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org

High-Fat Diet-Induced Lysosomal Dysfunction and

Impaired Autophagic Flux Contribute to Lipotoxicity in
the Kidney
Takeshi Yamamoto,* Yoshitsugu Takabatake,* Atsushi Takahashi,* Tomonori Kimura,*
Tomoko Namba,* Jun Matsuda,* Satoshi Minami,* Jun-ya Kaimori,† Isao Matsui,*
Taiji Matsusaka,‡ Fumio Niimura,§ Motoko Yanagita,| and Yoshitaka Isaka*
Departments of *Nephrology and †Advanced Technology for Transplantation, Osaka University Graduate School of
Medicine, Suita, Osaka, Japan; ‡Institute of Medical Sciences and Department of Molecular Life Sciences and
§
Department of Pediatrics, Tokai University School of Medicine, Isehara, Kanagawa, Japan; and |Department of
Nephrology, Kyoto University Graduate School of Medicine, Kyoto, Japan

ABSTRACT
Excessive fat intake contributes to the progression of metabolic diseases via cellular injury and
inflammation, a process termed lipotoxicity. Here, we investigated the role of lysosomal dysfunction and
impaired autophagic flux in the pathogenesis of lipotoxicity in the kidney. In mice, a high-fat diet (HFD)
resulted in an accumulation of phospholipids in enlarged lysosomes within kidney proximal tubular cells
(PTCs). In isolated PTCs treated with palmitic acid, autophagic degradation activity progressively stagnated
in association with impaired lysosomal acidification and excessive lipid accumulation. Pulse-chase experiments
revealed that the accumulated lipids originated from cellular membranes. In mice with induced PTC-specific
ablation of autophagy, PTCs of HFD-mice exhibited greater accumulation of ubiquitin-positive protein ag-
gregates normally removed by autophagy than did PTCs of mice fed a normal diet. Furthermore, HFD-mice
had no capacity to augment autophagic activity upon another pathologic stress. Autophagy ablation also
exaggerated HFD-induced mitochondrial dysfunction and inflammasome activation. Moreover, renal
ischemia-reperfusion induced greater injury in HFD-mice than in mice fed a normal diet, and ablation
of autophagy further exacerbated this effect. Finally, we detected similarly enhanced phospholipid accu-
mulation in enlarged lysosomes and impaired autophagic flux in the kidneys of obese patients compared
with nonobese patients. These findings provide key insights regarding the pathophysiology of lipotoxicity
in the kidney and clues to a novel treatment for obesity-related kidney diseases.

J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016070731

In the last several decades, the increased prevalence
of obesity has been considered a serious health and
economic burden worldwide.1 A growing body of ev-
idence suggests that excess lipid intake induces cell in-
jury and organ dysfunction (termed lipotoxicity).2,3
Lipotoxicity has been intensively studied in organs
that have evolved specialized lipid handling functions,
such as adipose tissue and liver. Although Moorhead
proposed the hypothesis that lipid abnormalities con-
tribute to the progression of kidney injury in 1982,4
and increasing evidence has supported the lipid neph-
rotoxicity hypothesis, studies on lipid metabolism in
the kidney are scarce.
The mitochondrial b-oxidation of free fatty
acids (FFAs) is a major source of renal ATP produc-
tion, particularly in proximal tubular cells (PTCs),
Received July 8, 2016. Accepted November 7, 2016.
T.Y. and Y.T. contributed equally to this work.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Yoshitsugu Takabatake, Department of
Nephrology, Osaka University Graduate School of Medicine, Box
D11, 2-2 Yamada-oka, Suita, Osaka 585-0871, Japan. Email:
takaba@kid.med.osaka-u.ac.jp
Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 28: ccc–ccc, 2016 ISSN : 1046-6673/2805-ccc 1

 BASIC RESEARCH www.jasn.org
which have a high energy demand.5–7 PTCs take up circulating
FFAs dissociated from albumin through specific membrane
proteins, such as fatty acid (FA) translocase (CD36) and
FA-binding protein.8 In addition, PTCs retrieve albumin-
bound FFAs from the glomerular filtrate by receptor-mediated
albumin endocytosis.
Autophagy is one of the major cellular degradation path-
ways, along with the ubiquitin–proteasome system. 9–11
Recently, a close relationship between autophagic activity
and lipid metabolism has been recognized. Firstly, lipid drop-
lets are sequestered by autophagosomes and eventually broken
down by lysosomes through a process termed lipophagy.12–14
Secondly, several saturated and unsaturated FFAs appear to
modulate autophagic activity. For example, palmitic acid
(PA) activates autophagy by downregulation of the mechanis-
tic target of rapamycin (mTOR) signal15 or by the phosphor-
ylation of the double-stranded RNA-dependent protein kinase
c-Jun N-terminal kinases 1 pathway.16 Moreover, PA causes
the disruption of inhibitory signal transducer and activator of
transcription 3–RNA-dependent protein kinase interactions, which
facilitates autophagy induction.17 In contrast, a high-fat diet (HFD)
downregulates autophagy by reducing autophagosome/lysosome
fusion,18 as well as by decreasing the number and the acidity
of lysosomes.19 The conflicting results may be attributed to
differences in cell types, observation period, FFAs concentra-
tion and, more likely, difficulty in monitoring autophagic flux
in vivo.
We have previously demonstrated that enhanced autopha-
gic activity in the kidney proximal tubules plays a protective
role in several pathologic settings.20–24 More recently, we have
established methods for monitoring autophagic activity in
vivo.25 Given that autophagic activity can be downregulated
by lipid overload, cellular functions may be jeopardized in
pathologic settings in which properly enhanced autophagy
should exert a compensatory or protective role. On the
basis of this background information, we investigated (1)
the pathophysiology of lipotoxicity in the PTCs with a focus
on lysosomes and mitochondria, (2) lipid overload-mediated
alterations in autophagic activity in vivo and in vitro, and (3)
the effects of autophagy deficiency on kidney morphology and
function during lipid overload.
RESULTS
HFD Induces Phospholipid Accumulation in the PTC
Lysosomes
Eight-week-old mice were fed a normal diet (ND) or a HFD for
2 months (hereafter, referred to as nonobese and obese mice,
respectively). Cytosolic vacuolar formation was induced in the
PTCs of the cortex by HFD, but not by ND (Figure 1A).
Twenty-four hours of starvation increased vacuole size in
the PTC of obese mice, but not in nonobese mice. There
were very few Oil Red O-positive vacuoles in the PTCs of
both obese and nonobese mice, whereas 24 hours of starvation
2 Journal of the American Society of Nephrology
led to Oil Red O-positive lipid droplet formation, especially in
nonobese mice (Supplemental Figure 1A). In contrast, almost
all vacuoles in obese mice were positively stained with Nile red,
which detects all neutral lipids, and with toluidine blue stain-
ing, which detects phospholipids (Supplemental Figure 1, B
and C). Electron microscopy revealed that the vacuoles con-
tain multilamellar, onion skin-like structures indicative of
phospholipids in agreement with a previous report (Supple-
mental Figure 1, D–K).26 In addition, the margins of the
vacuoles were immunostained with lysosomal-associated
membrane protein 1 (LAMP1) (Supplemental Figure 1L).
HFD/PA Induces Lysosomal Dysfunction with
Impairment in Acidification
Inspired by the above observations, we postulated that
HFD affects lysosomal machinery and/or autophagic degrada-
tion in the PTCs. Eight-week-old green fluorescent protein-
conjugated microtubule-associated protein 1 light chain 3
(GFP-MAP1LC3) transgenic mice, in which GFP-positive
puncta reflect autophagosomes,27 were fed a ND or a HFD
for 2 months. We found lysosomal vacuoles containing GFP-
positive phospholipid accumulation, which should not be ob-
served in acidic environment (Figure 1B).28 We then treated
cultured PTCs stably expressing GFP-MAP1LC3 with
PA-bound BSA. Colocalization of GFP and LysoTracker
Red was significantly increased in PA-treated PTCs compared
with BSA-treated controls, suggesting impairment in lyso-
somal acidification (Figure 1C). Furthermore, we utilized
the LysoSensor Yellow/Blue reagent, a pH-sensitive probe
that emits predominantly yellow fluorescence in acidic organ-
elles and blue fluorescence in less acidic organelles. Treatment
of PTCs with PA significantly increased the blue/yellow ratio in
the acidic vesicular organelles, reflecting an increase in pH
(Figure 1D). We also found a time-dependent decrease in cel-
lular ATP levels in PTCs treated with PA (Figure 1E). Electron
microscopy analysis revealed a massive vacuolization contain-
ing undigested proteins and organelles, and degenerated mito-
chondria in the PTCs treated with PA (Figure 1, F–M).
PA Treatment Mobilizes Phospholipids from Cellular
Membranes to Lysosomes via Autophagy
To investigate the origin of phospholipids and the role of auto-
phagy in phospholipid accumulation, we performed an FA
pulse-chase assay using phosphatidylcholine fluorescence-
tagged at its FA tail (FL HPC) (Figure 2A). Cultured PTCs
were initially labeled overnight with trace amounts of FL
HPC, which is known to integrate into various cellular mem-
branes, including the endoplasmic reticulum, mitochondria,
and Golgi apparatus. Cultured PTCs chased with BSA treat-
ment preserved the FL HPC signal in cellular membrane
structures (Figure 2B). In contrast, the FL HPC signal on
membranes was decreased in PA-treated PTCs. Colocaliza-
tion of FL HPC and LysoTracker Red was significantly in-
creased and enlarged in PA-treated PTCs compared with
BSA-treated controls, indicative of PA-induced redistribution
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Figure 1. HFD contributes to phospholipid accumulation in the dysfunctional lysosomes of PTCs. (A) PAS-stained kidney cortical regions
of nonobese or obese mice that were either fed or subjected to 24 hours of starvation (n=6–9 in each group). Magnified images are
shown in the insets (original magnification, 31000). (B) Kidney sections of nonobese and obese GFP-MAP1LC3 transgenic mice (n=6–9
in each group) were immunostained for LAMP1, a marker of lysosome, or LRP2/MEGALIN, a marker of proximal tubules (red), and
counterstained with DAPI (blue). (C) PTCs isolated from wild-type mice stably expressing GFP-MAP1LC3 were stained with LysoTracker

