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org

Kidney Proximal Tubule Lipoapoptosis Is Regulated by

Fatty Acid Transporter-2 (FATP2)
Shenaz Khan,* Pablo D. Cabral,† William P. Schilling,*† Zachary W. Schmidt,*
Asif N. Uddin,* Amelia Gingras,* Sethu M. Madhavan,* Jeffrey L. Garvin,† and
Jeffrey R. Schelling*
*Department of Medicine, The MetroHealth System and †Department of Physiology and Biophysics, Case Western
Reserve University, Cleveland, Ohio

ABSTRACT
Albuminuria and tubular atrophy are among the highest risks for CKD progression to ESRD. A parsimo-
nious mechanism involves leakage of albumin-bound nonesterified fatty acids (NEFAs) across the dam-
aged glomerular filtration barrier and subsequent reabsorption by the downstream proximal tubule,
causing lipoapoptosis. We sought to identify the apical proximal tubule transporter that mediates NEFA
uptake and cytotoxicity. We observed transporter-mediated uptake of fluorescently labeled NEFA in
cultured proximal tubule cells and microperfused rat proximal tubules, with greater uptake from the apical
surface than from the basolateral surface. Protein and mRNA expression analyses revealed that kidney
proximal tubules express transmembrane fatty acid transporter-2 (FATP2), encoded by Slc27a2, but not
the other candidate transporters CD36 and free fatty acid receptor 1. Kidney FATP2 localized exclusively
to proximal tubule epithelial cells along the apical but not the basolateral membrane. Treatment of mice
with lipidated albumin to induce proteinuria caused a decrease in the proportion of tubular epithelial cells
and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a22/2mice. Ex
vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic
apical proximal tubule exposure during glomerular injury revealed significantly reduced NEFA uptake
and palmitate-induced apoptosis in microperfused Slc27a22/2 proximal tubules and Slc27a22/2 or FATP2
shRNA-treated proximal tubule cell lines compared with wild-type or scrambled oligonucleotide–treated
cells, respectively. We conclude that FATP2 is a major apical proximal tubule NEFA transporter that
regulates lipoapoptosis and may be an amenable target for the prevention of CKD progression.

J Am Soc Nephrol 29: ccc–ccc, 2018. doi: https://doi.org/10.1681/ASN.2017030314

Over 26 million people in the United States are af-

flicted with CKD,1 and risks for CKD progression
and mortality are albuminuria and decreased GFR.2
Although albuminuria reflects glomerular filtration
barrier dysfunction, downstream tubular atrophy
Received March 21, 2017. Accepted August 8, 2017.
Published online ahead of print. Publication date available at
www.jasn.org.
Correspondence: Dr. Jeffrey R. Schelling, Division of Nephrology,
MetroHealth Medical Center, 2500 MetroHealth Drive, Rammelkamp
Center for Education and Research, R425, Cleveland, OH 44109-
1998. Email: jeffrey.schelling@case.edu
Copyright © 2018 by the American Society of Nephrology
Significance Statement
Albuminuria and tubular atrophy are significant risks
for CKD progression to ESRD. We have proposed that,
in progressive, proteinuric renal diseases, filtration of
albumin-bound nonesterified fatty acids (NEFAs) across
damaged glomeruli leads to proximal tubule NEFA
reabsorption, causing tubular epithelial cell death and
tubular atrophy. Using a variety of approaches, in-
cluding microperfused tubules, cell culture, and ge-
netically manipulated mouse models, we localized fatty
acid transporter-2 (FATP2) to the proximal tubule lu-
minal membrane and showed that FATP2 mediates
proximal tubule NEFA uptake and cytotoxicity. We
conclude that FATP2 may, therefore, represent a po-
tential therapeutic target for the prevention of CKD
progression.

