Vous êtes sur la page 1sur 11
BASIC RESEARCH www.jasn.org Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2) Shenaz

BASIC RESEARCH www.jasn.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 nonesteri ed fatty acids (NEFAs) across the dam- aged glomerular ltration barrier and subsequent reabsorpt ion 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 uorescently 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 Slc27a2 2 / 2 mice. Ex vivo microperfusion and in vitro experiments with NEFA-bound albumin at concentrations that mimic apical proximal tubule exposure during glomerular injury revealed signi cantly reduced NEFA uptake and palmitate-induced apoptosis in microperfused Slc27a2 2 / 2 proximal tubules and Slc27a2 2 / 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 ma jor 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- icted with CKD, 1 and risks for CKD progression and mortality are albuminuria and decreased GFR. 2 Although albuminuria reects glomerular ltration 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

Signi cance Statement

Albuminuria and tubular atrophy are signi cant risks for CKD progression to ESRD. We have proposed that, in progressive, proteinuric renal diseases, ltration of albumin-bound nonesteri ed fatty acids (NEFAs) across damaged glomeruli leads to proximal tubule NEFA reabsorption, causing tubula r 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.

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org Figure 1. NEFAs are absorbed by the proximal tubule in vitro . (A)

Figure 1. NEFAs are absorbed by the proximal tubule in vitro . (A) LLC-PK 1 cells were grown to con uence on permeable supports and then incubated with apical (AP) or basolateral (BL) BODIPY-labeled C16-NEFA (2.5 m M) complexed with albumin (0.2%) in buffer prewarmed to 37°C. BODIPY uptake was measured by uorescence microscopy 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-PK 1 maintained on permeable supports (mean6SEM from n=3).

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 FATPfamily. 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 lipoapoptosis.

RESULTS

NEFAs Are Absorbed by the Proximal Tubule

due to tubular epithelial cell apoptosis is superior to glomerular pathology as a predictor of CKD progression. 3 6 Infusion of albumin in animal models 7 9 or exposure of proximal tubule cells to albumin in vitro 10 12 results in apoptosis. However, cytotoxicity is not observed with delipidated albumin admin- istration, 8,10,12,13 implying that albumin-bound fatty acids cause apoptosis. Mouse models of CKD show increased con- centrations of NEFAs and NEFA metabolites in the kidney, which are hypothesized to cause renal function deterioration. 12,14 Proposed mechanisms leading to proximal tubule lipid ac- cumulation include increased uptake, increased synthesis, and diminished b -oxidation of NEFA, 15 17 but the relative impor- tance of each mechanism has not been determined. Increased NEFA uptake is plausible, because hyperlipidemia commonly coexists with CKD, resulting in enhanced extracellular NEFA supply for transport and intracellular storage by nonadipose tissues, such as in liver, skeletal muscle, and kidney. 18 More importantly, in proteinuric renal diseases, such as diabetic nephropathy, the damaged glomerulus permits albumin- bound NEFA to be ltered and gain access to the previously unexposed proximal tubule luminal surface, where aberrant NEFA reabsorption could then occur. Under normal circumstances, fatty acids are the preferred substrate for proximal tubule ATP generation. 19,20 In plasma, esteri ed fatty acids circulate as triglycerides, whereas water- insoluble NEFAs are solubilized by complexing with albumin. NEFAs dissociate from albumin at the plasma membrane and are taken up by saturable, basolateral membrane transporters in proximal tubules. 21 24 Under pathologic circumstances, the possibility of simultaneous apical and basolateral proxi- mal tubule NEFA uptake combined with increased NEFA synthesis 15,16 and diminished b -oxidation 17 could lead to

To screen for proximal tubule apical NEFA uptake, experiments were init ially conducted in proximal tubule cell lines. Figure 1A shows rapid basolateral NEFA absorption in polarized LLC-PK 1 cells, with time-dependent uptake. The rate and magnitude of NEFA uptake were con- siderably greater from the apical surface. Figure 1B shows concentration-dependent apical NEFA uptake. To test NEFA uptake under native conditions, microdissec- ted rat proximal tubules were perfused with uorescently la- beled NEFA using established methods. 26 Figure 2, A and B depicts a proximal tubule immediately and then 10 minutes after luminal microperfusion with boron-dipyrromethene (BODIPY)-tagged NEFA, respectively. Figure 2C shows rapid time-dependent uptake and approximately vefold greater NEFA internalization from the apical compared with the basolateral surface, consistent with Figure 1A in vitro data. Figures 1 and 2 collectively show that proximal tubule epithe- lial cells reabsorb NEFA from the apical surface and by a mag- nitude that exceeds basolateral uptake.

Proximal Tubule FATP2 Expression

FATPs (gene name Slc27 ) are a family of transmembrane span- ning proteins, which contain six members with distinct tissue expression patterns. Of the FATP isoforms expressed in kid- ney, FATP1 and FATP4 are present in very small amounts 25 (S. Khan et al. , unpublished observations). FATP2 is expressed exclusively in kidney and liver and has previously been de- tected in total kidney and proximal tubule. 25,27 32 Figure 3A shows FATP2 mRNA expression in wild-type mouse kidney, which is diminished in Slc27a2 +/ 2 and absent in Slc27a2 2 / 2 control kidneys. Figure 3, B D reveals that FATP2 protein expression in kidney is restricted to proximal tubule. No FATP2 staining was detected in Slc27a2 2 / 2 kidneys (not shown). Figure 3E con rms FATP2 mRNA expression in

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH Figure 2. NEFA are absorbed from the proximal tubule apical surface. Fluorescence image

Figure 2. NEFA are absorbed from the proximal tubule apical surface. Fluorescence image of a rat proximal tubule (A) immediately and (B) 10 minutes after lumen perfusion with BODIPY-labeled NEFAs. (C) BODIPY-NEFA (2.5 mM; complexed with 0.2% al- bumin) uptake in a microdissected rat proximal tubule after luminal perfusion (left panel), addition of BODIPY-NEFA to the basolateral bath (center panel), and luminal perfusion of BODIPY-NEFA (500 nM) in Hepes (10-mM) buffered saline (right panel).