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of phospholipids from cellular membranes into lysosomes
(Figure 2B). The active involvement of autophagy in FL
HPC transfer from cellular membranes was shown by immu-
nostaining for MAP1LC3 and LAMP1 in autophagy-deficient
and -competent cultured PTCs (Figure 2, C and D). Under PA
treatment, FL HPC was occasionally found in autophago-
somes in addition to lysosomes (Figure 2C). This FL HPC/
autophagosome/lysosome colocalization was inhibited by
autophagy deficiency (Figure 2D).
HFD-Loaded Kidney Is More Reliant on Autophagy for
the Degradation of Increased Substrates
Next, HFD-mediated changes in demand and activity of basal
autophagy were evaluated in the recently established tamoxifen-
inducible PTC-specific autophagy-deficient mice (Atg5F/F;N-myc
downstream regulated gene 1 [NDRG1]).25,29 Eight-week-old
mice (Atg5F/F;NDRG1 and Atg5F/F control mice) were fed a
ND or HFD for 2 months. Three weeks before euthanasia,
ND-fed nonobese control and HFD-induced obese mice re-
ceived tamoxifen to induce genetic ablation of Atg5. The accu-
mulation of SQSTM1/p62 and ubiquitin represents the
amount of substrate requiring degradation that accumulated
over the 3-week period, because SQSTM1/p62 is the ubiquitin-
and MAP1LC3-binding protein that regulates the formation of
protein aggregates and is removed by autophagy, thus
serving as an index of autophagic degradation.30,31 Auto-
phagy deficiency was confirmed (Supplemental Figure 2).
PTCs of obese Atg5F/F;NDRG1 mice treated with tamoxifen
exhibited mild cytosolic swelling compared with those
of nonobese Atg5F/F;NDRG1 mice (Figure 3A). Moreover,
3-week ablation of autophagy triggered increased accumula-
tion of SQSTM1/p62- and ubiquitin-positive protein aggre-
gates in obese Atg5F/F;NDRG1 mice, indicating that obese
mice utilize autophagy for the degradation of increasing
substrates (Figure 3B).
HFD Alters Autophagic Flux in Response to Starvation
To assess HFD-mediated alterations in autophagic flux, GFP-
MAP1LC3 transgenic mice were administered chloroquine
6 hours before euthanasia. GFP-positive puncta were rarely
observed in the fed, chloroquine-free nonobese mice,
whereas a substantial number of GFP-positive puncta were
observed in the LRP2/MEGALIN-positive PTCs of the fed,
chloroquine-free obese mice (Figure 3, C and D). Chloroquine
administration significantly increased the number of GFP-
positive puncta in obese mice, whereas the number of puncta
remained unchanged in nonobese mice, suggesting high basal
autophagic activity in obese mice and low basal autophagy in
nonobese mice. Next, autophagic flux was assessed in response
to 24 hours of starvation. Chloroquine administration signif-
icantly increased GFP-positive puncta in starved nonobese
mice, whereas the increase was blunted in obese mice (Figure
3, C and D). Western blot analysis (MAP1LC3, SQSTM1/p62,
and ubiquitinated proteins) using whole kidney lysates
roughly confirmed this fluorescence study (Supplemental Fig-
ure 2, B and C). Overall, these results demonstrate that the
PTCs in nonobese mice exhibit low basal autophagic activity
and high starvation-induced autophagic flux, whereas those in
obese mice exhibit high basal autophagic flux and stagnated
autophagic flux by starvation.
PA Treatment Stimulates Autophagic Activity and
Subsequently Induces Downstream Suppression of
Autophagic Flux
We next investigated the effects of PA overload on autophagic
activity in cultured PTCs. BSA treatment promoted the con-
version of MAP1LC3-I to MAP1LC3-II, which was augmented
in the presence of bafilomycin A1 at all indicated times (Sup-
plemental Figure 3A). Consistently, BSA treatment increased
the degradation of SQSTM1/p62 protein in a time-dependent
manner, as shown by the reduction of SQSTM1/p62 turnover
index (defined as the proportion of the levels of SQSTM1/p62
of the indicated sample to the levels of SQSTM1/p62 of the
untreated controls at the baseline), indicating BSA-induced
upregulation of autophagic flux, as decreased SQSTM1/p62
protein levels are associated with autophagy activation (Sup-
plemental Figure 3B). In contrast, PA treatment strongly in-
creased the conversion of MAP1LC3-I to MAP1LC3-II with no
additional increase in MAP1LC3-II observed in the presence
of bafilomycin A1 (Supplemental Figure 3A). Consistently, PA
treatment significantly increased the degradation of SQSTM1/
p62 protein at early stages (3 hours), and thereafter, a significant
rebound at a late stage was observed (12 hours) (Supplemental
Figure 3B). The PA-induced rebound of the SQSTM1/p62 turn-
over index was presumably because of the downstream suppres-
sion of autophagic flux, as SQSTM1/p62 mRNA expression was
unchanged (Supplemental Figure 3C).
Mitophagy Could Protect PTCs from PA-Induced
Mitochondrial Injury
To determine the role of autophagy during lipid overload, we
exposed autophagy-deficient and -competent cells to PA or
BSA. The mitochondrial membrane potential, assessed by
tetramethylrhodamine ethyl ester (TMRE), was significantly