J Am Soc Nephrol 29: ccc–ccc, 2018 ISSN : 1046-6673/2901-ccc 1

 BASIC RESEARCH www.jasn.org

intracellular NEFA accumulation and tubular

atrophy through a lipotoxicity mecha-
nism.12,14
Our goal was to identify the apical mem-
brane proximal tubule NEFA transporter,
so that it could ultimately be exploited as a
therapeutic target to prevent CKD progres-
sion. There are several candidate transporter
classes, most notably the FATP family. FATP2
is exclusively expressed in kidney and liver,
and it is the most abundant isoform in kid-
ney.25 Using in vitro, ex vivo, and in vivo ap-
proaches, we show that FATP2 regulates
proximal tubule apical NEFA transport and
Figure 1. NEFAs are absorbed by the proximal tubule in vitro. (A) LLC-PK1 cells were lipoapoptosis.
grown to confluence on permeable supports and then incubated with apical (AP) or
basolateral (BL) BODIPY-labeled C16-NEFA (2.5 mM) complexed with albumin (0.2%) in
buffer prewarmed to 37°C. BODIPY uptake was measured by fluorescence microscopy
RESULTS
using a QBT kit (Molecular Devices; excitation/emission l=488/510 nm; mean6SEM
from 30 cells; n=3). (B) Concentration-dependent apical uptake of BODIPY-labeled
NEFAs in LLC-PK1 maintained on permeable supports (mean6SEM from n=3).
NEFAs Are Absorbed by the Proximal
Tubule
To screen for proximal tubule apical NEFA
due to tubular epithelial cell apoptosis is superior to glomerular uptake, experiments were initially conducted in proximal
pathology as a predictor of CKD progression.3–6 Infusion of tubule cell lines. Figure 1A shows rapid basolateral NEFA
albumin in animal models7–9 or exposure of proximal tubule absorption in polarized LLC-PK1 cells, with time-dependent
cells to albumin in vitro10–12 results in apoptosis. However, uptake. The rate and magnitude of NEFA uptake were con-
cytotoxicity is not observed with delipidated albumin admin- siderably greater from the apical surface. Figure 1B shows
istration,8,10,12,13 implying that albumin-bound fatty acids concentration-dependent apical NEFA uptake.
cause apoptosis. Mouse models of CKD show increased con- To test NEFA uptake under native conditions, microdissec-
centrations of NEFAs and NEFA metabolites in the kidney, ted rat proximal tubules were perfused with fluorescently la-
which are hy pothesized to cause renal function beled NEFA using established methods.26 Figure 2, A and B
deterioration. 12,14 depicts a proximal tubule immediately and then 10 minutes
Proposed mechanisms leading to proximal tubule lipid ac- after luminal microperfusion with boron-dipyrromethene
cumulation include increased uptake, increased synthesis, and (BODIPY)-tagged NEFA, respectively. Figure 2C shows rapid
diminished b-oxidation of NEFA,15–17 but the relative impor- time-dependent uptake and approximately fivefold greater
tance of each mechanism has not been determined. Increased NEFA internalization from the apical compared with the
NEFA uptake is plausible, because hyperlipidemia commonly basolateral surface, consistent with Figure 1A in vitro data.
coexists with CKD, resulting in enhanced extracellular NEFA Figures 1 and 2 collectively show that proximal tubule epithe-
supply for transport and intracellular storage by nonadipose lial cells reabsorb NEFA from the apical surface and by a mag-
tissues, such as in liver, skeletal muscle, and kidney.18 More nitude that exceeds basolateral uptake.
importantly, in proteinuric renal diseases, such as diabetic
nephropathy, the damaged glomerulus permits albumin- Proximal Tubule FATP2 Expression
bound NEFA to be filtered and gain access to the previously FATPs (gene name Slc27) are a family of transmembrane span-
unexposed proximal tubule luminal surface, where aberrant ning proteins, which contain six members with distinct tissue
NEFA reabsorption could then occur. expression patterns. Of the FATP isoforms expressed in kid-
Under normal circumstances, fatty acids are the preferred ney, FATP1 and FATP4 are present in very small amounts25
substrate for proximal tubule ATP generation.19,20 In plasma, (S. Khan et al., unpublished observations). FATP2 is expressed
esterified fatty acids circulate as triglycerides, whereas water- exclusively in kidney and liver and has previously been de-
insoluble NEFAs are solubilized by complexing with albumin. tected in total kidney and proximal tubule.25,27–32 Figure 3A
NEFAs dissociate from albumin at the plasma membrane and shows FATP2 mRNA expression in wild-type mouse kidney,
are taken up by saturable, basolateral membrane transporters which is diminished in Slc27a2+/2 and absent in Slc27a22/2
in proximal tubules.21–24 Under pathologic circumstances, the control kidneys. Figure 3, B–D reveals that FATP2 protein
possibility of simultaneous apical and basolateral proxi- expression in kidney is restricted to proximal tubule. No
mal tubule NEFA uptake combined with increased NEFA FATP2 staining was detected in Slc27a2 2/2 kidneys (not
synthesis15,16 and diminished b-oxidation17 could lead to shown). Figure 3E confirms FATP2 mRNA expression in