Data are representative of experiments from 12 tubules.

Human Renal Proximal Tubule (HRPT) and HK-2 human proximal tubule cell lines. FATP2 transcripts were weakly de- tectable by RT-PCR in LLC-PK 1 cells (Figure 3E), perhaps re ecting nucleotide sequence differences between human and porcine mRNA (porcine cDNA sequence is unknown). Because FATP2 may not be the sole FATP in the proximal tubule, screens for other plausible transporters were under- taken. CD36 has been reliably isolated in mouse kidney, 33 but it was not detectable by immunohistochemistry in proximal tubules (Supplemental Figure 1) 12,34 or by RT-PCR in HK-2 cells (not shown). The G protein coupled GP40 and GP120 long-chain NEFA transporters have recently been deorphan- ized and termed FFA1 and FFA4, respectively; only FFA1 is expressed in kidney. 35,36 Supplemental Figure 2 shows FFA1 expression in a glomerular epithelial cell (podocyte) pattern but not within tubules, consistent with the previously de- scribed role of podocyte FFA1-mediated NEFA uptake in the pathogenesis of the nephrotic syndrome. 37 Unlike FATP2, CD36 and FFA1 are, therefore, not candidate transporters for regulation of proximal tubule NEFA reabsorption.

FATP2 Is Expressed in Proximal Tubule Apical Membranes

Like other members of the FATP family, FATP2 is predicted to be a membrane-associated transporter containing two trans-

be a membrane-associated transporter containing two trans- Figure 3. FATP2 is expressed in proximal tubules. (A)

Figure 3. FATP2 is expressed in proximal tubules. (A) Kidney FATP2 and GAPDH mRNA expression by RT-PCR. Immunohistochemical localization of (B) FATP2, (C) T. purpureus lectin staining, and (D) merged image in mouse kidney cortex. Gl, glomerulus. *Distal tu- bule. (E) FATP2 and GAPDH mRNA expression by RT-PCR in proximal tubule cell lines.

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-PK 1 and HK-2 proximal tubule cell membranes. Immunocytochemistry ex- periments in post xed HK-2 cells revealed FATP2 expression in a plasma membrane distribution (Figure 4B). Immunoprecipi- tation of biotin surface labeled FATP2 also showed abundant expression in LLC-PK 1 , HK-2, and HRPT proximal tubule cell lines (Figure 4C), indicating that FATP2 is ex- pressed on the plasma membrane. To establish FATP2 localization, human

kidney sections were probed with FATP2 and g -glutamyl transferase-1 (a brush bor- der enzyme) antibodies. Figure 5 reveals FATP2 and g -glutamyl transferase-1 colocalization in an apical membrane pattern. To conrm FATP2 membrane domain localization, mouse kidney sections were colabeled with Glut5 (a luminal brush border protein) and Na + /K + -ATPase (to demarcate the basolateral membrane) antibodies. Supplemental Figure 3, AD shows the diffuse FATP2 cytosolic pattern, although colocalization was also observed with Glut5, indicating that FATP2 is expressed on the proximal tubule apical membrane. In contrast, no FATP2 coloc- alization with Na + /K + -ATPase was noted (Supplemental Figure 3, EG), indicating that FATP2 is not expressed in basolateral mem- branes and would, therefore, not mediate basolateral NEFA uptake (Figure 6C). Taken together, Figures 4 and 5 and Supplemental Figure 3 show that proximal tubule FATP2 is expressed most prominently within the apical plasma membrane.

Proximal Tubule FATP2-Regulated NEFA Uptake

FATP2-dependent NEFA uptake was initially evaluated in experiments with microdissected proximal tubules from wild-type and Slc27a2 2 / 2 mice, which were perfused with BODIPY-labeled NEFA. No difference was observed between male and female tubules, and therefore, results from both sexes are combined. Figure 6, A and B shows enhanced time- and concentration-dependent NEFA uptake from lumi- nal perfusion of wild-type compared with Slc27a2 2 / 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 Slc27a2 2 / 2 proxi- mal tubules (Figure 6C), indicating that basolateral NEFA uptake does not require FATP2. NEFA uptake was also greater in

proximal tubule cell lines derived from wild-type versus Slc27a2 2 / 2 mice (Figure 6, D and E), similar to results from tubules perfused ex vivo (Figure 6, A and B). These

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org Figure 4. FATP2 is expressed in proximal tubule membranes. (A) Lysates from crude

Figure 4. FATP2 is expressed in proximal tubule membranes. (A) Lysates from crude

membrane preparations were immunoblotted with anti-FATP2 antibodies (upper panel). Membranes were stripped and reprobed with anti Na + /K + -ATPase antibodies as a loading control (lower panel). (B) HK-2 c ells on coverslips were probed with anti- FATP2 antibodies and post xed in paraformaldehyde (4%, 10 minutes at room temperature), and primary antibody detection was ampli ed with Alexa Fluor 568 conjugated goat anti-rabbit IgG. (C) Proxim al tubule cell lines were surface labeled with biotin, and lysates wer e precipitated with streptavidin-coated beads, eluted, resolved by SDS-PAGE, and immunoblot ted with anti-FATP2 antibodies (upper panel). Membranes were stripped and reprobed with anti-Glut5 antibodies as a loading

control (lower panel).

results show that a large proportion of apical (but not basolat- eral) proximal tubule NEFA uptake is mediated by FATP2.