Red, and counterstained with DAPI (blue) after treatment with either 0.25% BSA or 0.25 mM PA for 12 hours (n=5). (D) PTCs were stained with
the pH indicator LysoSensor Yellow/Blue after treatment with either 0.25% BSA or 0.25 mM PA for 12 hours (n=5). The blue/yellow ratio
is presented (right). (E) Cellular ATP levels after treatment with either 0.25% BSA or 0.25 mM PA for 12 hours. (n=5). (F–M) Electron
micrographs of PTCs treated with 0.25% BSA (F–I) or with 0.25 mM PA (J–M) (n=2). (M’ and I’) Outer mitochondrial membranes are traced
(solid lines). Bars: 50 mm (A), 10 mm (B, C, and D), 5 mm (F and J), and 500 nm (G–I and K–M). Data are provided as mean6SEM. Statistically
significant differences (*P,0.05) are indicated. All images are representative of multiple experiments. M, mitochondria.

4 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2016
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Figure 2. PA treatment mobilizes phospholipids from cellular membranes via autophagy in the PTCs. (A) Schematic illustration of
fluorescence FA pulse-chase assay. (A and B) FL-HPC loaded PTCs isolated from wild-type mice were chased after treatment with either
0.25% BSA or 0.25 mM PA for 6 hours (n=5), and the subcellular localization of FL-HPC was determined by staining with LysoTracker
Red. To determine phospholipid accumulations in lysosomes, the number and size of merged dots were measured. (C and D) FL-HPC

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reduced in both cell lines with PA treatment compared with
those with BSA; however, the reduction was more prominent
in autophagy-deficient PTCs (Figure 4A). Furthermore, PA
significantly increased mitochondrial reactive oxygen species
(ROS), assessed by MitoSOX Red staining, in autophagy-
deficient PTCs but not in autophagy-competent PTCs (Figure
4B). PA significantly decreased mitochondrial DNA/nuclear
DNA (mtDNA/nDNA) ratios, which was more pronounced
in autophagy-deficient PTCs than autophagy-competent
PTCs (Figure 4C). Consistent with these observations, PA
decreased cell survival of both PTCs, which was prominent
in autophagy-deficient PTCs (Supplemental Figure 4A). PA
significantly decreased mRNA of mitochondrial oxidative
phosphorylation gene in both autophagy-competent and
-deficient PTCs and increased mRNAs of FA b-oxidation
genes in autophagy-competent PTCs but not in autophagy-
deficient PTCs (Supplemental Figure 4B). These data suggest
that PA places substantial stress on mitochondria, which is
alleviated by autophagy.
We next examined whether PA induces autophagy to selec-
tively eliminate impaired mitochondria (mitophagy). Staining
mitochondria with MitoTracker Red FM in GFP-MAP1LC3–
transfected PTCs demonstrated that more autophagosomes
were colocalized with mitochondria in PTCs treated with PA
than BSA (Figure 4D). Furthermore, to investigate whether
ubiquitinated mitochondria were recognized as a target of
autophagic degradation, we stained GFP-MAP1LC3 and
mStrawberry-ubiquitin double-transfected PTCs with
MitoTracker Deep Red FM. More autophagosomes were
localized with mitochondria and ubiquitin when treated
with PA than with BSA (Figure 4E). The phosphatase and tensin
homolog-induced putative kinase 1 (PINK1) and Parkin RBR E3
ubiquitin protein ligase (PARK2/Parkin) pathway is critical in
controlling mitophagy.32 Western blot analysis revealed that PA
increased the expression levels of PINK1 and PARK2/Parkin
but decreased level of cytochrome c oxidase subunit IV
(COX4), which confirmed PA-mediated mitophagy induction
and mitochondrial depletion (Figure 5M). Together, although
causal relationship between autophagy deficiency and mito-
chondrial dysfunction remains to be examined, mitophagy
could protect proximal tubules from PA-induced mitochon-
drial damage.
Autophagy Deficiency Aggravates HFD-Induced
Mitochondrial Dysfunction, Inflammation, and Fibrosis
We next examined the role of autophagy during lipid overload
using PTC-specific Atg5-deficient mice (Atg5F/F;kidney an-
drogen regulated protein [KAP] mice). Cytosolic vacuolar for-
mation was almost completely resolved in obese Atg5F/F;KAP
loaded autophagy-competent and -deficient PTCs were immunostained for MAP1LC3 (red) and LAMP1 (white), and counterstained with
DAPI (blue) after treatment with either 0.25% BSA or 0.25 mM PA for 6 hours (n=3). Magnified images are shown in the insets (original
magnification, 37200). Bars: 10 mm. Data are provided as mean6SEM. Statistically significant differences (*P,0.05) are indicated. All
images are representative of multiple experiments.
6 Journal of the American Society of Nephrology
mice (Figure 5A). This remarkable finding confirms the in
vitro data that PA-induced autophagy is involved in shuttling
FL HPC from cellular membranes into lysosomes where phos-
pholipids accumulate (Figure 2, A–D). In the kidneys of obese
Atg5F/F control littermates, massive lysosomal accumulations
containing multilamellar, onion skin-like structures indicative
of phospholipids were confirmed (Figure 5, B and C), whereas
mitophagy was occasionally observed (Figure 5D) and there
was no remarkable morphologic change in the mitochondria
(Figure 5E). In contrast, we found massive aggregation of
fragmented and swollen mitochondria with derangement of
the cristae in obese Atg5F/F;KAP mice, although no remark-
able morphologic change in lysosomes was observed (Figure
5, F–I). COX and succinate dehydrogenase (SDH) staining
were performed to evaluate HFD-induced alterations in mito-
chondrial respiration activity. PTCs of nonobese Atg5F/F;KAP
mice showed less intense COX and SDH staining than those of
nonobese controls (Supplemental Figure 5, A and B). Tubular
COX and SDH staining was somewhat decreased in obese Atg5F/F
control littermates compared with nonobese Atg5F/F control
littermates; however, a more prominent decline in COX and
SDH staining was observed in obese Atg5F/F;KAP mice. We
found increased staining for oxidative stress markers, including
4-hydroxy-2-nonenal (HNE), dityrosine, and N-carboxymethyl
lysine (CML), in obese Atg5F/F;KAP kidneys (Supplemental
Figure 5C). Nonobese Atg5F/F;KAP mice exhibited patchy
macrophage infiltration into the outer stripe of the outer
medulla, which was significantly exaggerated by HFD (Fig-
ure 5J). This macrophage infiltration was associated with
change in the levels of mRNA expression of inflammatory
cytokines TNFa, chemokine CC motif ligand 2 (CCL2), and
CCL3, as well as inflammasome-related molecules nod-like
receptor, pyrin containing 3 (NLRP3); apoptosis-associated
speck-like protein containing; and caspase-1 (Figure 5,
K and L). Western blot analysis (NLRP3, caspase-1, IL-1b,
and IL-18) confirmed HFD-induced inflammasome activa-
tion (Figure 5M). Obese Atg5F/F control mice showed in-
creased expression of IL-1b in F4/80-positive macrophages,
which was significantly exaggerated by autophagy deficiency
(Figure 5, M and N). Additionally, to investigate the long-
term consequences of HFD-induced lipotoxicity, we ob-
served Atg5F/F;KAP mice fed a HFD for up to 10 months.
Obese Atg5F/F;KAP mice exhibited severe brush border loss,
tubular dilation, cast formation, enhanced macrophage in-
filtration, and interstitial fibrosis (Supplemental Figure 6,
A–C). Collectively, autophagy protects kidney PTCs from
HFD-induced reduction in mitochondrial function, sup-
presses macrophage infiltration and inflammasome activa-
tion, and slows HFD-induced renal fibrosis.
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Figure 3. HFD induces autophagy dependency and alteration in autophagic flux in the PTCs. (A and B) The HFD-loaded kidney is more reliant on
autophagy for the degradation of increased substrates (n=4–5 in each group). (A) PAS-stained kidney cortical regions of vehicle- or tamoxifen-treated
nonobese and obese Atg5F/F and Atg5F/F;NDRG1 mice. (A, i and ii) Magnified images from tamoxifen-treated nonobese and obese Atg5F/F;
NDRG1 mice are presented (original magnification, 31000). (B) Kidney cortical regions of nonobese and obese Atg5F/F and Atg5F/F;NDRG1 mice
were immunostained for SQSTM1/p62 and ubiquitin after treatment with vehicle or tamoxifen 3 weeks before euthanasia. Magnified images
(original magnification, 31600) from tamoxifen-treated Atg5F/F;NDRG1 mice are presented. The number of SQSTM1/p62- or ubiquitin-positive