2 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc, 2018
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membrane domains. 38,39 To screen for

FATP2 membrane expression, crude mem-
brane fractions from cultured proximal tu-
bules were probed by immunoblotting.
Figure 4A reveals FATP2 expression in
LLC-PK1 and HK-2 proximal tubule cell
membranes. Immunocytochemistry ex-
periments in postfixed HK-2 cells revealed
FATP2 expression in a plasma membrane
distribution (Figure 4B). Immunoprecipi-
Figure 2. NEFA are absorbed from the proximal tubule apical surface. Fluorescence tation of biotin surface–labeled FATP2 also
image of a rat proximal tubule (A) immediately and (B) 10 minutes after lumen perfusion showed abundant expression in LLC-PK1,
with BODIPY-labeled NEFAs. (C) BODIPY-NEFA (2.5 mM; complexed with 0.2% al- HK-2, and HRPT proximal tubule cell lines
bumin) uptake in a microdissected rat proximal tubule after luminal perfusion (left (Figure 4C), indicating that FATP2 is ex-
panel), addition of BODIPY-NEFA to the basolateral bath (center panel), and luminal pressed on the plasma membrane.
perfusion of BODIPY-NEFA (500 nM) in Hepes (10-mM)–buffered saline (right panel). To establish FATP2 localization, human
Data are representative of experiments from 12 tubules. kidney sections were probed with FATP2
and g-glutamyl transferase-1 (a brush bor-
Human Renal Proximal Tubule (HRPT) and HK-2 human der enzyme) antibodies. Figure 5 reveals FATP2 and g-glutamyl
proximal tubule cell lines. FATP2 transcripts were weakly de- transferase-1 colocalization in an apical membrane pattern. To
tectable by RT-PCR in LLC-PK1 cells (Figure 3E), perhaps confirm FATP2 membrane domain localization, mouse kidney
reflecting nucleotide sequence differences between human sections were colabeled with Glut5 (a luminal brush border
and porcine mRNA (porcine cDNA sequence is unknown). protein) and Na+/K+-ATPase (to demarcate the basolateral
Because FATP2 may not be the sole FATP in the proximal membrane) antibodies. Supplemental Figure 3, A–D shows the
tubule, screens for other plausible transporters were under- diffuse FATP2 cytosolic pattern, although colocalization was also
taken. CD36 has been reliably isolated in mouse kidney,33 but observed with Glut5, indicating that FATP2 is expressed on the
it was not detectable by immunohistochemistry in proximal proximal tubule apical membrane. In contrast, no FATP2 coloc-
tubules (Supplemental Figure 1)12,34 or by RT-PCR in HK-2 alization with Na+/K+-ATPase was noted (Supplemental Figure 3,
cells (not shown). The G protein–coupled GP40 and GP120 E–G), indicating that FATP2 is not expressed in basolateral mem-
long-chain NEFA transporters have recently been deorphan- branes and would, therefore, not mediate basolateral NEFA uptake
ized and termed FFA1 and FFA4, respectively; only FFA1 is (Figure 6C). Taken together, Figures 4 and 5 and Supplemental
expressed in kidney.35,36 Supplemental Figure 2 shows FFA1 Figure 3 show that proximal tubule FATP2 is expressed most
expression in a glomerular epithelial cell (podocyte) pattern prominently within the apical plasma membrane.
but not within tubules, consistent with the previously de-
scribed role of podocyte FFA1-mediated NEFA uptake in the Proximal Tubule FATP2-Regulated NEFA Uptake
pathogenesis of the nephrotic syndrome.37 Unlike FATP2, FATP2-dependent NEFA uptake was initially evaluated in
CD36 and FFA1 are, therefore, not candidate transporters experiments with microdissected proximal tubules from
for regulation of proximal tubule NEFA reabsorption. wild-type and Slc27a22/2 mice, which were perfused with
BODIPY-labeled NEFA. No difference was observed between
FATP2 Is Expressed in Proximal Tubule Apical male and female tubules, and therefore, results from both
Membranes sexes are combined. Figure 6, A and B shows enhanced
Like other members of the FATP family, FATP2 is predicted to time- and concentration-dependent NEFA uptake from lumi-
be a membrane-associated transporter containing two trans- nal perfusion of wild-type compared with Slc27a22/2 tubules.
In contrast, basolateral NEFA was dimin-
ished compared with apical uptake, and the
magnitude and kinetics of basolateral
NEFA transport were identical in micro-
perfused wild-type and Slc27a22/2 proxi-
mal tubules (Figure 6C), indicating that
basolateral NEFA uptake does not require
FATP2. NEFA uptake was also greater in
Figure 3. FATP2 is expressed in proximal tubules. (A) Kidney FATP2 and GAPDH mRNA proximal tubule cell lines derived from
2/2
expression by RT-PCR. Immunohistochemical localization of (B) FATP2, (C) T. purpureus wild-type versus Slc27a2 mice (Figure
lectin staining, and (D) merged image in mouse kidney cortex. Gl, glomerulus. *Distal tu- 6, D and E), similar to results from tubules
bule. (E) FATP2 and GAPDH mRNA expression by RT-PCR in proximal tubule cell lines. perfused ex vivo (Figure 6, A and B). These

