Nephrol 29: 1223–1237, 2018. doi: https://doi.org/10.1681/ASN.2017070802

Age-related changes in kidney function, structure,
and their relationships have been extensively recog-
nized recently.1 The effects of aging on the kidneys
are the most dramatic among any other organs
Received July 26, 2017. Accepted January 3, 2018.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Prof. Hae Young Chung, Molecular In-
flammation Research Center for Aging Intervention (MRCA) and
Department of Pharmacy, College of Pharmacy, Pusan National
University, 2, Busandaehak-ro, 63beon-gil, Geumjeong-Gu, Bu-
san 46241, Republic of Korea. Email: hyjung@pusan.ac.kr
Copyright © 2018 by the American Society of Nephrology
Significance Statement
Defective renal lipid metabolism has been implicated in
the development of kidney disease. Defects in the fatty
acid oxidation (FAO) pathway may play a crucial role
during renal fibrosis progression. This paper describes
the important roles of PPARa and the FAO pathway
during age-related renal fibrosis. The studies demon-
strate that PPARa and the FAO pathway are signifi-
cantly decreased with severe interstitial fibrosis during
kidney aging. The decreased PPARa was associated
with increased miR-21 during renal aging, which
showed the inhibitory effect on PPARa in the renal
epithelial cells. The importance of PPARa and the FAO
pathway was further confirmed in antiaging calorie re-
striction and PPARa knockout aging mouse models.
Collectively, targeting PPARa may be useful for pre-
venting age-associated renal fibrosis and CKD.

J Am Soc Nephrol 29: 1223–1237, 2018 ISSN : 1046-6673/2904-1223 1223

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because of diverse factors that can accelerate the changes.1
Epidemiologic studies have suggested that age-related renal
changes may be associated mainly with systemic hypertension,
diabetes, dyslipidemia, and other environmental causes such
as smoking.2,3 With aging, most subjects show a progressive
functional decline that includes decreases in GFR and renal
blood flow as well as increases in urinary excretion of proteins
such as albumin.4,5 The prevalence of CKD is significantly
higher in older people, with more than one-third of those
aged 70 or older exhibiting moderate or severe CKD.6 Inter-
estingly, these functional changes occur in concert with struc-
tural changes, which gives more accurate information on the
status of renal disease.7 Morphologic changes of aging kidneys
include loss of renal mass (primarily in the cortical region),
development of glomerular sclerosis, tubular atrophy, and
tubulointerstitial fibrosis.7
Fibrosis is characterized by a loss of parenchymal tissue with
the accumulation of fibrillary collagens produced by activated
myofibroblasts. Although glomerular lesions are significantly
associated with renal functional decline and particular renal
diseases, renal fibrosis is always accompanied in all forms of
CKD.8 In renal tubulointerstitial fibrosis, tubular epithelial
cells play an important role.9 Loss of epithelial integrity causes
cell cycle arrest and dedifferentiation, leading to increased ex-
pression of mesenchymal markers.10 During renal aging, it has
been well established that structural changes occur with glo-
merulosclerosis, interstitial fibrosis, and tubular atrophy.11
Interestingly, a recent study has shown that age-related
changes in the renal structure occur earlier than functional
changes.12 The authors found a strong relationship between
age and nephrosclerosis in healthy adults without functional
changes in the kidney, thus implicating the importance of
structural changes.
Alterations in lipid metabolism have been implicated in
various metabolic diseases.13 Although the kidney is not clas-
sified as a metabolic organ, renal tubular cells have high basal
levels of baseline energy consumption and they prefer fatty
acid oxidation (FAO) as a major energy source.14,15 The high
energy yield of FAO (106 ATP equivalents per fatty acid [FA]),
which is produced in the mitochondria and peroxisomal com-
partments, makes it possible to support the high energy con-
sumption of cells. Recently, defects in the FAO pathway have
received substantial attention in the context of acute and
chronic kidney diseases.15,16 It has been suggested that a de-
fective FAO pathway induces abnormal lipid accumulation of
triglycerides (TG), resulting in lipotoxicity that contributes to
the development of kidney diseases.
Peroxisome proliferator–activated receptor a (PPARa) is
the key transcriptional factor that regulates intracellular lip-
ids through direct transcriptional control of genes involved
in peroxisomal and mitochondrial FAO pathways, FA up-
takes, and TG catabolism. 17,18 Accumulating evidence
supports a link between PPARa and metabolic diseases in-
cluding diabetes, obesity, dyslipidemia, and fatty liver.18 Be-
cause the kidney is a metabolically active tissue that uses FAs
1224 Journal of the American Society of Nephrology
as a major energy source, the role of PPARa in the develop-
ment of renal diseases has been extensively investigated re-
cently.15,19–21 It has been suggested that defects in PPARa
and the FAO pathway play important roles during renal in-
terstitial fibrosis development. Defective FAO in tubule ep-
ithelial cells has been associated with ATP depletion, cell
death, dedifferentiation, and lipid accumulation, which are
the phenotypes resembling fibrosis.15 Furthermore, others
have suggested that miR-21-mediated downregulation of
PPARa contributes to fibrogenesis and epithelial injury
in a mouse fibrosis model.22
In this study, we hypothesized as to whether age-associated
renal fibrosis is associated with defective FAO in the renal
tubule epithelium. We found that levels of PPARa and FAO-
associated enzymes were reduced in aged rat kidneys with
significantly increased lipid accumulation and interstitial fi-
brosis. Reduced PPARa was associated with increases of
miRNAs that regulate PPARa translation. Further animal
studies using antiaging calorie restriction (CR) and aged
PPARa2/2 mouse models demonstrated the role of PPARa
in the regulation of age-associated renal fibrosis.
RESULTS
Aging Increases Renal Fibrosis, Functional Changes,
and Epithelial Damage
First, we verified renal fibrosis, functional changes, and
epithelial damages that accompany aging. To investigate
age-related fibrosis in our aging rat model, four different-
aged kidneys (6, 12, 18, and 24 months) were examined.
The expression levels of extracellular matrix (ECM) genes in-
cluding Acta2, Fn, Col1a, Col3a1, Col4a, and Col11a1 were all
significantly increased at 24 months of aging (Figure 1A). We
next checked markers for mesenchymal and epithelial cells.
Interestingly, only markers of mesenchymal cells (Vim and
Fsp) were increased during aging, whereas epithelial cell
markers (Krt8 and Cdh1) were not affected by aging (Supple-
mental Figure 1A). The ECM protein levels were further in-
vestigated. The ECM proteins started to increase at 18 months
of age, and the extent was dramatically increased at 24 months
(Figure 1B). A histologic analysis also confirmed age-associ-
ated ECM accumulation and fibrosis. Immunohistochemistry
(IHC) staining of collagen I was dramatically increased in the
interstitial region of aged kidneys (Figure 1C). However, the
expression of collagen III did not show any differences be-
tween young and aged kidneys (Supplemental Figure 1B). The
accumulation of ECM proteins also showed an increase when
detected by Sirius red and Masson’s trichrome staining (Fig-
ure 1D). Fibrotic-positive regions were significantly increased
at 24 months of aging (Figure 1E). We next measured serum
urea and creatinine levels to assess age-related kidney func-
tion decline. Serum urea levels significantly increased at 24
months of aging (Figure 1F); however, creatinine levels did
not change significantly during aging (Supplemental Figure 1C).
J Am Soc Nephrol 29: 1223–1237, 2018
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Figure 1. Aged kidneys show increased renal fibrosis, function decline, and epithelial damages. Four different-aged kidneys (6, 12, 18,
and 24 months, n=8) were examined to examine the effect of aging on renal changes. (A) Gene levels of ECM proteins were measured
by qPCR. *P,0.05 versus 6 months. (B) Protein levels of ECM proteins were measured by western blotting. Actin was used as the
loading control. (C) Collagen I expression in young and aged kidney was visualized by IHC. Scale bar=50 mm. (D) Masson’s trichrome
(MT) and Sirius red (SR) staining were performed to visualize fibrosis in kidney. Scale bar=100 mm. (E) Quantification of SR-stained
fibrosis in kidneys. *P,0.05 versus 6 months. (F) Urea levels were measured by biochemical method in serum to measure kidney
function. *P,0.05 versus 6 months. (G) KIM-1 levels were detected in serum by ELISA to measure renal epithelial damages. *P,0.05
versus 6 months.