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HFD Enhances the Vulnerability toward Ischemia-
Reperfusion Injury in Autophagy-Deficient Mice
We exposed mice to ischemia-reperfusion (I/R) injury to deter-
mine whether HFD-induced chronic lipotoxicity leads to vulner-
ability to ischemic and/or oxidative stresses. More severely injured
tubules with massive tubular sediments and vacuolation
were observed in I/R-injured obese Atg5F/F mice compared with
I/R-injured nonobese Atg5F/F mice (Figure 6A). The number of
apoptotic, terminal deoxynucleotidyl transferase–mediated
digoxigenin-deoxyuridine nick-end labeling (TUNEL)-positive
tubular cells markedly increased in obese Atg5F/F mice after I/R
injury (Figure 6B). These injuries were significantly exacerbated
by autophagy deficiency. Renal function data, as assessed by plasma
urea nitrogen and creatinine, verified the morphologic results,
although not statistically significant presumably because mice
were subjected to unilateral (not bilateral) I/R injury (Figure 6C).
Phospholipid Accumulation in the Enlarged Lysosomes
and Impaired Autophagic Flux in the Kidneys of Obese
Patients
Kidney biopsy samples taken from obese and nonobese pa-
tients with various CKDs (n=5, respectively) were analyzed
(Figure 7, A and B). Clinical characteristics of these patients
are shown in Supplemental Table 1. All patients showed overt
proteinuria. PTCs of all obese patients exhibited cytosolic vac-
uolar formation (Figure 7A). The margins of the vacuoles were
positive for LAMP1. SQSTM1/p62 accumulation was ob-
served in the PTCs from all these obese patients. In contrast,
the LAMP1-positive vacuoles or SQSTM1/p62-positive aggre-
gates were rarely observed in the nonobese patients. The vac-
uoles observed in the PTCs of patient 2 were positively stained
with toluidine blue (Figure 7B). Electron microscopy from
patient 4 (Figure 7, C–M) revealed that the vacuoles contain
multilamellar, onion skin-like structures (Figure 7, F–K). A
massive vacuolization containing undigested proteins and or-
ganelles (Figure 7L), and aberrant and degenerated mitochon-
drial morphology (Figure 7M) were also observed in the PTCs.
These data suggest that phospholipid accumulation in the en-
larged lysosomes and impaired autophagic flux were recapit-
ulated in the kidneys of obese patients.
DISCUSSION
In this study, we found a close association between autophagy
and lipotoxicity in the kidney proximal tubule, as (1) lipid
overload stimulates autophagy; (2) elevated autophagy
plays a crucial role in counteracting lipotoxicity through mi-
tochondrial quality control; (3) long-term lipid overload and
subsequent autophagic activation places a burden on the ly-
sosomal system, resulting in phospholipid accumulation and
inadequate acidification; (4) lysosomal dysfunction, in turn,
stagnates autophagic flux, which can lead to vulnerability to I/R
injury; and (5) some of these findings are recapitulated in pa-
tients with obesity-related nephropathy. We are convinced that
our findings are novel and clinically important to elucidate a
molecular mechanism of lipid overload-mediated kidney injury.
Accumulating evidence has suggested that several saturated
and unsaturated FAs appear to either activate or inactivate
autophagy.15–19 The significant variation in the literature
could be attributed to differences in the cells used in the ex-
periments, observation periods, concentration of FFAs, and
most importantly, the methods used to monitor autophagic
activity. In this study, we explored the effect of lipid overload
on autophagic activity in vitro and in vivo by using methods
developed in our previous report25 and demonstrated com-
plex alterations in autophagic activity, with several important
findings. A recent report has revealed that mTOR, a potent sup-
pressor of autophagy,33 was activated in the proximal tubules of
obese mice.34 It is also widely known that high levels of FFA lead
to constant activation of mTOR signaling, a process related to the
development of diseases such as diabetes and obesity.35 Indeed,
in our study, mTOR complex 1 activation, as determined by
positive staining for phosphorylated ribosomal protein S6
(Ser235/236), was observed in part of the PTCs and distal neph-
ron segments of obese GFP-MAP1LC3 transgenic mice (Sup-
plemental Figure 7). This obesity-mediated mTOR complex
1 activation can certainly suppress canonical (nonselective)
autophagy induction; however, it should be noted that auto-
phagy, especially noncanonical (selective) autophagy, can be in-
duced by a variety of stresses such as ROS, endoplasmic reticulum
stress, and hypoxia independently of mTOR activation.36 In
agreement with this notion, recent reports have demonstrated
that PA induces autophagy independent of mTOR activity.37,38
Then, what triggers lipid overload-induced autophagy in-
dependent of mTOR activation? What is degraded by lipid
overload-induced autophagy? We found that mitochondria
are a putative substrate. Indeed, other groups have previously
reported the close association of mitochondria and lipid excess.
High levels of FFAs evoke mitochondrial DNA (mtDNA) dam-
age and impair mitochondrial dynamics and morphology in
INS1 cells.39,40 FFAs modulate mitochondrial ROS production

dots was counted in at least ten high-power fields (original magnification, 3400). (C and D) GFP-positive puncta formation was assessed in
the proximal tubules of nonobese or obese GFP-MAP1LC3 transgenic mice that were either fed or subjected to 24 hours of starvation, with
or without chloroquine administration 6 hours before euthanasia (n=6–9 in each group). (C) Kidney sections were immunostained for LRP2/
MEGALIN, a marker of proximal tubules (red), and counterstained with DAPI (blue). (D) The number of GFP-positive puncta per proximal
tubule under each condition was counted in at least ten high-power fields (original magnification, 3600) (each high-power field contained
10–15 proximal tubules). Bars: 50 mm (A) and 10 mm (B and C). Data are provided as mean6SEM. Statistically significant differences
(*P,0.05) are indicated. All images are representative of multiple experiments. F/F, Atg5F/F mice; F/F:NDRG1, Atg5F/F;NDRG1 mice.