J Am Soc Nephrol 29: ccc–ccc, 2018 Proximal Tubule FATP2 3

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Proximal Tubule Luminal FATP2-

Mediated NEFA Uptake Is Cytotoxic
Accumulation of NEFAs and NEFA metab-
olites has been shown to be cytotoxic to
proximal tubule epithelial cells (Supple-
mental Figure 5)8,10,12 and may contribute
to tubular atrophy and CKD progres-
Figure 4. FATP2 is expressed in proximal tubule membranes. (A) Lysates from crude
membrane preparations were immunoblotted with anti-FATP2 antibodies (upper
sion.12,14 To directly test whether FATP2
panel). Membranes were stripped and reprobed with anti–Na+/K+-ATPase antibodies regulates lipotoxicity, BODIPY-tagged
as a loading control (lower panel). (B) HK-2 cells on coverslips were probed with anti- NEFA uptake was measured in FATP2
FATP2 antibodies and postfixed in paraformaldehyde (4%, 10 minutes at room shRNA-silenced versus shRNA-scrambled
temperature), and primary antibody detection was amplified with Alexa Fluor 568– sequence-treated HRPT human proximal
conjugated goat anti-rabbit IgG. (C) Proximal tubule cell lines were surface labeled tubule cell lines (Figure 8, A and B). Figure
with biotin, and lysates were precipitated with streptavidin-coated beads, eluted, 8C depicts cells with oil red O–stained lipid
resolved by SDS-PAGE, and immunoblotted with anti-FATP2 antibodies (upper droplets as an indirect measure of NEFA
panel). Membranes were stripped and reprobed with anti-Glut5 antibodies as a loading uptake and qualitatively confirms the effi-
control (lower panel).
cacy of functional FATP2 knockdown with
two of the three shRNA constructs. Oil red
results show that a large proportion of apical (but not basolat- O quantification revealed reduced staining in FATP2 shRNA
eral) proximal tubule NEFA uptake is mediated by FATP2. compared with scrambled nucleotide sequence control cells
(Figure 8D), once again indicating that proximal tubule
FATP2 Mediates Tubulointerstitial Disease FATP2 mediates NEFA uptake. Figure 8, E and F shows that
To determine the pathophysiologic significance of proximal sustained NEFA exposure caused apoptosis, as shown by
tubule FATP2, wild-type and Slc27a22/2 mice were treated terminal deoxynucleotidyl transferase–mediated digoxigenin-
with daily intraperitoneal injections of lipidated albumin to deoxyuridine nick-end labeling (TUNEL) and caspase-2
induce albuminuria (Figure 7A) and tubulointerstitial in- activation, which was significantly reduced in FATP2 shRNA-
jury.8,40 Quantitative histomorphometric analysis revealed expressing cells compared with scrambled nucleotide–treated
modest tubular atrophy and interstitial fibrosis, although tu- cells. NEFA-induced apoptosis was also observed in proximal
bular epithelial cell number and interstitial and tubular lumen tubule cells lines derived from wild-type mice and significantly
areas were significantly different between wild-type and blunted in Slc27a22/2 mouse proximal tubule cells (Figure
Slc27a22/2 mouse kidneys (Figure 7, B–D). Similar differ- 8G). Taken together, these loss of function approaches show
ences in tubular epithelial cell density were also observed in that FATP2 regulates lipoapoptosis.
wild-type versus Slc27a22/2 mice after induction of albumin-
uria with daily LPS injections (Supplemental Figure 4).41,42
The pathophysiology of tubular atrophy is complex, but one DISCUSSION
proposed pathway is apoptosis, in the absence of compensa-
tory hyperplasia.43 Figure 7E shows increased tubular epithe- CKD associated with heavy albuminuria accounts for the vast
lial cell apoptosis in lipidated albumin–injected mice, which majority of causes of ESRD. There has been significant progress
was significantly attenuated in Slc27a22/2 compared with in understanding the role of glomerular cells, particularly the
wild-type mice. Taken together, the in vivo data suggest that glomerular epithelial cell (podocyte), in the pathophysiology of
apical proximal tubule FATP2-mediated NEFA uptake con- albuminuria, but it is often overlooked that tubular atrophy
tributes to tubular atrophy and interstitial fibrosis. more accurately predicts CKD progression compared with
glomerular pathology.3–6 Because each al-
bumin molecule that leaks across the dam-
aged glomerular filtration barrier has the
capacity to bind up to seven NEFA mole-
cules, it has been suggested that proxi-
mal tubule reabsorption and intracellular
accumulation of NEFAs are cytotoxic and
contribute to tubular atrophy pathogen-
12,14 Using combined in vitro and ex
Figure 5. FATP2 is expressed in proximal tubule apical membranes. Formalin-fixed esis.
paraffin sections from human kidneys were probed with (A) anti-FATP2 and Alexa Fluor vivo approaches, we show that proximal
488 secondary antibodies or (B) anti–g-glutamyl transferase-1 (GGT1) and Alexa Fluor tubules rapidly reabsorb luminal NEFA
568–labeled secondary antibodies. (C) Nuclei were labeled with DAPI in the mounting from the apical surface and by a magnitude
medium. (D) Colocalization from merged images is depicted in yellow. that is several fold greater than from the