Renal periodic acid–Schiff staining and evaluation were further
implemented in aged kidney. Aged rat showed significantly
increased renal damages in both glomerular and tubule regions
(Supplemental Figure 1, D–F). To evaluate renal epithelial
damage, we measured kidney injury molecule-1 (KIM-1) levels
in the serum and kidney. Serum and kidney KIM-1 levels were
also significantly increased during aging (Figure 1G, Supple-
mental Figure 1, G and H). Taken together, we concluded that
aging significantly increases renal fibrosis and damage with
impaired renal function.
Aging Increases Renal Lipid Accumulation
To investigate the relationship between increased fibrosis and
changes in lipid metabolism during aging, lipid metabolism–
associated changes were further evaluated. Levels of extracted
TG were significantly increased at 24 months of aging (Figure
2A). Accumulated TG levels were further visualized via oil red
O–staining analysis. As expected, renal lipid accumulation was
observed in 24-month-old kidneys (Figure 2B). Oil red
O–positive regions were mostly located in renal tubular epithe-
lial cells (Figure 2B). We further detected an overall increase of

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Figure 2. Aging increases lipid accumulation with alteration of various lipid metabolism–associated transcriptional factors. Four dif-
ferent-aged kidneys (6, 12, 18, and 24 months, n=8) were examined to see the effect of aging on renal changes. (A) TGs were
quantified in aging kidney model. *P,0.05 versus 6 months. (B) Kidneys (6 months and 24 months) were stained with oil red O to