8 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2016
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Figure 4. PA-induced mitophagy protects PTCs from mitochondrial injury. (A and B) The mitochondrial membrane potential (A) and the
mitochondrial ROS (B) of autophagy-competent and -deficient PTCs treated with either 0.25% BSA or 0.25 mM PA for 12 hours were
assessed by TMRE and MitoSOX Red stainings, respectively (n=4–6). Quantitative data of relative intensity were also provided. (C) The
mtDNA copy number of autophagy-competent and -deficient kidney PTCs was quantified after treatment with either 0.25% BSA or
0.25 mM PA for 12 hours. (A–C) Values are normalized by the signal intensity of BSA-treated autophagy-competent PTCs. (D and E)
PTCs stably expressing GFP-MAP1LC3 (D) or GFP-MAP1LC3 and mStrawberry-ubiquitin (E) were stained with MitoTracker Red FM (D)

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by several mechanisms, such as interaction with components of
the respiratory chain, thereby inhibiting the electron transport.41
Our observation that PA significantly increased mitochon-
drial ROS and decreased mitochondrial membrane potential
in autophagy-deficient PTCs, both of which could trigger
mitophagy,42 suggests that autophagy could be triggered to
target dysfunctional mitochondria during lipid overload. In-
deed, PA-induced mitophagy has been recently reported in
human aortic endothelial cells.43 In keeping with suppressed mi-
tochondrial bioenergetics, we found a time-dependent decrease
in cellular ATP levels in PTCs treated with PA, which may explain
the impairment of lysosomal acidification, as H+ pump activity is
highly sensitive to decreases in ATP concentration.44 Collectively,
our findings demonstrate that, during lipid overload, PINK1-
PARK2/Parkin–mediated mitophagy continues to protect
proximal tubules from increasing mitochondrial stress; however,
persistent mitophagy inevitably places a burden on the lyso-
somal system (as manifested by impaired acidification and phos-
pholipids accumulation), leading to impaired autophagic flux.
It has been previously reported that HFD-mice have prox-
imal tubule injury with enlarged LAMP1-positive lysosomes
containing phospholipids as multilaminar inclusions.26 In
addition, a significant increase in the levels of specific glomer-
ular and tubular lipid species, including phospholipids, has
been recently reported in diabetes.45 Our study confirmed
these findings, not only in HFD-loaded mouse kidney, but
also in the clinical setting (obesity-related nephropathy). Al-
though lipotoxicity in organs that have evolved specialized
lipid handling functions has been generally attributed to FFAs
and/or triglyceride accumulation, in our study, phospholipid
accumulation has emerged as being central to lipotoxicity in
the kidney. The origin of phospholipids appears to be the
mitochondrial membrane. Phospholipids play an important
role in cellular homeostasis by engagement in various cellular
signaling pathways.46 However, the mechanism underlying the
accumulation of undigested phospholipids in the lysosome
during lipid overload and consequent alterations in cellular
function remain to be elucidated.
We have previously demonstrated that autophagy deficiency
exacerbates kidney injury in many mouse models. In this study,
we demonstrated that impairment in autophagic activity is
observable in patients as well as genetically engineered mice.
Although largely overlooked and not fully understood relative
to the process of phagophore formation, the mechanism
through which autophagosomes fuse with lysosomes is a crit-
ical aspect of macroautophagy. Similar to this study, we have
previously observed that, in the aged kidney, basal autophagic
activity is enhanced to cope with increasing “garbage” with age,
but upregulation of autophagic flux in response to additional
stress is blunted partly through lysosomal dysfunction (e.g.,
lipofustin).25 In other words, aging and lipid overload have a
common mechanistic platform, lysosomal dysfunction, and
consequent impaired autophagic flux. The interesting obser-
vation that may strengthen the close mechanistic relationship
between aging and lipid overload are that caloric restriction
potentially promotes longevity in many mammalian mod-
els,47 whereas high-fat and/or -carbohydrate diets accelerates
aging.48 Furthermore, ischemic injury in the elderly and pa-
tients with hyperlipidemia is a major risk factor for ESRD.49,50
In this point, therapeutic effort to restore lysosomal function
and autophagic flux will pave the way to improving the clinical
outcome of kidney injury related to aging or HFD.
The NLRP3 inflammasome senses obesity-associated dan-
ger signals, such as intracellular ceramide-induced caspase-1
cleavage, in macrophages and adipose tissue and contributes to
obesity-induced inflammation and insulin resistance.51 In an-
other report, PA stimulation results in AMP-activated protein
kinase inhibition, leading to defective autophagy in myeloid
cells, which activate inflammasomes.52 In addition, inflamma-
some activation in hematopoietic cells impairs insulin signal-
ing in several target tissues to reduce glucose tolerance and
insulin sensitivity.52 In this study, we extend these previous
observations to the kidney and have demonstrated that HFD-
induced impaired autophagic flux evokes macrophage infiltra-
tion and inflammasome activation, followed by renal fibrosis. It
remains to be elucidated whether lipid overload-induced inflam-
masome activation could lead to reduced glucose tolerance and
insulin sensitivity.
In conclusion, we report that HFD-induced elevation in
autophagy plays a crucial role in counteracting lipotoxicity
in the kidney through mitochondrial quality control.
Furthermore, a reduced capacity for upregulation of autopha-
gic flux is associated with HFD-induced vulnerability to I/R
injury. Strategies aimed at modulating autophagy hold promise
for treating obesity-related kidney disease.
CONCISE METHODS
Mice
GFP-MAP1LC3 transgenic mice and Atg5F/F;NDRG1 and Atg5F/F;KAP
mice on a C57BL/6 background have been described previously.20,25,27

or MitoTracker Deep Red FM (E) after treatment with either 0.25% BSA or 0.25 mM PA for 6 hours (n=5). (D) Yellow dots in merged images
represent colocalization of mitochondria and autophagosomes. Colocalization was assessed by Pearson correlation. (E) White dots in
merged images represent colocalization of ubiquitinated mitochondria and autophagosomes. (F) Western blot analysis (PINK1, PARK2/Parkin,
and COX4) (n=4). Actin, b (ACTB) was used as loading control. The mean value of untreated controls at the baseline was adjusted to “1” as a
reference. Bars: 10 mm. Data are provided as mean6SEM. Statistically significant differences: *P,0.05 versus autophagy-competent cells of
corresponding treatment; #P,0.05 versus BSA-treated control cells (A–C); *P,0.05 (D and E). Atg5(+), autophagy-competent PTC; Atg5(2),
autophagy-deficient PTC. All images are representative of multiple experiments.

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Figure 5. Autophagy deficiency aggravates HFD-induced mitochondrial dysfunction and inflammation, leading to renal fibrosis. (A) PAS-stained
kidney cortexes of nonobese and obese Atg5F/F;KAP mice and Atg5F/F control littermates (n=10–14 in each group). Magnified images are shown
in the insets (original magnification, 31000). (B–I) Electron micrographs of obese Atg5F/F (B–E) and Atg5F/F;KAP mice (F–I) (n=3 in each group).
Arrow indicates the lysosome and asterisk indicates the nucleus. (J–N) Immunostaining for F4/80, a macrophage marker (J), mRNA expression
levels of inflammatory cytokine- (K) and inflammasome- (L) related genes, Western blot analysis of NLRP3, caspase-1, IL-1b, and IL-18 (M), and
immunostaining for F4/80 (green) and IL-1b (red) (N) in the kidney outer medulla of Atg5F/F;KAP and Atg5F/F control mice fed ND or HFD for
2 months. n=7–9 (J) and 6–7 (K–N) in each group. Bars: 50 mm (A and J), 10 mm (B, F and N), 5 mm (F and J), and 500 nm (C–E and G–I). Data are
expressed as the fold change relative to the mean value of nonobese Atg5F/F mice. Values represent the mean6SEM. Statistically significant
differences: *P,0.05 versus treatment-matched Atg5F/F control littermates; #P,0.05 versus nonobese mice. (N) Sections were counterstained
with DAPI (blue). All images are representative of multiple experiments. F/F, Atg5F/F mice; F/F;KAP, Atg5F/F;KAP mice; M, mitochondria.