FATP2 Mediates Tubulointerstitial Disease

To determine the pathophysiologic signi cance of proximal tubule FATP2, wild-type and Slc27a2 2 / 2 mice were treated with daily intraperitoneal injections of lipidated albumin to induce albuminuria (Figure 7A ) and tubulointerstitial in- jury. 8,40 Quantitative histomorphometric analysis revealed modest tubular atrophy and interstitial brosis, although tu- bular epithelial cell number and interstitial and tubular lumen areas were signi cantly different between wild-type and Slc27a2 2 / 2 mouse kidneys (Figure 7, B D). Similar differ- ences in tubular epithelial cell density were also observed in wild-type versus Slc27a2 2 / 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 proposed pathway is apoptosis, in the absence of compensa- tory hyperplasia. 43 Figure 7E shows increased tubular epithe- lial cell apoptosis in lipidated albumin injected mice, which was signi cantly attenuated in Slc27a2 2 / 2 compared with wild-type mice. Taken together, the in vivo data suggest that apical proximal tubule FATP2-mediated NEFA uptake con- tributes to tubular atrophy and interstitial brosis.

DISCUSSION

to tubular atrophy and interstitial fi brosis. DISCUSSION Figure 5. FATP2 is expressed in proximal tubule

Figure 5. FATP2 is expressed in proximal tubule apical membranes. Formalin- xed paraf n sections from human kidneys were probed with (A) anti-FATP2 and Alexa Fluor 488 secondary antibodies or (B) anti g -glutamyl transferase-1 (GGT1) and Alexa Fluor 568 labeled secondary antibodies. (C) Nuclei were labeled with DAPI in the mounting medium. (D) Colocalization from merged images is depicted in yellow.

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- sion. 12,14 To directly test whether FATP2 regulates lipotoxicity, BODIPY-tagged NEFA uptake was measured in FATP2 shRNA-silenced versus shRNA-scrambled sequence-treated HRPT human proximal tubule cell lines (Figure 8, A and B). Figure 8C depicts cells with oil red O stained lipid droplets as an indirect measure of NEFA uptake and qualitatively con rms the ef -

cacy of functional FATP2 knockdown with two of the three shRNA constructs. Oil red

O quanti cation revealed reduced staining in FATP2 shRNA

compared with scrambled nucleotide sequence control cells (Figure 8D), once again indicating that proximal tubule FATP2 mediates NEFA uptake. Figure 8, E and F shows that sustained NEFA exposure caused apoptosis, as shown by terminal deoxynucleotidyl transferasemediated digoxigenin- deoxyuridine nick-end labeling (TUNEL) and caspase-2 activation, which was signicantly reduced in FATP2 shRNA- expressing cells compared with scrambled nucleotidetreated cells. NEFA-induced apoptosis was also observed in proximal tubule cells lines derived from wild-type mice and signicantly

blunted in Slc27a2 2 / 2 mouse proximal tubule cells (Figure 8G). Taken together, these loss of function approaches show that FATP2 regulates lipoapoptosis.

CKD associated with heavy albuminuria accounts for the vast

majority of causes of ESRD. There has been signi cant progress

in understanding the role of glomerular cells, particularly the

glomerular epithelial cell (podocyte), in the pathophysiology of

albuminuria, but it is often overlooked that tubular atrophy more accurately predicts CKD progression compared with glomerular pathology. 3 6 Because each al- bumin molecule that leaks across the dam- aged glomerular ltration 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-

esis. 12,14 Using combined in vitro and ex vivo approaches, we show that proximal tubules rapidly reabsorb luminal NEFA from the apical surface and by a magnitude that is several fold greater than from the

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH Figure 6. Proximal tubule FATP2-regulated NEFA uptake is time- and concentration- dependent.

Figure 6. Proximal tubule FATP2-regulated NEFA uptake is time- and concentration-

dependent. (A) BODIPY-NEFA (500 nM NEFA complexed with 0.2% albumin in Hepes [10-mM]buffered saline) uptake after luminal perfusion in wild-type versus Slc27a2 2 / 2 tubules microdissected from 12-week-old mice. A representative tracing (from n=5) is shown. (B) BODIPY-NEFA (202500 nM NEFA in vefold dilutions complexed with 0.2% albumin in Hepes [10-mM]buffered saline) uptake after luminal perfusion in wild-type versus Slc27a2 2 / 2 tubules microdissected from 12-week-old mice (mean6SEM from n=8). (C) BODIPY-NEFA uptake from the basolateral bath. Representative tracings (from n=3) are shown for clarity due to overlapping error bars. (D) BODIPY-NEFA uptake in temperature-sensitive SV40 immortalized proximal tubule cell lines derived from wild- type and Slc27a2 2 / 2 mice (mean6SEM; n=4). (E) Concentration-dependent uptake of BODIPY-labeled NEFA in wild-type versus Slc27a2 2 / 2 mouse proximal tubule cell lines

(mean from n=4).