1226 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1223–1237, 2018

vacuoles in renal tubules during aging (Figure 2, C and D).
These data indicated that lipid accumulation was increased
during renal aging, and the accumulation was especially in-
creased in the tubular epithelial region.
In the regulation of lipid metabolism, various transcrip-
tional factors play important roles.23 We first checked the
mRNA levels of these transcription factors in the four dif-
ferent-aged kidneys; however, there were no significant
changes during aging (Figure 2E). Because it is generally
accepted that the localization of transcription factors is im-
portant, we next checked the nuclear protein expression of
these transcriptional factors. Surprisingly, unlike mRNA lev-
els, there were remarkable changes in protein expression of
the transcription factors in the nucleus. Nuclear expression
of PPARa and Farnesoid X receptor (FXR) were dramatically
decreased in 24-month-old rat kidneys, whereas SREBP1
and ChREBP were rather increased during aging (Figure 2,
F and G). Taken together, these data implicated that dysre-
gulated lipid metabolism during aging might be associated
with changes in nuclear expression of transcriptional factors
that regulate lipid metabolism.
Aging Decreases PPARa and FAO Pathway in Renal
Tubule Epithelial Region
Recent studies have suggested that defective FAO by impaired
PPARa in renal tubular epithelial cells plays a pivotal role in
kidney fibrosis development.15 On the basis of our results and
those of previous studies, we hypothesized that impairments
in PPARa and the FAO pathway may be important in the
development of age-associated renal fibrosis. First, we
checked whether the decreased nuclear expression of PPARa
was due to defects in translocation. However, cytosolic frac-
tions of aged kidney also showed decreased PPARa expression
(Figure 3A). We next checked the expression levels of well
known PPARa gene targets by quantitative PCR (qPCR).
Most of the PPARa target genes (Cpt1a, Acox1, Lcad, Mcad,
Acad10, and Hadh) were significantly decreased at 24 months
of aging (Figure 3B). However, other lipid metabolism–asso-
ciated genes that play important roles in FA synthesis and TG
accumulation did not show significant changes (Supplemen-
tal Figure 2, A and B). We further checked carnitine palmi-
toyltransferase 1a (CPT1a) and acyl-coenzyme A oxidase 1
(ACOX1) protein levels. Aged rat kidneys showed signifi-
cantly decreased CPT1a and ACOX1 protein levels (Figure
3C). The decreased levels of PPARa and its target protein,
ACOX1, were further verified by IHC. PPARa and ACOX1
primarily localized to the tubular epithelial regions where the
visualize lipid accumulation. Scale bar=50 mm. (C) Representative H&E staining shows increased vacuoles in renal tubules during aging. (D)
Number of vacuoles was quantified by counting vacuoles per tubule. *P,0.05 versus 6 months. (E) Gene levels of transcription factors
associated with lipid metabolism were measured by qPCR. *P,0.05 versus 6 months. (F) Nuclear protein expressions of transcription
factors associated with lipid metabolism were measured by western blotting. TFIIB was used as the loading control. (G) Western blot
results from 4 independent experiments were quantified by densitometry. *P,0.05 versus 6 months.
J Am Soc Nephrol 29: 1223–1237, 2018
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lipids accumulated (Figure 3D). Furthermore, the expression
of PPARa and ACOX1 detected by IHC was also decreased
during aging (Figure 3D). Taken together, we found that the
levels of PPARa and its target proteins, CPT1a and ACOX1,
were significantly decreased during aging.
Because there were discrepancies between gene and pro-
tein levels of PPARa during aging, we next checked the
upstream signaling of PPARa. However, the levels of
AMP-activated protein kinase (AMPK) and activated
(phosphorylated) AMPK were both increased during aging
(Figure 3E). The protein levels of PGC1a, which is an im-
portant coactivator of PPARa, were not changed during
aging (Figure 3E). We further checked various miRNAs
that can silence PPARa translation by epigenetic mecha-
nisms. Interestingly, among the seven miRNAs that are
known to suppress PPARa translation, levels of miR-21,
miR-34a, and miR-155 were significantly increased during
aging (Figure 3F). In summary, decreased PPARa expres-
sion during aging may be affected by miRNA that regulates
the translation of PPARa epigenetically.
PPARa Translation and FAO Are Suppressed by miR-21
in Renal Tubular Epithelial Cells
We next performed in vitro experiments to examine whether
increased miRNA levels during aging suppress PPARa ex-
pression and affect FAO in renal tubular epithelial cells.
NRK52E renal tubular epithelial cells were used to examine
the effects of miRNAs on PPARa and the FAO pathway.
Among the three miRNAs (miR-21, miR-34a, and miR-
155) that were increased during aging, only miR-21 effi-
ciently decreased PPARa protein expression in NRK52E cells
without changes in PPARa gene levels (Figure 4A, Supple-
mental Figure 3A). Furthermore, miR-21 also decreased
PPRE binding activity as deduced from a luciferase assay
(Figure 4B). PPARa target gene levels were further analyzed
under miR-21 treatment conditions. Most, but not all, of the
PPARa target genes were downregulated by an miR-21 treat-
ment (Figure 4C). Decreased CPT1a and ACOX1 levels were
further confirmed by measuring their protein levels (Figure
4D). We next measured oxygen consumption rate (OCR)
under the miR-21 treatment conditions. miR-21 treatment
decreased OCR levels both under the normal and oleic acid
treatment conditions (Figure 4E). Furthermore, consistent
with decreased OCR, miR-21 treatment reduced ATP pro-
duction in cells (Supplemental Figure 3B). However, treat-
ment with miR-21 resulted in marginal changes in the total
lipid content of the cells (Figure 4, F and G). Next, we
PPARa and Age-Associated Renal Fibrosis 1227
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Figure 3. Aging decreases PPARa and FAO signaling pathways in the renal tubule epithelial region. Four different-aged
kidneys (6, 12, 18, and 24 months, n=8) were examined to see the effect of aging on renal changes. (A) Nuclear and cytosolic
PPARa levels were measured in the rat kidney. TFIIB was used as the loading control for the nuclear fraction, and actin was
used as the loading control for the cytosolic fraction. (B) Gene levels of PPARa target proteins were measured by qPCR.
*P,0.05 versus 6 months. (C) Protein expression of CPT1a and ACOX1 was measured by western blotting. Actin was used as
the loading control. (D) PPARa and ACOX1 expression in young and aged kidneys was visualized by IHC. Scale bar=50 mm.
(E) Protein expression levels of signals upstream of PPARa (AMPK, p-AMPK, and PGC1a) were measured by western blotting.
Actin was used as a loading control. (F) The miRNA levels that are known to regulate PPARa expression were detected by
qPCR. *P,0.05 versus 6 months.