We used two different mice models, tamoxifen-inducible (Atg5F/F;
NDRG1) and conditional (Atg5F/F;KAP) PTC-specific autophagy-
deficient mice; Atg5F/F;NDRG1 mice were used only for the experi-
ment assessing HFD-mediated changes in demand and activity of
basal autophagy because using corn oil to dissolve tamoxifen could
modify HFD-mediated changes such as metabolic profiles, and Atg5F/F;KAP
mice were used for most of the experiments for evaluating the effects
of autophagy deficiency during lipid overload. Atg5F/F;KAP mice
were generated by crossing KAP-Cre transgenic mice, in which Cre
recombinase is expressed under control of the KAP gene promoter,
and mice bearing an Atg5 flox allele (Atg5F/F mice). KAP-Cre/CAG-
CAT-Z male mice, in which b-galactosidase expression reflects Cre

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Figure 6. HFD enhances the vulnerability against I/R injury to autophagy-deficient mice. (A and B) Representative images of PAS-staining (A)
and TUNEL staining (B) on kidney cortexes of nonobese and obese Atg5F/F;KAP mice and Atg5F/F control littermates 2 days after unilateral
I/R injury or sham operation. (A) The tubular injury score was shown. (B) The number of TUNEL-positive PTCs was calculated in at least ten
high-power fields (original magnification, ☓400). Bars: 500 mm. (C) Plasma urea nitrogen and creatinine were measured (n=6–8 in each
group). Values represent the mean6SEM. Statistically significant differences: *P,0.05 versus treatment-matched Atg5F/F control littermates;
#
P,0.05 versus nonobese mice; ‡P,0.05 versus sham-operated mice. F/F, Atg5F/F mice; F/F;KAP, Atg5F/F;KAP mice.

activity, exhibited LacZ-positive tubules in the cortex and the outer
medulla, whereas LacZ-positive tubules were absent in female
mice.20 Therefore, only male mice were used in this study. Eight-
week-old mice were housed in box cages, maintained on a 12-hour
light/dark cycle, and fed an ND (12.8% of kilocalories from fat: 5%
fat, 23% protein, and 55% carbohydrate) or HFD (62.2% of kilocalories
from fat: 35% fat, 23% protein, and 25% carbohydrate) (Oriental Yeast,
Tokyo, Japan) for 2 or 10 months. For the induction of Atg5 deletion in
Atg5F/F;NDRG mice, we intraperitoneally injected tamoxifen (Sigma-
Aldrich, St. Louis, MO) (1 mg/10 g body wt) dissolved in corn oil
(Sigma-Aldrich) at a concentration of 10 mg/ml three times every other
day. In the experiment assessing the autophagic flux in vivo, chloroquine
(Sigma-Aldrich; 50 mg/g body wt) was injected intraperitoneally and
mice were euthanized 6 hours after the injection.
Antibodies
We used the following antibodies: antibodies for LRP2/MEGALIN
(a gift from T. Michigami, Department of Bone and Mineral Research,
Osaka Medical Center and Research Institute for Maternal and Child
Health, Osaka, Japan), COL1A1 (Abcam, Inc., Cambridge, MA),
ATG5 (MBL, Nagoya, Japan), SQSTM1 (MBL), ubiquitin (Cell Sig-
naling Technology, Danvers, MA), MAP1LC3 (Cell Signaling Tech-
nology), IL-1b (Cell Signaling Technology), IL-18 (Abcam, Inc.),
NLRP3 (Adipogen, Seoul, Korea), caspase-1 (Santa Cruz Biotechnology,

12 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2016
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Figure 7. Phospholipid accumulation and impaired autophagic flux in the kidneys of obese patients. (A) Representative images of kidney
specimens obtained from obese and nonobese patients with overt proteinuria (n=5, respectively). Kidney sections were immunostained for LAMP1
and SQSTM1/p62, and counterstained with hematoxylin. Magnified images are shown in the insets (original magnification, 31000). (B)
Representative images of toluidine blue staining from obese patient 2 (original magnification, 31000). (C–M) Representative images of
electron micrographs from obese patient 4. A lysosome with normal morphology is shown (D and E). Magnified images of lysosomes are
presented (original magnification, 33000). Bars: 50 mm (A and B), 5 mm (C, F, and I), and 500 nm (D, E, G, H, J–M). Arrows indicate the
lysosome and asterisks indicate the nucleus. BM, basement membrane; M, mitochondria; TL, tubular lumen.