4 Journal of the American Society of Nephrology J Am Soc Nephrol 29: ccc–ccc, 2018
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that both FATP2 functions may be operant

in the proximal tubule. Using in vitro and
ex vivo approaches, we also conclude that
apical uptake of NEFA was largely mediated
by membrane-localized FATP2.
Apical NEFA uptake capacity at equilib-
riumwas significantly greater than basolateral
uptake, suggesting that the apical-directed
NEFA compartment size is different and
larger than for basolateral uptake. Because
fatty acids are a major energy source for the
proximal tubule, we propose that, under nor-
mal circumstances, NEFA uptake across the
basolateral membrane might be targeted to a
subcellular compartment associated with me-
tabolism, whereas damaged glomeruli filter
albumin-bound NEFAs, which are transpor-
ted by apical FATP2, and then may traffic to a
compartment that ultimately initiates apo-
Figure 6. Proximal tubule FATP2-regulated NEFA uptake is time- and concentration- ptosis. Thus, disparate cytotoxicityafter apical
dependent. (A) BODIPY-NEFA (500 nM NEFA complexed with 0.2% albumin in Hepes versus basolateral NEFA uptake could be ex-
[10-mM]–buffered saline) uptake after luminal perfusion in wild-type versus Slc27a22/2 plained by differential NEFA partitioning. It
tubules microdissected from 12-week-old mice. A representative tracing (from n=5) is is also noteworthy that apical NEFA uptake
shown. (B) BODIPY-NEFA (20–2500 nM NEFA in fivefold dilutions complexed with 0.2% at equilibrium was significantly lower in
albumin in Hepes [10-mM]–buffered saline) uptake after luminal perfusion in wild-type Slc27a22/2 compared with wild-type proxi-
versus Slc27a22/2 tubules microdissected from 12-week-old mice (mean6SEM from mal tubules, suggesting that metabolism and
n=8). (C) BODIPY-NEFA uptake from the basolateral bath. Representative tracings (from distribution to intracellular pools may differ
n=3) are shown for clarity due to overlapping error bars. (D) BODIPY-NEFA uptake in after FATP2- and non–FATP2-mediated up-
temperature-sensitive SV40 immortalized proximal tubule cell lines derived from wild-
take. Plasma membrane FATPs are coupled
type and Slc27a22/2 mice (mean6SEM; n=4). (E) Concentration-dependent uptake of
2/2 to specific long-chain acyl CoA synthetases,
BODIPY-labeled NEFA in wild-type versus Slc27a2 mouse proximal tubule cell lines
(mean from n=4). which accelerate NEFA flux and shuttle fatty
acids to binding proteins (FABPs) that then
channel NEFAs to specific intracellular sites.
basolateral surface. Uptake was achieved by complexing NEFA We speculate that FATP/acyl CoA synthetase/FABP isoform
with albumin at a concentration of 2 g/L, which corresponds to combinations could also segregate NEFA to different compart-
apical proximal tubule exposure of albumin under conditions ments after transport across plasma membrane domains.44,45
that mimic glomerular filtration barrier dysfunction. Our data We have previously shown that proximal tubule cells un-
support the notion that aberrantly filtered NEFAs are effi- dergo apoptosis after exposure to NEFA (palmitate) under
ciently reabsorbed by the proximal tubule and contribute to conditions that mimic apical albumin and NEFA concentra-
cytotoxicity by expediting lipoapoptosis. tions in the context of glomerular filtration barrier injury.12
A major advance in this report is the detailed characteriza- This concept of lipoapoptosis was invoked 35 years ago as a
tion of the proximal tubule transporter, FATP2, which medi- cause of tubular atrophy by Moorhead et al.,14 but the specific
ates apical NEFA uptake and lipoapoptosis. We confirmed that mechanisms that regulate tubular epithelial cell lipoapoptosis
FATP2 is expressed in kidney and localizes to proximal tubule had not been previously described. At a molecular level, lip-
in an apical plasma membrane and cytoplasmic distribution. oapoptosis has been associated primarily with long-chain
Subcellular identification was not pursued in detail in our (C12–C22) NEFA accumulation, and cytotoxicity is propor-
studies, but the murine FATP2 cytosolic and plasma membrane tional to acyl chain length and carbon bond saturation.46,47 In
immunohistochemical staining pattern is remarkably similar addition, the quantity of bound NEFA per albumin molecule
to the peroxisome and plasma membrane FATP2 expression may regulate toxicity as evidenced in clinical studies of min-
pattern described in liver.31 Hepatic FATP2 exhibits peroxi- imal change disease, which does not progress due to absence of
somal very long–chain (.22-carbon chain length) acyl CoA tubular atrophy and interstitial fibrosis. Despite massive albu-
synthetase activity and plasma membrane transport of long- minuria, the ultrafiltrate albumin was markedly NEFA deplete
chain (12–22 carbons) NEFAs, which ultimately undergo compared with serum in minimal change disease. In contrast,
b-oxidation in mitochondria.32 The diffuse cytosolic and api- the ultrafiltrate NEFA content was relatively unchanged in
cal membrane pattern observed in Figures 3 and 4 suggests other types of nephrotic syndrome,48 suggesting that the