pretreated cells with 200 mM of oleic acid to induce lipid
stress in the epithelial cells. Treatment with oleic acid signif-
icantly increased intracellular lipid content when measured
quantitatively or visualized by oil red O staining (Figure 4,
F and G). The miR-21 treatment further increased the accu-
mulation of intracellular lipids (Figure 4, F and G). We
further checked FAO-related gene changes in the same con-
ditions. Although oleic acid treatment regulated FAO-related
gene expressions differently, miR-21 constantly reduced
those gene expressions regardless of oleic acid treatment
(Supplemental Figure 3C). Furthermore, oleic acid with
miR-21 treatment induced a decrease in cell viability,

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Figure 4. miR-21 suppresses PPARa translation and FAO in NRK52E cells. Suppression of PPARa translation and FAO by miR-21
in renal tubular epithelial cells. NRK52E renal epithelial cells were used to investigate the role of miRNAs in PPARa and FAO
signaling. (A) NRK52E cells were transfected with selected miRNAs (miR-21, miR-34a, and miR-155) and protein expression of
PPAR was measured by western blotting. Actin was used as the loading control. (B) NRK52E cells were transfected with selected
miRNAs (miR-21, miR-34a, and miR-155) and PPAR activities were measured by the PPRE luciferase activity. (C) Gene levels of
PPARa target proteins were measured by qPCR after miR-21 transfection in NRK52E cells. *P,0.05 versus nontreated control.
(D) Protein expression of CPT1a and ACOX1 was measured by western blotting in miR-21-treated NRK52E cells. Actin was used
as the loading control. (E) OCR was measured under normal and miR-21-treated conditions. Oleic acid (200 mM) and oligomycin
(1 mM) were treated at designated times. (F) Cellular TG levels were extracted and quantified in NRK52E cells treated with 200
mM oleic acid and/or miR-21. #P,0.05 versus nontreated control. *P,0.05 versus oleic acid treatment. (G) Cellular TG was
visualized by oil red O staining in NRK52E cells treated with 200 mM oleic acid and/or miR-21. Scale bar=50 mm.

suggesting the role of FAO in regulating lipotoxicity under
the stress condition (Supplemental Figure 3D). These data
suggest that miR-21 suppresses the translation of PPARa in
renal epithelial cells, thus inhibiting FAO and inducing lipid
accumulation.
The FAO Pathway Is Important during TGF-b-Induced
Fibrosis Signaling in Renal Tubular Epithelial Cells
It has been suggested that TGF-b-induced miR-21 plays an
important role during the mediation of fibrosis signaling in
renal epithelial cells.24 To this end, we further investigated
the roles of miR-21-mediated PPARa silencing and an
impaired FAO pathway during fibrosis signaling in renal ep-
ithelial cells. First, we measured miR-21 expression changes
on TGF-b-treated NRK52E cells. TGF-b significantly in-
duced miR-21 expression as previously described (Figure
5A). We next checked whether TGF-b treatment affected
lipid accumulation that was induced by oleic acid. TGF-b
treatment further increased oleic acid–induced lipid accu-
mulation (Figure 5, B and C). To further demonstrate the
role of miR-21-mediated PPARa silencing and the FAO
pathway on TGF-b, an miR-21 inhibitor (anti-miR-21 oligo)
was utilized. TGF-b treatment reduced PPARa expression in
epithelial cells, and anti-miR-21 abrogated this reduction

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Figure 5. FAO signaling is important in TGF-b-induced fibrosis signaling in renal tubular epithelial cells. NRK52E renal epithelial cells
were transfected with miR-21 under TGF-b treatment conditions to investigate the role of FAO signaling in fibrosis. (A) The miR-21
levels were measured by qPCR under TGF-b treatment conditions. (B) Cellular TG was visualized by oil red O staining in NRK52E cells
treated with 200 mM oleic acid or/and TGF-b. Scale bar=50 mm. (C) Cellular TG levels were extracted and quantified in NRK52E cells
treated with 200 mM oleic acid or/and TGF-b. #P,0.05 versus nontreated control. *P,0.05 versus oleic acid treatment. (D) Protein
expression of PPARa, CPT1a, and ACOX1 was measured by western blotting in TGF-b and anti-miR-21–treated NRK52E cells. Actin
was used as the loading control. (E) Gene levels of PPARa target proteins were measured by qPCR after TGF-b and anti-miR-21
treatment in NRK52E cells. #P,0.05 versus nontreated control. *P,0.05 versus TGF-b treatment. (F) OCR was measured under
designated conditions. Oleic acid (200 mM) and oligomycin (1 mM) were treated at designated times. (G) Cellular TG was visualized by
oil red O staining in NRK52E cells treated with designated conditions. Scale bar=50 mm. (H) Cellular TG levels were extracted and
quantified in NRK52E cells treated with designated conditions. #P,0.05 versus nontreated control. *P,0.05 versus TGF-b and anti-
miR-21 treatment. (I) Changes in expression of ECM genes in NRK52E cells. Gene expression levels of ECM proteins were measured by
qPCR under oleic acid or/and TGF-b treatment conditions. #P,0.05 versus nontreated control. *P,0.05 nontreated control versus
TGF-b, oleic acid, and anti-miR-21 treatment.