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 BASIC RESEARCH www.jasn.org
Santa Cruz, CA), PINK1 (Abcam, Inc.), PARK2/Parkin (Cell Signaling
Technology), COX4 (MBL), phosphorylated ribosomal protein
S6 (Ser235/236; Cell Signaling Technology), ACTB (Sigma-Aldrich),
F4/80 (AbD Serotec, Oxford, UK), LAMP1 (BD Biosciences, San Jose,
CA), 4HNE (Japan Institute for the Control of Aging, Fukuroi, Japan),
dityrosine (Japan Institute for the Control of Aging), CML (Transgenic,
Kobe, Japan), biotinylated secondary antibodies (Vector Laboratories,
Burlingame, CA), horseradish peroxidase-conjugated secondary anti-
bodies (DAKO, Glostrup, Denmark), and Alexa Fluor 555- and Alexa
Fluor 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA).
Histologic Analysis
Histologic analysis was performed as previously described, with mod-
ification.25 To examine the number of GFP-positive dots, postfixed
frozen kidney was sectioned and immunostained for LRP2/MEGALIN
(PTC maker). The number of GFP-MAP1LC3 dots per proximal tubule
were counted in at least ten high-power fields (3600) (each high-power
field contained 10–15 proximal tubules). The fluorescence images
were collected using confocal microscopy (FV1000-D; Olympus, Tokyo,
Japan). Immunohistochemical staining for SQSTM1/p62, ubiquitin,
COL1A1/collagen type I, HNE, dityrosine, CML, and IL-1b was
performed on paraffin-embedded sections after antigen retrieval
via autoclaving in 0.01 mM citrate buffer (pH 6.0) for 10 minutes
at 120°C and blocking with 1.5% BSA (Sigma-Aldrich) in PBS for
60 minutes. Immunohistochemical staining for F4/80 (or double stain-
ing for F4/80 and IL-1b) was performed on paraffin-embedded sections
after enzyme pretreatment using proteinase K solution (20 mg/ml) in TE
buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) for 3 minutes at room
temperature. For double staining for SQSTM1 (or ubiquitin) and LRP2/
MEGALIN, each molecule was first visualized using a horseradish
peroxidase-DAB compound (Nichirei, Chuo-ku, Tokyo, Japan), after
detection of LRP2/MEGALIN using alkaline phosphatase and nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate Stock Solution
(Roche Diagnostics, Basel, Switzerland). The percentage of the
immune-positive area for COL1A1/collagen type I (or F4/80) was
calculated using a digital image-analyzing software, ImageJ (available at
http://rsbweb.nih.gov/ij/index.html; National Institutes of Health).
For each kidney, at least ten high-power fields (3400) were analyzed.
For electron microscopy, kidney specimens were fixed with 2.5%
glutaraldehyde (Wako, Osaka, Japan) and observed using a Hitachi
H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).
Characterization of tubular vacuole accumulation was performed
using the toluidine blue staining on 4% paraformaldehyde (PFA)-fixed
paraffin-embedded sections and using the Nile Red staining (Wako) on
4% PFA-fixed frozen sections. In all quantitative or semiquantitative
analyses of histologic staining, at least ten high-power fields were re-
viewed for each tissue by two nephrologists (T.Y. and A.T.) in a blinded
manner.
Oil Red O Staining
Sections (5 mm thick) of frozen, 4% PFA-embedded tissue were dried
for 1 hour at room temperature and rinsed in PBS, and placed in 60%
isopropylene for 1 minute. They were stained in 60% Oil Red O so-
lution (100% solution: 0.3 g of Oil Red O [Sigma-Aldrich] dissolved
in 100 ml of isopropylene) for 20 minutes at 37°C, placed in 60%
14 Journal of the American Society of Nephrology
isopropylene for 1 minute, and washed with PBS three times. Tissue
sections were counterstained with hematoxylin.
COX/SDH Staining
COX/SDH staining were performed as previously described.23 Briefly,
we used fresh cryosections (5 mm thick) of kidney tissues. For COX
staining, the sections were incubated in the solution composed of
2 mg/ml of catalase (Sigma-Aldrich), 1 mg/ml of cytochrome c
(Sigma-Aldrich), and 0.5 mg/ml of diaminobenzidine (Wako) in
0.1 M of phosphate buffer (pH 7.4) for 1 hour at 37°C. The SDH
staining was performed according to the modified method of Martin
et al.53 In brief, we prepared nitro blue tetrazolium stock composed of
KCN 6.5 mg (Nacalai, Kyoto, Japan), EDTA 185 mg (Sigma-Aldrich),
and nitro blue tetrazolium 100 mg (Nacalai) in 100 ml of 0.1 M
phosphate buffer (pH 7.6) and 500 mM of sodium succinate solution
(Nacalai) diluted in distilled water. Then the sections were incubated
in a solution mixed with 2 ml of nitro blue tetrazolium stock solution,
0.2 ml of succinate solution, and 0.7 mg of phenazine methosulfate
(Nacalai) for 15 minutes at 37°C. The intensity of the positive staining
area was measured using the imaging software, ImageJ. For each
kidney, at least ten high-power fields (3400) were analyzed.
Cell Culture
Autophagy-deficient and autophagy-competent PTC lines were as
previously described.20 In brief, Atg5-deficient kidney PTCs (Atg5-
negative PTCs) were isolated from 3-week-old Atg5F/F;KAP mice
and immortalized by using pEF321-T, an SV-40 large T antigen ex-
pression vector. As a control, we generated Atg5-positive PTCs by
stably transfecting pMRX-IRES-Atg5-bsr (Atg5-expressing retroviral
vector cassette) to Atg5-negative PTCs. We also isolated PTCs from
wild-type mice and immortalized cell lines were established by trans-
formation using pEF321-T, an SV-40 large T antigen expression vector.
We transfected the pEGFP-MAP1LC3 vector (a gift from T. Yoshimori,
Department of Genetics, Osaka University) to the wild-type PTC lines
and then constructed GFP-MAP1LC3 transgenic PTC lines. Cells were
cultured in DMEM (Sigma-Aldrich) containing 5% FBS at 37°C under
a humidified atmosphere of 5% CO2 and 95% air. Lipid-containing
media (PA-BSA) were prepared by conjugation of PA with essentially
FA-free, low endotoxin BSA (Sigma-Aldrich). Sodium palmitate
(Sigma-Aldrich) were dissolved in 50% ethanol and vigorously mixed
with BSA in PBS at a molar ratio of 6.6:1,54 filter-sterilized, and added to
culture media at the final FA (PA) concentration of 0.25 mM. In
this experimental condition, the BSA concentration was 2.5 mg/ml, or
37.88 mM. This concentration of FFA was used because the molar ratio
of FFA to albumin could reach up to 8.59,55 and the albumin concen-
tration in the proximal tubular lumen could be as high as 2.9 mg/ml, or
43 mM.56 Twenty-four hours after the seeding, the cells were treated
with PA-BSA or 2.5 mg/ml BSA for 6–24 hours before harvest or anal-
ysis. According to previous studies, we used 2.5 mg/ml BSA treatment
as a control for 0.25 mM PA treatment because it has been reported
that, at a higher concentration, FA-free BSA has several cytotoxic effects,
including ROS production, mitochondrial damage, and endoplasmic
reticulum stress,57,58 all of which could induce autophagy and apoptosis
in the PTCs.59 To assess autophagic activity, PTCs were treated with
200 nM of bafilomycin A1 (Wako) for 1 hour at 37°C before harvest.
J Am Soc Nephrol 28: ccc–ccc, 2016