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Figure 7. FATP2 mediates tubulointerstitial injury. Wild-type and Slc27a22/2 mice were treated with daily intraperitoneal lipidated
albumin (or saline control) injections to induce albuminuria and tubulointerstitial disease as described in Concise Methods. After 3
weeks, mice were euthanized and evaluated for tubulointerstitial injury using previously described quantitative histomorphometry
methods. (A) Coomassie Blue–stained gel showing albuminuria after the 3-week protocol was completed. Quantification of (B) tubular
epithelial cells, (C) interstitium, (D) tubular lumen, and (E) apoptotic proximal tubular epithelial cells within the tubulointerstitial
compartment. Data are expressed as mean6SEM (n=5). P values were generated using ANOVA with Bonferroni test for post hoc
between-group comparisons. IP, intraperitoneal; NS, not significant (P.0.05).

quantity and composition of NEFA bound to filtered albumin
dictate whether albuminuric renal diseases progress or AKI
develops after a relapse. With this caveat in mind, our in
vivo and in vitro experiments were performed using albumin
that was saturated with palmitate, the most toxic long-chain
fatty acid. Slc27a22/2 mice developed less reduction in tubular
epithelial cell number and interstitial fibrosis in the albumin
overload model of inducible proteinuria, in which mice
received daily intraperitoneal injections of palmitate-loaded
albumin. Furthermore, Slc27a22/2 and shRNA-treated prox-
imal tubule cell lines were almost completely protected from
palmitate-induced apoptosis, suggesting that FATP2 plays a
major pathophysiologic role in the apical uptake of filtered
NEFAs that result from glomerular injury.
Proximal tubule lipotoxicity is likely to result from a com-
bination of increased uptake, increased synthesis, and de-
creased catabolism of NEFA. The intracellular pathways are
complex and may not be accessible to small molecule pharma-
cologic inhibitors, and therefore, they represent less rational
therapeutic targets compared with apical FATP2. High-
throughput screens of small molecule libraries for FATP2 in-
hibitors have been conducted to identify lead molecules for the
treatment of hepatosteatosis. 49,50 Some candidates have
emerged, although enthusiasm has been dampened, because
many of the compounds with the best inhibition profiles (e.g.,
anesthetics and tricyclic antidepressants) could cause nonspe-
cific membrane disruption rather than specific inhibition of
FATP2 and at high IC50 values (midmicromolar), which may
be unattainable in blood. However, pharmacodynamics stud-
ies have not been conducted, and if these drugs are filtered
and/or secreted by the kidneys, low doses could result in suf-
ficiently high luminal concentrations to be effective inhibitors
of proximal tubule FATP2 and lipoapoptosis-dependent tubu-
lar atrophy.
There are six FATP isoforms, all of which contain a signature
Y/FIY/FTSGTTG AMP binding motif within a cytoplasmic
loop, and they are predicted to contain two extracellular do-
mains, which form an NEFA binding region after dimeriza-
tion.25 As a group, FATPs are efficient transporters, with Km
ranging from 0.3 to 20 mM,30,51,52 which is consistent with our
observations for proximal tubule NEFA transport. However,
the low- to midmicromolar concentrations used in our exper-
iments represent total concentration, whereas circulating free
NEFA concentration is in the low nanomolar range.53 There

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

transporter localized to proximal tubule.

CD36 has been implicated as a candidate
kidney NEFA transporter in humans,33,34
but in mouse studies, it has reliably been
shown only in kidney macrophages and in-
consistently in proximal tubule.12,33,34,55
FABPpm is expressed in proximal tubule
but localizes to mitochondria,56 and it is,
therefore, not a likely candidate for NEFA
uptake from the apical proximal tubule
membrane. FATP1 and FATP4 have been
detected in kidney, although at very low
levels, and localization within the nephron
has not been described. Nevertheless, these
FATP isoforms could represent residual
proximal tubule NEFA transporters.
In conclusion, intracellular accumula-
tion of NEFA and NEFA metabolites leads
Figure 8. FATP2 mediates proximal tubule lipoapoptosis. (A) HRPT cells were stably to tubular atrophy and CKD progression.
transfected with three FATP2 shRNA constructs or scrambled oligonucleotide (control). Normal proximal tubule metabolism is de-
Lysates from a crude membrane fraction were probed by immunoblot for FATP2 ex- pendent on basolateral NEFAuptake, which
pression. (B) Coomassie Blue–stained membrane as loading control. (C) Images of is regulated by an FATP2-independent
transfected HRPT cells exposed to apical 0.5% delipidated BSA complexed with mechanism, whereas apical NEFA uptake is
palmitate (100 mM) for 24 hours. Fixed cells were stained with oil red O and coun-
largely mediated by FATP2. We speculate
terstained with hematoxylin. (D) Oil red O was eluted with 100% propanol from
shRNA- or scrambled oligonucleotide sequence (control)–transfected HRPT cells and
that apical FATP2-directed NEFA uptake in
quantitated by spectrophotometry (l=492 nm; mean6SEM; n=3). FATP2 shRNA or conjunction with increased NEFA synthesis
scrambled sequence–treated HRPT proximal tubule cells were incubated with 0.2% and decreased NEFA metabolism represent
albumin + palmitate (100 mM; 24 hours), and apoptosis was determined by (E) TUNEL mechanisms of pathologic proximal tubule
or (F) immunoblot with rat antiactive (cleaved) caspase-2 IgG and a-tubulin loading NEFA accumulation in proteinuric renal
control (n=3). (G) Proximal tubule cell lines derived from wild-type and Slc27a22/2 disease, such as diabetic nephropathy. Using
mice were incubated with 100 mM palmitate complexed with 0.2% fatty acid–free in vivo, ex vivo, and in vitro techniques, we
albumin (Palm) or albumin-only control (BSA) for 24 hours and then assayed for apo- show that FATP2 is a major apical proximal
ptosis by TUNEL (n=3). *P,0.05 compared with the BSA-treated group by t test; tubule NEFA transporter, which regulates cy-
**P,0.05 compared with other groups by ANOVA. totoxicity. Because of this newly established
role as a mediator of lipoapoptosis, we pro-
are two FATP2 splice variants: one with intrinsic acyl CoA pose that FATP2 deserves attention as a potentially attractive tar-
synthetase activity (FATP2a), as previously mentioned, and get for the design of treatments for tubular atrophy and CKD
one without acyl CoA synthetase activity (FATP2b); both progression.
splice variants are capable of transporting long-chain
NEFA.32 Only FATP2a is detectable in kidney,32 which we
confirmed by RT-PCR. CONCISE METHODS
Apical NEFA uptake in Slc27a22/2 proximal tubules and
cell lines was significantly diminished, although not elimi- Animals
nated, and basolateral NEFA was similar between wild-type Sprague–Dawley rats weighing 100–150 g were purchased from
and FATP2 knockout mice, implying the presence of other Charles River Breeding Laboratories (Wilmington, MA). Wild-type
functional NEFA transporters in the proximal tubule. Consid- and 129S-Slc27a2tm1Kds/J (Slc27a22/2) mice were purchased from
ering the importance of NEFA b-oxidation to the proximal Jackson Laboratories (Bar Harbor, ME). All protocols and procedures
tubule metabolism, such redundancy is not surprising. One were approved by the Institutional Animal Care and Use Committee
implication of this observation is that, if FATP2 is targeted as a of Case Western Reserve University in accordance with the National
therapy to reduce lipoapoptosis, the residual NEFA uptake by Institutes of Health Guide for the Care and Use of Laboratory
FATP2-independent mechanisms should be sufficient to sup- Animals.
port proximal tubule survival as evidenced by the viability of
Slc27a22/2 cells (Figure 8) and previous observations that Human Samples
Slc27a22/2 mice exhibit no overt renal phenotype.54 We Formalin-fixed, paraffin-embedded normal human kidney sections
show that FFA1 and CD36 are expressed in kidney, but neither were obtained from Imgenex Corp. (San Diego, CA). All studies