(Figure 5D). PPARa target genes and protein expression
changes were also reduced by TGF-b treatment and blocked
by anti-miR-21 treatment (Figure 5, D and E). We next mea-
sured the effect of anti-miR-21 on OCR under the TGF-b
treatment condition. Treatment with anti-miR-21 blocked
TGF-b-induced OCR level decrease (Figure 5F) and rescued
ATP levels (Supplemental Figure 4). In addition, anti-miR-
21 treatment significantly reduced oleic acid–induced lipid
accumulation under the TGF-b treatment conditions
(Figure 5, G and H). We next checked whether lipid stress
increases TGF-b-induced fibrosis-related signaling in cells.
Although oleic acid treatment itself did not alter fibrosis-
related gene expression, oleic acid under the TGF-b treat-
ment conditions significantly increased fibrosis-related
gene expression (Supplemental Figure 5). The effect of oleic
acid was partially blocked by anti-miR-21 treatment
(Figure 5I). Collectively, these data implicated the impor-
tance of the FAO pathway in TGF-b-induced fibrosis sig-
naling in renal tubular epithelial cells, especially under
excessive lipid stresses.

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CR Restores PPARa Expression and Slows Renal
Fibrosis
CR is one of the most powerful antiaging regimens that
can retard aging and age-related diseases.25 To investigate
whether CR can restore defective lipid metabolism during
aging, calorie-restricted aged rats were used. CR significantly
rescued aging-induced decreases in PPARa expression (Figure 6A).
Furthermore, CPT1a and ACOX1 expression levels were also
increased in response to CR (Figure 6A). The increased PPARa
and ACOX1 levels were confirmed by IHC analysis. Increased
expressions of PPARa and ACOX1 were detected especially in
the epithelial region of CR kidneys (Figure 6B). Other PPARa
target genes that modulate FAO were also increased by CR
(Figure 6C). The increased expression of FAO regulators was
associated with decreased lipid accumulation in the CR kid-
neys (Figure 6, D and E). In contrast, other lipid metabolism–
associated genes were not affected by CR (Supplemental Fig-
ure 6, A and B). We further detected lipid vacuoles in renal
tubules during CR (Figure 6, F and G). CR significantly re-
duced the number of vacuoles in tubules. PPARa-silencing
miRNAs were further investigated. CR resulted in significant
reductions in the levels of all three miRNAs (miR-21, miR-
34a, miR-155) that showed increase during aging (Figure 6H).
Together, these data suggested that CR restored PPARa ex-
pression through the regulation of miRNAs, implicating the
importance of the FAO pathway in the regulation of renal
lipid accumulation.
Because defective FAO is known to play a role in the devel-
opment of fibrosis, we further examined the effects of CR on
age-related fibrosis development. CR significantly reduced gene
expression of several ECM components (Figure 7A). CR also
reduced protein expression of ECM components including
procollagen I, collagen I, and collagen IV (Figure 7B). IHC
staining of collagen I, Sirius red, and Masson’s trichrome stain-
ing also showed decreased expression of ECM in the interstitial
region of the kidneys (Figure 7C). Consistently, fibrotic scores
were decreased in the CR group when the area positive for
Sirius red staining was calculated (Figure 7D). Furthermore,
CR improved renal function with decreased epithelial damage
(Figure 7, E and F, Supplemental Figure 6, C–G). In summary,
CR significantly delayed aging-induced renal fibrosis with the
restoration of PPARa expression and FAO-associated proteins.
PPARa Deficiency Increases Renal Lipid Accumulation
and Accelerates Renal Fibrosis
To confirm the role of PPARa in aging-induced renal fibrosis,
we subjected PPARa2/2 mice and sex-matched control litter-
mates to aging. We first compared changes in lipid metabo-
lism. As expected, aged control mice showed decreased PPARa
and FAO-associated gene expression (Figure 8, A and B).
Compared with aged control littermates, PPARa2/2 mice
showed reduced expression of FAO-associated proteins and
genes (Figure 8, A and B). In contrast, other lipid metabolism
genes were not significantly changed in aged PPARa2/2
mice except for Scd (Supplemental Figure 7, A and B). Aged
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PPARa2/2 mice showed significantly higher lipid accumula-
tion compared with littermates (Figure 8C). Interestingly,
aged PPARa2/2 mouse kidneys showed distinct histologic
structures in the renal tubule region when analyzed by
hematoxylin and eosin (H&E) staining (Figure 8D). Aged
PPARa2/2 mouse kidneys showed increased vacuoles in tu-
bules (Supplemental Figure 8, A and B). These distinct histo-
logic structures were found to be due to excessive lipid droplet
formation when the kidneys were subjected to oil red
O staining (Figure 8D). These data suggested that PPARa de-
ficiency significantly decreases FAO with high lipid accumu-
lation in the tubule epithelial region during aging.
We further investigated whether PPARa-deficient mice ex-
hibit accelerated renal fibrosis during aging. Aged PPARa2/2
mice developed significantly more interstitial fibrosis than
control littermates as detected by mRNA and protein expres-
sion levels of ECM proteins (Figure 8, E and F). IHC staining
of collagen I, Sirius red, and Masson’s trichrome staining were
also higher in the interstitial region of aged PPARa2/2 kidneys
(Figure 8G). Consistently, fibrotic scores were increased in
aged PPARa2/2 kidneys when calculated by using Sirius
red–staining–positive area (Figure 8H). Serum urea levels
and histologic damages were also increased only in the aged
PPARa2/2 mice group (Figure 8I, Supplemental Figure 8, C–E).
Similarly, the epithelial injury marker KIM-1 was also expressed
at high levels in the aged PPARa2/2 mouse serum and kidneys
(Figure 8J, Supplemental Figure 8, F and G). Collectively, these
data indicated that PPARa deficiency not only increases renal
lipid accumulation but also accelerates renal interstitial fibro-
sis, suggesting the importance of PPARa on the development of
age-associated renal fibrosis.
DISCUSSION
The role of lipid metabolism has been recently implicated in
various renal diseases.26,27 The kidney is not a major metabolic
organ that actively participates in the systemic regulation of
lipid metabolism. However, recent evidence suggests that lipid
accumulation in nonadipose tissues can contribute to organ
injury.28 Clinical observations have implicated a potential as-
sociation between renal lipid accumulation and CKD devel-
opment.29 Furthermore, there is an abundance of animal data
demonstrating an association between renal lipid accumula-
tion and kidney dysfunction, including models of metabolic
and nonmetabolic renal diseases.15,30 In addition, several pre-
vious reports have examined the effects of renal lipid metab-
olism on age-associated renal diseases.31,32 Aged animals show
higher lipid accumulation with increased glomerulosclerosis
and tubulointerstitial fibrosis. However, although the occur-
rence of renal lipid accumulation in aged kidneys was coinci-
dent in our aged model, the regions of lipid accumulation were
different compared with those reported by previous studies.
Our results clearly indicated that the site of lipid accumulation
was the renal tubular epithelial region.
PPARa and Age-Associated Renal Fibrosis 1231
 BASIC RESEARCH www.jasn.org