Assessment of Lysosomal Acidification
To assess autolysosome/lysosomal acidification, PTCs stably expressing
GFP-MAP1LC3 were stained with 50 nM LysoTracker Red DND-99
(Invitrogen) for 30 minutes at 37°C. Colocalization of autophago-
somes and lysosome was calculated using ImageJ. The lysosomal
acidification was determined by staining with 1 mM of LysoSensor
Yellow/Blue DND-160 (PDMPO) (Invitrogen) for 1 minute at 37°C.
The fluorescence images were collected at the wavelength range from
510 to 641 nm (yellow) and at the wavelength range from 404 to
456 nm (blue) with the Olympus FV1000-D (Olympus). The mean
fluorescence intensity in the fluorescence-positive area was mea-
sured using ImageJ, for yellow and blue fluorescence, respectively.
The blue/yellow ratio was calculated by division process in each
section. The average blue/yellow ratio of a given sample was calcu-
lated from five sections. Intensity was measured using at least three
independent experiments (.150 cells per experiment).
Fluorescence FA Pulse-Chase Assay
Fluorescence FA pulse-chase assay was performed as previously de-
scribed, with slight modification. 60 PTCs were incubated with
DMEM with 5% FBS containing 2 mM b-BODIPY FL C12-HPC
(FL HPC) (Invitrogen) for 16 hours. Cells were then washed three
times with DMEM with 5% FBS, incubated for 1 hour to allow the
fluorescent lipids to incorporate into cellular membranes, and then
chased for the indicated times under PA-BSA treatment or 0.25%
BSA treatment. FL-HPC–loaded and chased PTCs were stained
with 50 nM LysoTracker Red DND-99 for 30 minutes at 37°C
or stained with 25 nM of MitoTracker Red FM (Invitrogen) for
15 minutes at 37°C immediately before imaging, or fixed with 4%
PFA for 15 minutes at room temperature for immunocytochemistry.
The number and size of phospholipid/lysosome-merged dots were
measured using ImageJ “analyze particles” function in threshold
images.
Measurement of Mitochondrial Membrane Potential,
ROS Production, and ATP
Cells cultured on 35 mm glass-bottomed dishes were subjected to the
confocal analysis. The mitochondrial membrane potential was deter-
mined by staining with 50 nM of TMRE (Invitrogen) for 30 minutes at
37°C. To determine mitochondrial ROS production, cells were incu-
bated with 0.3 mM MitoSOX Red (Invitrogen) for 10 minutes. The
fluorescence images for TMRE or MitoSOX Red were collected using
the Olympus FV1000-D (Olympus), and the mean fluorescence in-
tensity in the fluorescence-positive area was measured using ImageJ.
The average mean fluorescence intensity of a given sample was
calculated from five sections. Intensity was measured using at
least three independent experiments (.150 cells per experiment).
ATP was measured using ATP Colorimetric/Fluorometric Assay
Kit (BioVision, Inc., Milpitas, CA) according to manufacturer’s
instruction.
Assessment of Mitophagy
Assessment of mitophagy were performed as previously described.23
To prove mitophagy, PTCs stably expressing GFP-MAP1LC3 were
stained with 25 nM of MitoTracker Red FM for 15 minutes at 37°C.
J Am Soc Nephrol 28: ccc–ccc, 2016
www.jasn.org BASIC RESEARCH
Colocalization of autophagosomes and mitochondria was calculated
using ImageJ and represented as Pearson correlation between fluo-
rescence of GFP and MitoTracker Red FM. We also constructed GFP-
MAP1LC3 and mStrawberry-ubiquitin double-transfected PTCs to
assess the association between ubiquitin and mitophagy. GFP-
MAP1LC3 and mStrawberry-ubiquitin double-transfected PTCs
were stained with 25 nM of MitoTracker Deep Red FM (Invitrogen)
for 15 minutes at 37°C.
Relative Quantification of mtDNA Copy Numbers
Relative quantification of mtDNA copy numbers were performed as
previously described.25 In brief, total DNA (mtDNA and nuclear
DNA) was extracted from isolated proximal tubules with Wizard
Genomic DNA Purification Kit according to the manufacturer’s in-
struction (Promega, Madison, WI). Real-time quantitative PCR of
the mtDNA-encoded gene mt-Co1 (GenBank accession no.
NC_001807) and the single-copy nuclear gene Ndufv1 (NADH de-
hydrogenase [ubiquinone] flavoprotein 1) (GenBank accession no.
NM_133666) was carried out with an ABI PRISM 7900HT sequence
detection system (Applied Biosystems, Foster City, CA). Relative
quantification of mtDNA copy numbers were depicted as the ratio
of the relative concentrations of mtDNA/nDNA. The sequences of
primers for the MT-CO1 and Ndufv1 gene are as follows: mt-Co1-F,
59-tgctagccgcaggcattac-39; mt-Co1-R, 59-gggtgcccaaagaatcagaac-39;
Ndufv1-F, 59-cttccccactggcctcaag-39; Ndufv1-R, 59-ccaaaacccagtgatccagc-39).
Cell Proliferation Assay
MTS assays were performed using CellTiter 96 AQueous One Solution
Cell Proliferation Assay (Promega), according to the manufacturer’s
instructions, in a 96-well plate. Each experiment was performed in
triplicate and repeated at least three times. Cell viability in percentage
refers to the MTS value of the induced cells compared with the
value of the untreated control cells (cultured in DMEM containing
5% FBS).
Quantitative RT-PCR and Western Blot Analysis
Quantitative RT-PCR and Western blot analyses were performed as
previously described.61 The sequences of the primers used were as
follows: Cpt1-F, 59-accactggccgaatgtcaag-39; Cpt1-R, 59-agcgagtagcg-
catggtcat-39; Aox-F, 59-tcaacagcccaactgtgacttccatta-39; Aox-R, 59-
tcaggtagccattatccatctcttca-39; Scad-F, 59-ttacctggcctactccatcg-39;
Scad-R, 59-tgatccactgttgcttctgc-39; Mcad-F, 59-taatcggtgaaggag-
caggttt-39; Mcad-R, 59-ggcatacttcgtggcttcgt-39; Lcad-F, 59-gcatcaa-
catcgcagagaaa-39; Lcad-R, 59-acgcttgctcttcccaagta-39; Cox5b-F,
59-aggcagcttcaggcaccaag-39; Cox5b-R, 59-ggtggggcaccagcttgtaa-39;
Tnfa-F, 59-atggcctccctctcatcagt-39; Tnfa-F -R, 59-cttggtggtttgctac-
gacg-39; Ccl2-F, 59-cccaatgagtaggctggaga-39; Ccl2-R, 59-tctggacc-
cattccttcttg-39; Ccl3-F, 59-tgcccttgctgttcttctct-39; Ccl3-R,
59-gatgaattggcgtggaatct-39; Nlrp3-F, 59-atgctgcttcgacatctcct-
39; Nlrp3-R, 59-aaccaatgcgagatcctgac-39; Asc-F, 59-acagaagtg-
gacggagtgct-39; Asc-R, 59-ctccaggtccatcaccaagt-39; caspase-1-F,
59-cacagctctggagatggtga-39; caspase-1-R, 59-tctttcaagcttgggcactt-39;
Sqstm1-F, 59-ccccagagtcgaagtagctg-39; Sqstm1-R, 59-agtgagaa-
gaggctggtgga-39; Gapdh-F, 59-aactttggcattgtggaagg-39; Gapdh-R, 59-
acacattgggggtaggaaca-39.
Autophagy and Lipotoxicity 15
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Induction of Kidney I/R Injury
Kidney ischemia was induced as previously described.20 In brief, the
animals were anesthetized and were kept on a homeothermic table.
For unilateral clamping, back incisions were made to expose the kid-
ney pedicles to induce 35 minutes of kidney ischemia. The clamps
were released for reperfusion. Control animals were subjected to
sham operation without renal pedicle clamping. Kidney tubular
cells of the cortex were examined by two nephrologists (T.Y. and
A.T.) in a blind manner, and were scored from 0 to 10 according
to the percentage of damaged tubules. Tubular damage was defined
as cell necrosis, which was evaluated in this study by loss of brush
border, tubular dilation, and cast formation. At least ten high-power
fields (3400) were reviewed for each periodic acid–Schiff-stained
slide. Plasma urea nitrogen and creatinine were measured using
the BUN-Test-Wako (Wako), the CRE-EN Kainos (Kainos, Tokyo,
Japan), respectively.
TUNEL Staining
Apoptotic cells were detected using the TUNEL assay with an in situ
apoptosis detection kit (Takara, Kyoto, Japan). Tissue sections were
counterstained with Methyl Green Solution (Wako) and examined
under a microscope.
Kidney Biopsy Specimens
Human kidney biopsy specimens were obtained from patients with
overt proteinuria. All patients provided written informed consent.
Statistical Analyses
All results are presented as means6SEM. Statistical analyses were
conducted using JMP software (SAS Institute Inc., Cary, NC). Multiple-
group comparisons were performed using analysis of variance with
post-testing using the Tukey–Kramer test. The difference between
two experimental values was assessed by paired t test when appro-
priate. Statistical significance was defined as P,0.05.
Study Approval
All animal experiments were approved by the institutional committee
of the Animal Research Committee of Osaka University and the
Japanese Animal Protection and Management Law (No.25). In the
case of human studies, written informed consent was received from
participants before inclusion in the study.
ACKNOWLEDGMENTS
T.Y., A.T., Y.T., and Y.I. were involved in study design and writing
manuscript. The other authors and T.Y. performed experiments and
analyzed data. All authors contributed to the discussions. We thank
N. Mizushima, University of Tokyo, for Atg5F/F and GFP-MAP1LC3
mice; T. Michigami, Osaka Medical Center and Research Institute,
for LRP2/MEGALIN antibody; and N. Horimoto and for technical
assistance.
This work was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science and
Technology in Japan (15H06371 [to T.Y.], 24591196 and 15K09260
16 Journal of the American Society of Nephrology
[to Y.T.], and 24659416 [to Y.I.]), Takeda Medical Research Foun-
dation (to Y.T.), and Merck Sharp & Dohme (to Y.I.).
DISCLOSURES
None.
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This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2016070731/-/DCSupplemental.
J Am Soc Nephrol 28: ccc–ccc, 2016