basolateral surface. Uptake was achieved by complexing NEFA with albumin at a concentration of 2 g/L, which corresponds to apical proximal tubule exposure of albumin under conditions that mimic glomerular ltration barrier dysfunction. Our data support the notion that aberrantly ltered NEFAs are ef - ciently reabsorbed by the proximal tubule and contribute to cytotoxicity by expediting lipoapoptosis. A major advance in this report is the detailed characteriza- tion of the proximal tubule transporter, FATP2, which medi- ates apical NEFA uptake and lipoapoptosis. We con rmed that FATP2 is expressed in kidney and localizes to proximal tubule in an apical plasma membrane and cytoplasmic distribution. Subcellular identi cation was not pursued in detail in our studies, but themurine FATP2 cytosolic and plasmamembrane immunohistochemical staining pattern is remarkably similar to the peroxisome and plasma membrane FATP2 expression pattern described in liver. 31 Hepatic FATP2 exhibits peroxi- somal very long chain ( . 22-carbon chain length) acyl CoA synthetase activity and plasma membrane transport of long- chain (12 22 carbons) NEFAs, which ultimately undergo b -oxidation in mitochondria. 32 The diffuse cytosolic and api- cal membrane pattern observed in Figures 3 and 4 suggests

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 NEFAwas largely mediated by membrane-localized FATP2. Apical NEFA uptake capacity at equilib- riumwas signicantly 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 associatedwithme- tabolism, whereas damaged glomeruli lter albumin-bound NEFAs, which are transpor- ted by apical FATP2, and then may trafc to a compartment that ultimately initiates apo- ptosis.Thus,disparatecytotoxicityafterapical versus basolateral NEFA uptake could be ex- plained by differential NEFA partitioning. It is also noteworthy that apical NEFA uptake at equilibrium was signi cantly lower in Slc27a2 2 / 2 compared with wild-type proxi- mal tubules, suggesting that metabolism and distribution to intracellular pools may differ after FATP2- and nonFATP2-mediated up- take. Plasma membrane FATPs are coupled to specic long-chain acyl CoA synthetases,

which accelerate NEFA ux and shuttle fatty acids to binding proteins (FABPs) that then channel NEFAs to specic intracellular sites. We speculate that FATP/acyl CoA synthetase/FABP isoform combinations could also segregate NEFA to different compart- ments after transport across plasma membrane domains. 44,45 We have previously shown that proximal tubule cells un- dergo apoptosis after exposure to NEFA (palmitate) under conditions that mimic apical albumin and NEFA concentra- tions in the context of glomerular ltration barrier injury. 12 This concept of lipoapoptosis was invoked 35 years ago as a cause of tubular atrophy by Moorhead et al. , 14 but the speci c mechanisms that regulate tubular epithelial cell lipoapoptosis had not been previously described. At a molecular level, lip- oapoptosis has been associated primarily with long-chain (C12 C22) NEFA accumulation, and cytotoxicity is propor- tional to acyl chain length and carbon bond saturation. 46,47 In addition, the quantity of bound NEFA per albumin molecule may regulate toxicity as evidenced in clinical studies of min- imal change disease, which does not progress due to absence of tubular atrophy and interstitial brosis. Despite massive albu- minuria, the ultra ltrate albumin was markedly NEFA deplete compared with serum in minimal change disease. In contrast, the ultra ltrate NEFA content was relatively unchanged in other types of nephrotic syndrome, 48 suggesting that the

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org

BASIC RESEARCH www.jasn.org Figure 7. FATP2 mediates tubulointerstitial injury. Wild-type and Slc27a2 2 / 2

Figure 7. FATP2 mediates tubulointerstitial injury. Wild-type and Slc27a2 2 / 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. Quanti cation 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 mean 6 SEM ( n =5). P values were generated using ANOVA with Bonferroni test for post hoc between-group comparisons. IP, intraperitoneal; NS, not signi cant ( P . 0.05).

quantity and composition of NEFA bound to ltered 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. Slc27a2 2 / 2 mice developed less reduction in tubular epithelial cell number and interstitial brosis in the albumin overload model of inducible proteinuria, in which mice received daily intraperitoneal injections of palmitate-loaded albumin. Furthermore, Slc27a2 2 / 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 ltered 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 pro les ( e.g. , anesthetics and tricyclic antidepressants) could cause nonspe- ci c membrane disruption rather than speci c inhibition of FATP2 and at high IC 50 values (midmicromolar), which may be unattainable in blood. However, pharmacodynamics stud- ies have not been conducted, and if these drugs are ltered and/or secreted by the kidneys, low doses could result in suf- ciently 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 ef cient transporters, with K m ranging from 0.3 to 20 m M, 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

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH Figure 8. FATP2 mediates proximal tubule lipoapoptosis. (A) HRPT cells were stably

Figure 8. FATP2 mediates proximal tubule lipoapoptosis. (A) HRPT cells were stably transfected with three FATP2 shRNA constructs or scrambled oligonucleotide (control). Lysates from a crude membrane fraction were probed by immunoblot for FATP2 ex- pression. (B) Coomassie Blue stained membrane as loading control. (C) Images of transfected HRPT cells exposed to apical 0.5% delipidated BSA complexed with palmitate (100 m M) for 24 hours. Fixed cells were stained with oil red O and coun- terstained with hematoxylin. (D) Oil red O was eluted with 100% propanol from shRNA- or scrambled oligonucleotide sequence (control) transfected HRPT cells and quantitated by spectrophotometry ( l =492 nm; mean 6 SEM; n =3). FATP2 shRNA or scrambled sequence treated HRPT proximal tubule cells were incubated with 0.2% albumin + palmitate (100 m M; 24 hours), and apoptosis was determined by (E) TUNEL

or (F) immunoblot with rat antiactive (cleaved) caspase-2 IgG and a -tubulin loading control ( n =3). (G) Proximal tubule cell lines derived from wild-type and Slc27a2 2 / 2 mice were incubated with 100 mM palmitate complexed with 0.2% fatty acid free albumin (Palm) or albumin-only control (BSA) for 24 hours and then assayed for apo- ptosis by TUNEL ( n =3). * P , 0.05 compared with the BSA-treated group by t test;