J Am Soc Nephrol 29: ccc–ccc, 2018 Proximal Tubule FATP2 7

 BASIC RESEARCH www.jasn.org
involving human tissues were performed with approval of the insti-
tutional review board of the MetroHealth System, Case Western Re-
serve University.
Cell Lines
LLC-PK1 and HK-2 cell lines were purchased from ATCC (Manassas,
VA); HRPT cells were a gift from Lorraine Racusen (Johns Hopkins
University). LLC-PK1 and HRPT cells were maintained in DMEM-F12
(Invitrogen Life Technologies, Carlsbad, CA) plus 10% FBS (HyClone)
and 1% penicillin/streptomycin-fungizone (Sigma-Aldrich, St. Louis, MO)
as described previously.12 HK-2 cells were cultured in Keratinocyte-
SFM supplemented with 5 ng/ml EGF and 40 mg/ml bovine pituitary
extract (Gibco/Thermo Fisher Scientific, Waltham, MA).
Microperfusion Experiments
Microdissected proximal tubules were perfused with fluorescently
labeled NEFA using established methods.26 Animals were anesthe-
tized with ketamine (100 mg/kg body wt intraperitoneally) and xy-
lazine (20 mg/kg body wt intraperitoneally). The abdominal cavity
was opened, and the left kidney was superfused twice with ice cold
150 mM sodium chloride; then, it was removed and placed in phys-
iologic saline at 4°C. Coronal slices were cut, and proximal tubule
segments were isolated from the kidney cortex using microforceps
under a stereomicroscope at 4°C to 10°C. S2 segment tubules ranging
from 0.7 to 1.0 mm were transferred to a temperature-regulated
chamber and perfused using concentric glass pipettes at 37°C.
Tubules were perfused with BODIPY-conjugated 4,4-difuoro-5,7-
dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (final
concentration 2.5 mM; Invitrogen Life Technologies) complexed with
0.2% fatty acid–free albumin in buffer containing 130 mM NaCl,
2.5 mM NaH2PO4, 4 mM KCl, 1.2 mM MgSO4, 6 mM L-alanine,
1 mM trisodium citrate, 5.5 mM glucose, 2 mM calcium dilactate,
and 10 mM Hepes, pH 7.4 at 37°C. The fluorescence detection system
was mounted on a Nikon Diaphot inverted microscope (Nikon,
Tokyo, Japan). The intracellular BODIPY dye was excited at 490
nm, and emitted fluorescence was measured using a 510-nm dichroic
mirror. Images were recorded using a 403 immersion oil objective
and a Coolsnap HQ digital camera (Photometrics, Tucson, AZ), and
fluorescence measurements were recorded using Metafluor version 7
imaging software (Universal Imaging, Downingtown, PA).
NEFA Uptake in Proximal Tubule Cell Lines
Uptake studies were performed with minor modifications to pub-
lished methods.57 Cells were cultured to confluence on permeable
supports or 12-mm coverslips, which were mounted in a temperature-
controlled perfusion chamber of a Leica DMIRE2 inverted microscope
stage. Solutions containing BODIPY-conjugated dodecanoic acid
(QBT fatty acid uptake assay; Molecular Devices, Sunnyvale, CA;
final concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2.5 mM)
complexed with 0.2% essential fatty acid–free albumin were perfused
into the recording chamber. Excitation l=490-nm pulses were de-
livered and emission l=510-nm fluorescence was recorded at 20-second
intervals until steady-state uptake was achieved. Epifluorescence
was recorded using a SPOT-RT camera (Diagnostic Instruments,
Sterling Heights, MI), and images were acquired and analyzed using
8 Journal of the American Society of Nephrology
SimplePCI imaging software (Compix Inc., Cranberry Township,
PA). The uptake rate was defined as the maximum slope within
the first 10 seconds, which was determined using SigmaPlot 2000
software (SPSS, Chicago, IL).
RT-PCR
Total RNA was extracted from cell lines or mouse kidney cortex using
the RNeasy Mini kit (Qiagen, Hilden, Germany) in accordance with
the protocol described by the manufacturer. RNA concentrations were
determined using the NanoDrop 2000 Spectrophotometer (Thermo
Fisher Scientific). Reverse transcription was performed using Super-
Script IV VILO Master-mix (Invitrogen Life Technologies) according
to the manufacturer’s instructions. Each amplification reaction was
conducted in 25-ml volume using Choice Taq DNA Polymerase (Den-
ville Scientific Inc., Holliston, MA) according to the recommended
protocol and PCR cycling conditions. PCR products underwent 1%
agarose gel electrophoresis, and bands were identified by ethidium
bromide staining and photographed. Human FATP2 primer se-
quences were 59-GGAGATACATTCCGGTGGAA-39 (forward) and
59-TGATCTCAATGGTGTCCTGT-39 (reverse), yielding a 257-bp am-
plicon. Human CD36 primer sequences were 59-GGAACAGAGGCT-
GACAACTT-39 (forward) and 59-TCGCAGTGACTTTCCCAATAG-39
(reverse), yielding a 347-bp amplicon. Mouse GAPDH primer se-
quences were 59-CTGCCATTTGCAGTGGCAAAGTGG-39 (forward)
and 59-TTGTCATGGATGACCTTGGCCAGG-39 (reverse); human
GAPDH primer sequences were 59-GTCTTCACCACCATGGA-
GAAG-39 (forward) and 59-GCTTCACCACCTTCTTGATGT-
CATC-39 (reverse).
Immunohistochemistry
Methods have been described previously in detail.58 Mouse kidney
was frozen at 280°C. Samples were sectioned to 5-mm thickness by
cryostat, fixed in paraformaldehyde (4%; 10 minutes at room tem-
perature), rinsed in PBS, and then permeabilized with Triton X-100
(Sigma-Aldrich; 0.2% in PBS, 10 minutes at room temperature).
Sections were blocked with rabbit or goat serum (5% in PBS, 1
hour at room temperature) or Mouse on Mouse reagent (Vector,
Burlingame, CA). Primary antibodies were rabbit anti-FATP2 IgG
(GeneTex, Irvine, CA; 1:50, 16 hours, 4°C), rabbit polyclonal
FATP2 antisera31 (1:50, 16 hours, 4°C), rabbit anti-FFA1 IgG (Ala-
mone, Jerusalem, Israel; 1:200, 16 hours, 4°C), mouse anti-CD36 IgA
(BD Pharmingen, San Jose, CA; 1:200, 16 hours, 4°C), mouse anti–Na+
/K+-ATPase IgG (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100, 16
hours, 4°C), or goat anti-Glut5 IgG (Santa Cruz Biotechnology; 1:100,
16 hours, 4°C). Secondary Alexa Fluor 488 and 568 antibodies were used
(Invitrogen Life Technologies; 1:200, 1 hour at room temperature).
Proximal tubules were labeled with Texas red–conjugated Tetragonolobus
purpureus lectin as previously described.5 Sections were mounted in
Vectashield aqueous mounting media containing DAPI nuclear coun-
terstain (Vector) and viewed by confocal microscopy (Leica, Wetzlar,
Germany).
Immunoblot Analyses and Immunoprecipitation
Lysates were probed from whole cells or crude membrane fractions.
Crude membranes were harvested by incubation with hypotonic
J Am Soc Nephrol 29: ccc–ccc, 2018