Figure 6. CR restores PPARa expression and FAO pathways with decreased lipid accumulation in the kidneys. Young (6-month), old
(24-month), and calorie-restricted old (24-month) rat kidneys were used to investigate the effects of CR on age-related renal fibrosis
(n=7–8). (A) Nuclear protein expression of PPARa and cytosolic protein expression of CPT1a and ACOX1 were measured by western
blotting. TFIIB and actin were used as the loading controls, respectively. (B) PPARa and ACOX1 expression in kidney samples were
visualized by IHC. Scale bar=50 mm. (C) Gene levels of PPARa target proteins were measured by qPCR. #P,0.05 versus young.
*P,0.05 versus old. (D) TGs were quantified in the kidneys. #P,0.05 versus young. *P,0.05 versus old. (E) Kidneys were stained with
oil red O to visualize lipid accumulation. Scale bar=50 mm. (F) Representative H&E staining shows increased vacuoles in renal tubules
during aging and CR. (G) Number of vacuoles was quantified by counting vacuoles per tubule. #P,0.05 versus young. *P,0.05 versus
old. (H) The miRNA levels that were changed during aging were detected by qPCR. #P,0.05 versus young. *P,0.05 versus old.

1232 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1223–1237, 2018
 www.jasn.org BASIC RESEARCH

Figure 7. CR prevents the development of age-related renal fibrosis. Young (6-month), old (24-month), and CR-old (24-month) rat
kidneys were used to investigate the effects of CR on age-related renal fibrosis (n=7–8). (A) Gene levels of ECM proteins were mea-
sured by qPCR. #P,0.05 versus young. *P,0.05 versus old. (B) Protein levels of ECM proteins were measured by western blotting.
Actin was used as the loading control. (C) Collagen I expression was visualized by IHC, and Masson’s trichrome (MT) and Sirius red (SR)
staining were performed to visualize fibrosis. Scale bar=50 mm. (D) Quantification of SR-stained fibrosis in kidneys. #P,0.05 versus
young. *P,0.05 versus old. (E) Urea levels were measured by biochemical methods in the serum to measure kidney function. #P,0.05
versus young. *P,0.05 versus old. (F) KIM-1 levels were detected in the serum via ELISA to measure renal epithelial damages. #P,0.05
versus young. *P,0.05 versus old.

Recently, roles of the impaired FAO pathway in the develop-
ment of renal interstitial fibrosis have been investigated.15 The
authors found that renal tubular epithelial cells depend critically
on FAO as their energy source, and depressed FAO was associ-
ated with intracellular lipid accumulation. This observation was
consistent with previous reports demonstrating that mitochon-
drial b-oxidation of FAs is a major source for renal energy pro-
duction, which has a high energy demand with relatively low
glycolytic capacity.33 They also suggested that impaired FAO was
owing to a decreased PPARa-PGC1a axis during renal fibrosis
development. We also found that age-associated renal lipid
accumulation was associated with an impaired FAO pathway.
Critically, the protein levels of CPT1a, which is the rate-limiting
enzyme of FAO, showed great reduction in the tubular epithelial
region. However, we found that upstream regulators of PPARa,
AMPK and PGC1a, which were shown to be significantly re-
duced in the fibrosis model, were actually increased or un-
changed during aging. These differences led us to investigate
other mechanisms associated with decreased PPARa signaling
during aging.
miRNAs are endogenously encoded, evolutionarily con-
served small RNAs that regulate gene expression predominantly