** P , 0.05 compared with other groups by ANOVA.

are two FATP2 splice variants: one with intrinsic acyl CoA synthetase activity (FATP2a), as previously mentioned, and one without acyl CoA synthetase activity (FATP2b); both splice variants are capable of transporting long-chain NEFA. 32 Only FATP2a is detectable in kidney, 32 which we con rmed by RT-PCR. Apical NEFA uptake in Slc27a2 2 / 2 proximal tubules and cell lines was signi cantly diminished, although not elimi- nated, and basolateral NEFA was similar between wild-type and FATP2 knockout mice, implying the presence of other functional NEFA transporters in the proximal tubule. Consid- ering the importance of NEFA b-oxidation to the proximal tubule metabolism, such redundancy is not surprising. One implication of this observation is that, if FATP2 is targeted as a therapy to reduce lipoapoptosis, the residual NEFA uptake by FATP2-independent mechanisms should be suf cient to sup- port proximal tubule survival as evidenced by the viability of Slc27a2 2 / 2 cells (Figure 8) and previous observations that Slc27a2 2 / 2 mice exhibit no overt renal phenotype. 54 We show that FFA1 and CD36 are expressed in kidney, but neither

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 FABP pm 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 to tubular atrophy and CKD progression. Normal proximal tubule metabolism is de- pendent on basolateral NEFAuptake,which is regulated by an FATP2-independent mechanism, whereas apical NEFA uptake is largely mediated by FATP2. We speculate that apical FATP2-directed NEFA uptake in conjunction with increased NEFA synthesis and decreased NEFA metabolism represent mechanisms of pathologic proximal tubule NEFA accumulation in proteinuric renal disease, such as diabetic nephropathy. Using in vivo, ex vivo, and in vitro techniques, we show that FATP2 is a major apical proximal tubule NEFA transporter, which regulates cy-

totoxicity. Because of this newly established role as a mediator of lipoapoptosis, we pro- pose that FATP2 deserves attention as a potentially attractive tar- get for the design of treatments for tubular atrophy and CKD progression.

CONCISE METHODS

Animals

Sprague Dawley rats weighing 100 150 g were purchased from Charles River Breeding Laboratories (Wilmington, MA). Wild-type and 129S- Slc27a2 tm1Kds /J ( Slc27a2 2 / 2 ) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All protocols and procedures were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Human Samples

Formalin- xed, paraf n-embedded normal human kidney sections were obtained from Imgenex Corp. (San Diego, CA). All studies

BASIC RESEARCH www.jasn.org

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-PK 1 and HK-2 cell lines were purchased from ATCC (Manassas, VA); HRPT cells were a gift from Lorraine Racusen (Johns Hopkins University). LLC-PK 1 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 Scienti c, Waltham, MA).

Microperfusion Experiments

Microdissected proximal tubules were perfused with uorescently 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 ( nal concentration 2.5 m M; Invitrogen Life Technologies) complexed with 0.2% fatty acid free albumin in buffer containing 130 mM NaCl, 2.5 mM NaH 2 PO 4 , 4 mM KCl, 1.2 mM MgSO 4 , 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 uorescence detection system was mounted on a Nikon Diaphot inverted microscope (Nikon, Tokyo, Japan). The intracellular BODIPY dye was excited at 490 nm, and emitted uorescence was measured using a 510-nm dichroic mirror. Images were recorded using a 40 3 immersion oil objective and a Coolsnap HQ digital camera (Photometrics, Tucson, AZ), and uorescence measurements were recorded using Meta uor version 7 imaging software (Universal Imaging, Downingtown, PA).

NEFA Uptake in Proximal Tubule Cell Lines

Uptake studies were performed with minor modi cations to pub- lished methods. 57 Cells were cultured to con uence 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 BOD IPY-conjugated dodecanoic acid (QBT fatty acid uptake assay; Molecular Devices, Sunnyvale, CA; nal concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2.5 m M) complexed with 0.2% essential fatty acidfree albumin were perfused into the recording chamber. Excitation l=490-nm pulses were de- livered and emission l =510-nm uorescence was recorded at 20-second intervals until steady-state uptake was achieved. Epi uorescence was recorded using a SPOT-RT camera (Diagnostic Instruments, Sterling Heights, MI), and images were acquired and analyzed using

SimplePCI imaging software (Compix Inc., Cranberry Township, PA). The uptake rate was de ned as the maximum slope within the rst 10 seconds, which was determined using SigmaPlot 2000 software (SPSS, Chicago, IL).

RT-PCR

Total RNAwas 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 Scienti c). Reverse transcription was performed using Super- Script IV VILO Master-mix (Invitrogen Life Technologies) according to the manufacturer s instructions. Each ampli cation reaction was conducted in 25- ml volume using Choice Taq DNA Polymerase (Den- ville Scienti c Inc., Holliston, MA) according to the recommended protocol and PCR cycling conditions. PCR products underwent 1% agarose gel electrophoresis, and bands were identi ed by ethidium bromide staining and photographed. Human FATP2 primer se- quences were 5 9 -GGAGATACATTCCGGTGGAA-3 9 (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 5 9 -TTGTCATGGATGACCTTGGCCAGG-3 9 (reverse); human GAPDH primer sequences were 5 9 -GTCTTCACCACCATGGA- GAAG-3 9 (forward) and 5 9 -GCTTCACCACCTTCTTGATGT- CATC-3 9 (reverse).