buffer (1:10 dilution of PBS with protease inhibitor cocktail; Thermo
Fisher Scientific). Lysed cells were homogenized, and large particles were
removed by centrifugation at 5003g for 2 minutes. For immunoblots,
the supernatant was centrifuged at 14,0003g for 30 minutes, and the
resulting pellet was suspended in Laemmli buffer (125 mM Tris, pH 6.8,
2% SDS, and 5% glycerol), assayed for protein concentration by DC
protein assay (Bio-Rad, Hercules, CA), and frozen at 280°C.
For biotinylation experiments, PBS-washed cells were incubated
with sulfo-NHS-LC-Biotin (Thermo Fisher Scientific; 2 mM, 5 min-
utes, 4°C), washed again with PBS, and then incubated with glycine
quenching buffer (0.1 M, 5 minutes, 4°C). Cells were lysed in 1 ml
lysis buffer (25 mM Tris, pH 7.4, 50 mM NaCl, 25 mM NaF, 10%
glycerol, and 1% Triton X-100) and centrifuged (14,0003g, 10 min-
utes, 4°C), and supernatants were assayed for protein concentration
and stored at 280°C. Thawed samples of equal protein concentration
were mixed with streptavidin agarose beads (Thermo Fisher Scien-
tific; 20 ml bead volume) with gentle rocking for 3 hours at 4°C and
then centrifuged (10003g, 30 seconds, 4°C). The pelleted beads were
then washed with lysis buffer at 4°C.
Whole-cell, crude membrane, and streptavidin-precipitated sam-
ples were lysed and denatured in boiling SDS-PAGE buffer (125 mM
Tris, pH 6.8, 2% SDS, 5% glycerol, 1% b-mercaptoethanol, and
0.003% bromphenol blue) for 5 minutes. Immunoblot methods
have been described previously.12 Briefly, samples (20 mg protein
per lane) were resolved by SDS-PAGE and transferred to polyvinyli-
dine difluoride membranes. Blots were blocked in 5% nonfat dried
milk and probed with anti-FATP2 (GeneTex; 1:500, 16 hours, 4°C) or
anticaspase-2 (Abcam; 10 mg/ml, 16 hours, 4°C) IgG and then HRP-
conjugated IgG (1:10,000, 1 hour at room temperature). Band in-
tensity was detected by enhanced chemiluminescence. Blots were
exposed to stripping buffer (Thermo Fisher Scientific; 10 minutes
at room temperature) and then reprobed with anti–a-tubulin (Santa
Cruz Biotechnology; 1:3000, 1 hour at room temperature), anti–
Na+/K+-ATPase (Santa Cruz Biotechnology; 1:1000, 1 hour at room
temperature), or anti-Glut5 (Santa Cruz Biotechnology; 1:500, 1 hour
at room temperature) IgG followed by HRP-conjugated IgG (1:10,000,
1 hour at room temperature).
Immunocytochemistry
Methods have previously been described in detail.59
Cells were main-
tained on sterile glass coverslips within six-well plates, blocked with
5% low-IgG BSA and 0.2% Triton X-100 (Sigma-Aldrich) for 30
minutes at room temperature, incubated with rabbit anti-FATP2
IgG (1:50, 2 hours at room temperature), and then fixed in parafor-
maldehyde (4%, 10 minutes at room temperature). Postfixed cells
were incubated with Alexa Fluor 568 goat anti-rabbit IgG (1:200, 2
hours at room temperature). Coverslips were mounted in antifade,
aqueous media containing DAPI (Vectashield; Vector) on standard
microscope slides. Images were viewed using a Leica confocal micro-
scope with appropriate fluorescence filters.
shRNA Transfection
To achieve FATP2 knockdown, HRPT cells were transfected with
lentiviral shRNA vectors containing a puromycin selection cassette
and targeting human SLC27A2 (Ref-Seq NM_003645.3) according to
J Am Soc Nephrol 29: ccc–ccc, 2018
www.jasn.org BASIC RESEARCH
previously described methods.12 Oligonucleotides were purchased
from the Mission shRNA Gene Set and cloned into pLKO.1-puro
plasmid (Sigma-Aldrich), and lentiviral particles were produced in
HEK 293T packaging cells using the ViraPower Expression System
(Invitrogen Life Technologies). The viral supernatant was added to
HRPT monolayers grown on 10-cm plates, and stably transfected
cells were identified for further studies. Three constructs were
screened by immunoblotting for silencing of FATP2 expression:
ccggCCATACTTCTTCCAGGACATACTCGAGTATGTCCTGGAA-
GAAGTATGGtttttg (shRNA1), ccggGCTGATTACCTACCTAGT-
TATCTCGAGATAACTAGGTAGGTAATCAGCtttttg (shRNA2),
and ccggCCTATGACTGAGGACATCTATCTCGAGATAGATGTCCT-
CAGTCATAGGtttttg (shRNA3).
In Vivo Induction of Proteinuria
Proteinuria and tubulointerstitial disease were induced by adapted
protein overload8,40 and LPS41,42 protocols. Both protocols used 6- to
8-week-old wild-type and Slc27a22/2 mice on 129S genetic back-
grounds. For the protein overload model, the right and left abdomen
were alternately cleansed with isopropanol on consecutive days for in-
traperitoneal injections. Mice received daily (Monday through Friday)
sterile injections of endotoxin-free BSA (A-9430; Sigma-Aldrich; 10 mg/g
body wt) 5 d/wk for 3 weeks. For the LPS model, mice were injected with
endotoxin-free LPS (L-3137; Sigma-Aldrich; 10 mg per mouse) for 3
consecutive days. All mice were euthanized the day after protocol
completion. Urine was collected by bladder puncture, and kidneys
were harvested for quantitative histomorphometry analyses.
Urine Protein Electrophoresis
To determine albumin excretion, urine samples (10 ml per lane + 2 ml
63 SDS-PAGE buffer) were boiled for 10 minutes, loaded on 10%–
20% Tris-glycine gels, and electrophoresed at 150 V. Gels were then
stained overnight in solution containing 0.1% Coomassie Brilliant
Blue R-250, 50% methanol, and 70% glacial acetic acid. Gels were
then destained in 50% methanol + 7% acetic acid solution, and digital
images were obtained with a Canon Canoscan LiDE 120 scanner.
Quantitative Histomorphometry
Methods were conducted as previously described.12 Briefly, studies
were conducted by two observers blinded to experimental conditions
on images viewed at 403 magnification, which were overlaid with a
16322 grid within Adobe Photoshop (San Jose, CA) or ImageJ to
assess for tubular atrophy and interstitial fibrosis. Ten sections per
kidney were randomly selected for analysis. Coincidence of intersect-
ing grid lines with tubule (nucleus, cytoplasm, or brush border),
tubule lumen, or Masson trichrome–stained interstitium was coun-
ted, whereas glomeruli and blood vessels were omitted from calcula-
tions. The total of the three compartments was defined as 1.0, and
mean values between the two observers for the proportion of the total
composed of tubule cells, tubule lumen, or interstitium were com-
pared between experimental and control (saline-injected) mice.
Oil Red O Staining and Quantification
After incubation with palmitic acid (100 mM, 24 hours) complexed
with 0.2% albumin, cells were fixed with paraformaldehyde (4%, 10
Proximal Tubule FATP2 9
 BASIC RESEARCH www.jasn.org
minutes at room temperature), rinsed with PBS, and stained with the
Oil Red O (0.5%; Biovision; 5 minutes at room temperature) as pre-
viously described.12 Stained cells were rinsed with PBS and viewed
with a Leica Dmi8 inverted light microscope. For quantification, Oil
Red O was incubated for 30 minutes and washed with PBS and then
60% isopropanol. Oil Red O was eluted with isopropanol (100%, 10
minutes at room temperature) followed by eluate absorbance mea-
surement at 492 nm with a NanoDrop 2000 spectrophotometer
(Thermo Fisher Scientific).
TUNEL Assays
Proximal tubule epithelial cell lines were cultured on coverslips and
grown to near confluence. Ten to 12 random fields per coverslip were
assayed for apoptosis, which was assessed by TUNEL as previously
described.59 Nuclei of all cells were counterstained with DAPI, and
data are presented as percentage of apoptotic cells.
Statistical Analyses
Unless otherwise noted, all results are expressed as means6SEM.
Two-tailed paired t tests were used for statistical analysis between
two groups. Two-way ANOVA was used for comparisons between
more than two groups. Statistical significance is defined as P#0.05.
ACKNOWLEDGMENTS
Fatty acid transporter-2 antibodies were a gift from Dr. Andreas Stahl
(University of California at Berkeley).
This work was supported by National Institutes of Health grants
R01 HL128053 (to J.L.G.) and R01 DK067528 (to J.R.S.).
DISCLOSURES
None.
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Present address: Dr. Pablo D. Cabral, Miromatrix Medical, Eden Prairie,
Minnesota.
This article contains supplemental material online at http://jasn.asnjournals.
org/lookup/suppl/doi:10.1681/ASN.2017030314/-/DCSupplemental.
Proximal Tubule FATP2 11