J Am Soc Nephrol 29: 1223–1237, 2018 PPARa and Age-Associated Renal Fibrosis 1233
 BASIC RESEARCH www.jasn.org

Figure 8. PPARa deficiency increases renal lipid accumulation and accelerates renal fibrosis. PPARa2/2 mice and their littermates were
subjected to aging to investigate the effect of PPARa on renal lipid metabolism and fibrosis changes during aging (n=5–7). (A) Gene

1234 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 1223–1237, 2018
 www.jasn.org BASIC RESEARCH

by facilitating the degradation and translation inhibition of tar-
get mRNAs, thus having a powerful role in regulating various
biologic processes.34 Additionally, miRNAs have also been im-
plicated in regulating various kidney diseases, including renal
carcinoma, diabetic nephropathy, and AKI.35 We found a dis-
crepancy between the gene and protein levels of PPARa, which
suggests that there may be epigenetic mechanisms regulating
PPARa translation. Among the miRNAs that are known to
regulate PPARa, miR-21 showed great efficiency in regulating
PPARa expression in renal tubular epithelial cells. Interest-
ingly, miR-212/2 mice have been reported to exhibit far less
interstitial fibrosis in response to kidney injury, and the lipid
metabolism pathway regulated by PPARa has been implicated
in this model.22 In addition, transfection of an miR-21 shRNA
plasmid halted the progression of renal fibrosis in established
obstructive nephropathy.24 We demonstrated the importance of
the miR-21-PPARa axis in age-related renal fibrosis. Likewise,
miR-21-mediated PPARa silencing had significant effects in the
regulation of lipid metabolism and TGF-b response in renal
tubular epithelial cells. Given the previous reported data and
our results, miR-21 has been shown to play a role in the regula-
tion of PPARa and lipid metabolism as an important regulator
of renal fibrosis.
Through the regulation of various biologic processes rang-
ing from metabolic changes to epigenetic processes, CR is
known to regulate aging and age-related diseases in diverse
species.25 We hypothesized that changes in renal lipid metab-
olism are important in inducing age-associated renal fibrosis;
therefore, the effects of CR on renal lipid metabolism and
fibrosis were further investigated. We found that CR signifi-
cantly reduced renal lipid amounts with increased PPARa ex-
pression and FAO-associated genes. CR also reduced miRNAs
that regulate PPARa silencing during aging. These changes in
lipid metabolism were associated with decreased renal fibrosis
during aging. The effects of CR on age-associated fibrosis have
been reported in other studies. Although the suggested mech-
anisms vary between the model and tissues used, it is evident
that CR can alleviate age-related kidney, heart, liver, and aorta
fibrosis.32,36,37 As a strong metabolic modifier, CR can allevi-
ate renal fibrosis by regulating abnormal lipid metabolism
during aging.
It has been suggested that sirtuin 1 (SIRT1) is a main reg-
ulator that connects calorie intake and life span.38,39 Interest-
ingly, SIRT1 has been shown to directly or indirectly regulate
PPARa expression and activity, and is also associated with re-
nal fibrosis, aging, and other age-related diseases.40,41 Further-
more, SIRT1 is also regulated by miRNAs through epigenetic
regulation.42 Although we did not check the SIRT1 levels in
our animal models, previous studies have shown decreased
SIRT1 expression and activity during renal aging.43 Because
SIRT1 mediates various beneficial roles during CR, a possible
role of SIRT1 in age-related renal fibrosis and its association
with PPARa and CR should be further considered.
Increasing evidence has also demonstrated an association
between PPARa and aging.44,45 The decreased expression or
activity of PPARa has been detected during aging in several
tissues including kidney, heart, and spleen.46–48 More direct
evidence on the relationship between PPARa and aging comes
from observations of PPARa2/2 mice. Accelerated physiolog-
ically aged phenotypes were first reported in PPARa2/2 mice,
along with decreased longevity and increased age-dependent
lesions.49–51 In the liver, the roles of PPARa during aging have
also been shown. PPARa2/2 mice and hepatocyte-specific
PPARa2/2 mice showed significant alterations of systemic
lipid metabolism that lead to early hepatic steatosis during
aging.52 Although it is evident that PPARa deficiency acceler-
ates renal aging with defects in systemic lipid metabolism, the
phenotypes of aged PPARa2/2 mice regarding renal lipid
metabolism have not been previously reported. We found
that renal lipid metabolism was severely impaired in aged
PPARa2/2 mice with increased lipid accumulation. Impaired
lipid metabolism in the kidney was associated with early onset
of renal fibrosis in aged PPARa2/2 mice. However, we cannot
exclude other possibilities that can induce an early onset of the
fibrosis phenotype in aged PPARa2/2 mice, because we used
whole-body PPARa2/2 mice. Performing aging studies in kid-
ney epithelial–specific PPARa2/2 mice will be important.
To conclude, these studies identify PPARa and the FAO
pathway as important regulators in kidney lipid metabolism
that amplifies age-associated renal fibrosis (Supplemental
Figure 9). The post-transcriptional regulator of PPARa,
which is miR-21, critically decreased FAO pathways in renal