Immunohistochemistry

Methods have been described previously in detail. 58 Mouse kidney was frozen at 2 80°C. Samples were sectioned to 5- m m thickness by cryostat, xed 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 m inutes 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 antisera 31 (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 antiNa + /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 redconjugated 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

buffer (1:10 dilution of PBS with protease inhibitor cocktail; Thermo Fisher Scientic). Lysed cells were homogenized, and large particleswere 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 Scienti c; 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 (14,000 3 g , 10 min- utes, 4°C), and supernatants were assayed for protein concentration and stored at 2 80°C. Thawed samples of equal protein concentration were mixed with streptavidin agarose beads (Thermo Fisher Scien- ti c; 20 m l bead volume) with gentle rocking for 3 hours at 4°C and then centrifuged (1000 3 g , 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 Brie y, samples (20 m g protein per lane) were resolved by SDS-PAGE and transferred to polyvinyli- dine di uoride 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 Scienti c; 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 xed in parafor- maldehyde (4%, 10 minutes at room temperature). Post xed 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 uorescence lters.

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

www.jasn.org BASIC RESEARCH

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 identi ed 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 overload 8,40 and LPS 41,42 protocols. Both protocols used 6- to 8-week-old wild-type and Slc27a2 2 / 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 m l 6 3 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 Brie y, studies were conducted by two observers blinded to experimental conditions on images viewed at 40 3 magni cation, which were overlaid with a 16 3 22 grid within Adobe Photoshop (San Jose, CA) or ImageJ to assess for tubular atrophy and interstitial brosis. 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 de ned 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 Quantication

After incubation with palmitic acid (100 mM, 24 hours) complexed with 0.2% albumin, cells were xed with paraformaldehyde (4%, 10

BASIC RESEARCH www.jasn.org

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 quanti cation, 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 Scienti c).

TUNEL Assays

Proximal tubule epithelial cell lines were cultured on coverslips and grown to near con uence. Ten to 12 random elds 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 means 6 SEM. 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 signi cance is de ned 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.

REFERENCES

1. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van Lente F, Levey AS: Prevalence of chronic kidney disease in the United States. JAMA 298: 2038 2047, 2007

2. Tonelli M, Muntner P, Lloyd A, Manns BJ, James MT, Klarenbach S, Quinn RR, Wiebe N, Hemmelgarn BR; Alberta Kidney Disease Network:

Using proteinuria and estimated glomerular ltration rate to classify risk in patients with chronic kidney disease: A cohort study. Ann Intern Med 154: 12 21, 2011

3. Risdon RA, Sloper JC, De Wardener HE: Relationship between renal function and histological changes found in renal-biopsy specimens from patients with persistent glomerular nephritis. Lancet 2: 363 366,

1968

4. Schainuck LI, Striker GE, Cutler RE, Benditt EP: Structural-functional correlations in renal disease. II. The correlations. Hum Pathol 1: 631 641, 1970

5. Bohle A, Mackensen-Haen S, von Gise H: Signi cance of tubulointer- stitial changes in the renal cortex for the excretory function and con- centration ability of the kidney: A morphometric contribution. Am J Nephrol 7: 421 433, 1987

6. Schelling JR, Nkemere N, Kopp JB, Cleveland RP: Fas-dependent fratricidal apoptosis is a mechanism of tubular epithelial cell deletion in chronic renal failure. Lab Invest 78: 813 824, 1998

7. Thomas ME, Harris KPG, Walls J, Furness PN, Brunskill NJ: Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria. Am

J Physiol Renal Physiol 283: F640 F647, 2002

8. Kamijo A, Kimura K, Sugaya T, Yamanouchi M, Hase H, Kaneko T, Hirata Y, Goto A, Fujita T, Omata M: Urinary free fatty acids bound to albumin aggravate tubulointerstitial damage. Kidney Int 62: 1628 1637, 2002

9. van Timmeren MM, Bakker SJ, Stegeman CA, Gans RO, van Goor H:

Addition of oleic acid to delipidated bovine serum albumin aggravates renal damage in experimental protein-overload nephrosis. Nephrol Dial Transplant 20: 2349 2357, 2005

10. Arici M, Chana R, Lewington A, Brown J, Brunskill NJ: Stimulation of proximal tubular cell apoptosis by albumin-bound fatty acids mediated by peroxisome proliferator activated receptor- g . J Am Soc Nephrol 14:

17 27, 2003

11. Arici M, Brown J, Williams M, Harris KP, Walls J, Brunskill NJ: Fatty acids carried on albumin modulate proximal tubular cell bronectin pro- duction: A role for protein kinase C. Nephrol Dial Transplant 17: 1751 1757, 2002

12. Khan S, Abu Jawdeh BG, Goel M, Schilling WP, Parker MD, Puchowicz MA, Yadav SP, Harris RC, El-Meanawy A, Hoshi M, Shinlapawittayatorn K, Deschênes I, Ficker E, Schelling JR: Lipotoxic disruption of NHE1 interaction with PI(4,5)P2 expedites proximal tubule apoptosis. J Clin Invest 124: 1057 1068, 2014

13. Ruggiero C, Elks CM, Kruger C, Cleland E, Addison K, Noland RC, Stadler K: Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis. Am J Physiol Renal Physiol 306: F896F906, 2014

14. Moorhead JF, Chan MK, El-Nahas M, Varghese Z: Lipid nephrotoxicity in chronic progressive glomerular and tubulo-interstitial disease. Lan- cet 2: 1309 1311, 1982

15. Sun L, Halaihel N, Zhang W, Rogers T, Levi M: Role of sterol regulatory element-binding protein 1 in regulation of renal lipid metabolism and glomerulosclerosis in diabetes mellitus. J Biol Chem 277: 18919

18927, 2002

16. Proctor G, Jiang T, Iwahashi M, Wang Z, Li J, Levi M: Regulation of renal fatty acid and cholesterol metabolism, in ammation, and brosis in Akita and OVE26 mice with type 1 diabetes. Diabetes 55: 2502 2509,

2006

17. Kang HM, Ahn SH, Choi P, Ko YA, Han SH, Chinga F, Park AS, Tao J, Sharma K, Pullman J, Bottinger EP, Goldberg IJ, Susztak K: Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney brosis development. Nat Med 21: 37 46, 2015

18. Ruan XZ, Varghese Z, Moorhead JF: An update on the lipid nephro- toxicity hypothesis. Nat Rev Nephrol 5: 713 721, 2009

19. Nieth H, Schollmeyer P: Substrate-utilization of the human kidney. Nature 209: 1244 1245, 1966

20. Guder WG, Wagner S, Wirthensohn G: Metabolic fuels along the nephron: Pathways and intracellular mechanisms of interaction. Kidney Int 29: 41 45, 1986

21. Abumrad N, Harmon C, Ibrahimi A: Membrane transport of long-chain fatty acids: Evidence for a facilitated process. J Lipid Res 39: 2309

2318, 1998

22. McArthur MJ, Atshaves BP, Frolov A, Foxworth WD, Kier AB, Schroeder F: Cellular uptake and intracellular traf cking of long chain fatty acids.

J Lipid Res 40: 1371 1383, 1999

23. Glatz JF, Luiken JJ, Bonen A: Membrane fatty acid transporters as regulators of lipid metabolism: Implications for metabolic disease. Physiol Rev 90: 367 417, 2010

24. Trimble ME: Palmitate transport by rat renal basolateral membrane vesi- cles in the presence of albumin. J Am Soc Nephrol 3: 19201929, 1993

25. Hirsch D, Stahl A, Lodish HF: A family of fatty acid transporters con- served from mycobacterium to man. Proc Natl Acad Sci U S A 95: 8625 8629, 1998

26. Cabral PD, Hong NJ, Hye Khan MA, Ortiz PA, Beierwaltes WH, Imig JD, Garvin JL: Fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Hypertension 63: e68 e73, 2014

27. Anderson CM, Stahl A: SLC27 fatty acid transport proteins. Mol Aspects Med 34: 516 528, 2013

28. Johnson AC, Stahl A, Zager RA: Triglyceride accumulation in injured renal tubular cells: Alterations in both synthetic and catabolic pathways. Kidney Int 67: 2196 2209, 2005

29. Krammer J, Digel M, Ehehalt F, Stremmel W, Füllekrug J, Ehehalt R:

Overexpression of CD36 and acyl-CoA synthetases FATP2, FATP4 and ACSL1 increases fatty acid uptake in human hepatoma cells. Int J Med Sci 8: 599 614, 2011

30. Sandoval A, Fraisl P, Arias-Barrau E, Dirusso CC, Singer D, Sealls W, Black PN: Fatty acid transport and activation and the expression pat- terns of genes involved in fatty acid traf cking. Arch Biochem Biophys 477: 363 371, 2008

31. Falcon A, Doege H, Fluitt A, Tsang B, Watson N, Kay MA, Stahl A: FATP2 is a hepatic fatty acid transporter and peroxisomal very long-chain acyl- CoA synthetase. Am J Physiol Endocrinol Metab 299: E384E393, 2010

32. Melton EM, Cerny RL, Watkins PA, DiRusso CC, Black PN: Human fatty acid transport protein 2a/very long chain acyl-CoA synthetase 1 (FATP2a/Acsvl1) has a preference in mediating the channeling of ex- ogenous n-3 fatty acids into phosphatidylinositol. J Biol Chem 286:

30670 30679, 2011

33. Baines RJ, Chana RS, Hall M, Febbraio M, Kennedy D, Brunskill NJ:

CD36 mediates proximal tubular binding and uptake of albumin and is upregulated in proteinuric nephropathies. Am J Physiol Renal Physiol 303: F1006 F1014, 2012

34. Susztak K, Ciccone E, McCue P, Sharma K, Böttinger EP: Multiple metabolic hits converge on CD36 as novel mediator of tubular epi- thelial apoptosis in diabetic nephropathy. PLoS Med 2: e45, 2005

35. Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, Ellis C, Elshourbagy NA, Goetz AS, Minnick DT, Murdock PR, Sauls HR Jr. , Shabon U, Spinage LD, Strum JC, Szekeres PG, Tan KB, Way JM, Ignar DM, Wilson S, Muir AI: The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids. J Biol Chem 278: 11303 11311, 2003

36. Hirasawa A, Tsumaya K, Awaji T, Katsuma S, Adachi T, YamadaM, Sugimoto Y, Miyazaki S, Tsujimoto G: Free fatty acids regulate gut incretin glucagon- like peptide-1 secretion through GPR120. Nat Med 11: 9094, 2005

37. Chung JJ, Huber TB, Gödel M, Jarad G, Hartleben B, Kwoh C, Keil A, Karpitskiy A, Hu J, Huh CJ, Cella M, Gross RW, Miner JH, Shaw AS:

Albumin-associated free fatty acids induce macropinocytosis in podo- cytes. J Clin Invest 125: 2307 2316, 2015

38. Schaap FG, Hamers L, Van der Vusse GJ, Glatz JF: Molecular cloning of fatty acid-transport protein cDNA from rat. Biochim Biophys Acta 1354:

29 34, 1997

39. Lewis SE, Listenberger LL, Ory DS, Schaffer JE: Membrane topology of the murine fatty acid transport protein 1. J Biol Chem 276: 37042 37050, 2001