levels of PPARa target proteins were measured by qPCR. #P,0.05 versus 7-month wild type (WT). *P,0.05 versus 20-month WT. (B)
Nuclear protein expression of PPARa and cytosolic protein expression of CPT1a and ACOX1 were measured by western blotting. TFIIB
and actin were used as the loading controls, respectively. (C) TGs were quantified in the kidneys. #P,0.05 versus 7-month WT. *P,0.05
versus 20-month WT. (D) Kidneys were stained with H&E to visualize structural differences, and oil red O staining was used to visualize lipid
accumulation. Scale bar in H&E staining=100 mm. Scale bar in oil red O staining=50 mm. (E) Gene levels of ECM proteins were measured
by qPCR. #P,0.05 versus 7-month WT. *P,0.05 versus 20-month WT. (F) Protein levels of ECM proteins were measured by western
blotting. Actin was used as the loading control. (G) Collagen I expression was visualized by IHC, and Masson’s trichrome (MT) and Sirius red
(SR) staining were performed to visualize fibrosis. Scale bar=100 mm. (H) Quantification of SR-stained fibrosis in the kidneys. #P,0.05
versus 7-month WT. *P,0.05 versus 20-month WT. (I) Urea levels were measured by biochemical methods in the serum to measure kidney
function. #P,0.05 versus 7-month WT. *P,0.05 versus 20-month WT. (J) KIM-1 levels were detected in the serum by ELISA to measure
renal epithelial damages. #P,0.05 versus 7-month WT. *P,0.05 versus 20-month WT. ORO, oil red O; Ppara, Peroxisome proliferator-
activated receptor alpha.

J Am Soc Nephrol 29: 1223–1237, 2018 PPARa and Age-Associated Renal Fibrosis 1235
 BASIC RESEARCH www.jasn.org
epithelial cells with increased fibrotic responses. Antiaging
CR rescued age-associated PPARa and the FAO pathway
with slowed renal fibrosis. PPARa2/2 mice showed a severely
disrupted renal FAO pathway with high accumulation of lip-
ids, and showed earlier onset of renal fibrosis, demonstrating
the importance of PPARa in the development of age-related
renal fibrosis. Future studies may explore the effects of im-
paired PPARa and FAO pathways on human aged kidney
samples. It will also be necessary to assess the pharmacologic
interventions (e.g., PPARa agonist, miR-21 antagonist) for
the treatment of age-associated renal fibrosis. If so, this could
provide novel therapeutics to intervene in renal fibrosis and
functional decline during aging.
CONCISE METHODS
Animals
To investigate the changes in lipid metabolism and fibrosis during
aging, 6-, 12-, 18-, and 24-month-old SD rats (n=8) were used. To
compare the effects of CR on lipid metabolism and fibrosis
changes, young (6 months), aged (24 months), and CR (60% of
ad libitum for 1 month) aged (24 months) rats (n=7–8) were used.
To evaluate the effects of PPARa deficiency on age-associated re-
nal fibrosis, age-matched (3, 7, and 20 months) PPARa2/2 mice
and their wild-type littermates (n=5–7) were used. Animal studies
were designed by the Aging Tissue Bank, approved by the Insti-
tutional Animal Care Committee of Pusan National University,
and performed in accordance with the guidelines for animal ex-
perimentation issued by Pusan National University. Rats and mice
were maintained under controlled environmental conditions
under a 12-hour light/dark cycle, and allowed ad libitum access
to water and a standard laboratory diet except for the CR study.
PPARa2 /2 mice were genotyped by PCR as previously de-
scribed. 53 For all animal experiments, serum was collected for
biochemical analysis. Kidneys were collected and either frozen
immediately in liquid nitrogen for qPCR, western blotting, and
biochemical tests, or fixed in neutral-buffered formalin for his-
tochemical examination. For long-term storage, tissue samples
were moved to a 280°C deep freezer located at the Aging Tissue
Bank in Pusan National University.
Histologic Analysis
Kidneys were fixed in 10% neutral formalin and paraffin-embedded
sections were stained with H&E. Sirius red staining and Masson’s
trichrome staining were performed as described previously to deter-
mine the degree of aging-associated fibrosis.54
Statistical Analyses
The t test was used to analyze differences between two groups, and
ANOVA was used to analyze intergroup differences. P values of ,0.05
were considered statistically significant. The analysis was performed
using GraphPad Prism 5 (GraphPad software).
Detailed protocols for the other experiments are provided in the
Supplemental Material.
1236 Journal of the American Society of Nephrology
ACKNOWLEDGMENTS
The authors thank the Korean Aging Tissue Bank for providing re-
search materials. The authors also thank G.T.O. (Ewha Womans
University) for providing PPARa2/2 mice.
This work was supported by the National Research Foundation of
Korea (NRF) funded by the Korean government (MSIP: Minister of
Science, ICT, and Future Planning) (grant Nos. 2009-0083538,
2013M3A9B6076431 and 2015R1A2A2A01004137), and the Bio &
Medical Technology Development Program of the NRF funded by
the MSIP (grant No. 2015M3A9B8029074).
K.W.C. and H.Y.C. developed the concept and designed the ex-
periments. K.W.C., E.K.L., and M.K.L. performed the experiments.
K.W.C., B.P.Y., and H.Y.C. prepared the manuscript. All of the au-
thors analyzed the results and edited the manuscript.
DISCLOSURES
None.
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This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2017070802/-/DCSupplemental.
PPARa and Age-Associated Renal Fibrosis 1237