40. Eddy AA, Kim H, López-Guisa J, Oda T, Soloway PD: Interstitial brosis in mice with overload proteinuria: De ciency of TIMP-1 is not pro- tective. Kidney Int 58: 618 628, 2000

41. Reiser J, von Gersdorff G, Loos M, Oh J, Asanuma K, Giardino L, Rastaldi MP, Calvaresi N, Watanabe H, Schwarz K, Faul C, Kretzler M, Davidson A, Sugimoto H, Kalluri R, Sharpe AH, Kreidberg JA, Mundel P:

Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest 113: 1390 1397, 2004

42. Yanagida-Asanuma E, Asanuma K, Kim K, Donnelly M, Young Choi H, Hyung Chang J, Suetsugu S, Tomino Y, Takenawa T, Faul C, Mundel P:

Synaptopodin protects against proteinuria by disrupting Cdc42:

IRSp53:Mena signaling complexes in kidney podocytes. Am J Pathol 171: 415 427, 2007

43. Schelling JR: Tubular atrophy in the pathogenesis of chronic kidney disease progression. Pediatr Nephrol 31: 693 706, 2016

www.jasn.org BASIC RESEARCH

www.jasn.org BASIC RESEARCH

44. Black PN, Sandoval A, Arias-Barrau E, DiRusso CC: Targeting the fatty acid transport proteins (FATP) to understand the mechanisms linking fatty acid transport to metabolism. Immunol Endocr Metab Agents Med Chem 9: 11 17, 2009

45. Mashek DG, McKenzie MA, Van Horn CG, Coleman RA: Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells. J Biol Chem 281: 945 950, 2006

46. Riedel MJ, Light PE: Saturated and cis/trans unsaturated acyl CoA esters differentially regulate wild-type and polymorphic beta-cell ATP- sensitive K + channels. Diabetes 54: 2070 2079, 2005

47. Riedel MJ, Baczkó I, Searle GJ, Webster N, Fercho M, Jones L, Lang J, Lytton J, Dyck JR, Light PE: Metabolic regulation of sodium-calcium exchange by intracellular acyl CoAs. EMBO J 25: 4605 4614, 2006

48. Ghiggeri GM, Ginevri F, Candiano G, Oleggini R, Perfumo F, Queirolo C, Gusmano R: Characterization of cationic albumin in minimal change nephropathy. Kidney Int 32: 547 553, 1987

49. Li H, Black PN, Chokshi A, Sandoval-Alvarez A, Vatsyayan R, Sealls W, DiRusso CC: High-throughput screening for fatty acid uptake inhibitors in humanized yeast identi es atypical antipsychotic drugs that cause dyslipidemias. J Lipid Res 49: 230 244, 2008

50. Sandoval A, Chokshi A, Jesch ED, Black PN, Dirusso CC: Identi cation and characterization of small compound inhibitors of human FATP2. Biochem Pharmacol 79: 990 999, 2010

51. Stahl A, Evans JG, Pattel S, Hirsch D, Lodish HF: Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adi- pocytes. Dev Cell 2: 477 488, 2002

52. Trotter PJ, Ho SY, Storch J: Fatty acid uptake by Caco-2 human in- testinal cells. J Lipid Res 37: 336 346, 1996

53. Richieri GV, Kleinfeld AM: Unbound free fatty acid levels in human serum. J Lipid Res 36: 229 240, 1995

54. Heinzer AK, Watkins PA, Lu JF, Kemp S, Moser AB, Li YY, Mihalik S, Powers JM, Smith KD: A very long-chain acyl-CoA synthetase-de cient mouse and its relevance to X-linked adrenoleukodystrophy. Hum Mol Genet 12: 1145 1154, 2003

55. Souza AC, Bocharov AV, Baranova IN, Vishnyakova TG, Huang YG, Wilkins KJ, Hu X, Street JM, Alvarez-Prats A, Mullick AE, Patterson AP, Remaley AT, Eggerman TL, Yuen PS, Star RA: Antagonism of scavenger receptor CD36 by 5A peptide prevents chronic kidney disease pro- gression in mice independent of blood pressure regulation. Kidney Int 89: 809 822, 2016

56. Cechetto JD, Sadacharan SK, Berk PD, Gupta RS: Immunogold localiza- tion of mitochondrial aspartate aminotransferase in mitochondria and on the cell surface in normal rat tissues. Histol Histopathol 17: 353364, 2002

57. Goel M, Schilling WP: Role of TRPC3 channels in ATP-induced Ca 2+ signaling in principal cells of the inner medullary collecting duct. Am J Physiol Renal Physiol 299: F225 F233, 2010

58. Khan S, Lakhe-Reddy S, McCarty JH, Sorenson CM, Sheibani N, Reichardt LF, Kim JH, Wang B, Sedor JR, Schelling JR: Mesangial cell integrin a v b 8 provides glomerular endothelial cell cytoprotection by sequestering TGF- b and regulating PECAM-1. Am J Pathol 178: 609 620, 2011

59. Wu KL, Khan S, Lakhe-Reddy S, Wang L, Jarad G, Miller RT, Konieczkowski M, Brown AM, Sedor JR, Schelling JR: Renal tubular epithelial cell apoptosis is associated with caspase cleavage of the NHE1 Na + /H + exchanger. Am J Physiol Renal Physiol 284: F829 F839,

2003

Present address: Dr. Pablo D. Cabral, Miromatrix Medical, Eden Prairie, Minnesota.

This article contains supplemental materi al online at http://jasn.asnjournals. org/lookup/suppl/doi:10.1681/ ASN.2017030314/-/DCSupplemental.