Kidney Proximal Tubule Lipoapoptosis Is Regulated by Fatty Acid Transporter-2 (FATP2)

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Shenaz Khan,1 Pablo D. Cabral,2 William P. Schilling,1,2 Zachary W. Schmidt,1 Asif N. Uddin,1 Amelia Gingras,1 Sethu M. Madhavan,1 Jeffrey L. Garvin,2 and Jeffrey R. Schelling1

1Department of Medicine, The MetroHealth System and 2Department 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 damaged 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 2 2 and an increase in the proportion of interstitial space in kidneys from wild-type but not Slc27a2 / mice. 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 2/2 2/2 and palmitate-induced apoptosis in microperfused Slc27a2 proximal tubules and Slc27a2 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: 81–91, 2018. doi: https://doi.org/10.1681/ASN.2017030314

Over 26 million people in the United States are af- Significance Statement flicted with CKD,1 and risks for CKD progression and mortality are albuminuria and decreased GFR.2 Albuminuria and tubular atrophy are significant risks Although albuminuria reflects glomerular filtration for CKD progression to ESRD. We have proposed that, fi barrier dysfunction, downstream tubular atrophy in progressive, proteinuric renal diseases, ltration of albumin-bound nonesterified fatty acids (NEFAs) across damaged glomeruli leads to proximal tubule NEFA reabsorption, causing tubular epithelial cell death and Received March 21, 2017. Accepted August 8, 2017. tubular atrophy. Using a variety of approaches, including microperfused tubules, cell culture, and ge- Published online ahead of print. Publication date available at netically manipulated mouse models, welocalized fatty www.jasn.org. acid transporter-2 (FATP2) to the proximal tubule lu- Correspondence: Dr. Jeffrey R. Schelling, Division of Nephrology, minal membrane and showed that FATP2 mediates MetroHealth Medical Center, 2500 MetroHealth Drive, Rammelkamp proximal tubule NEFA uptake and cytotoxicity. We Center for Education and Research, R425, Cleveland, OH 44109- conclude that FATP2 may, therefore, represent a po- 1998. Email: [email protected] tential therapeutic target for the prevention of CKD progression. Copyright © 2018 by the American Society of Nephrology

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intracellular NEFA accumulation and tubular atrophy through a lipotoxicity mechanism.12,14 Our goal was to identify the apical membrane proximal tubule NEFA transporter, so that it could ultimately be exploited as a therapeutic target to prevent CKD progression. 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 kidney.25 Using in vitro, ex vivo,andin vivo approaches, 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 fl buffer prewarmed to 37°C. BODIPY uptake was measured by uorescence 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 Are Absorbed by the Proximal NEFAs in LLC-PK1 maintained on permeable supports (mean6SEM from n=3). 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 7–9 albumin in animal models 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 hypothesized 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, 2 2 2 are taken up by saturable, basolateral membrane transporters which is diminished in Slc27a2+/ and absent in Slc27a2 / 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 2 2 mal tubule NEFA uptake combined with increased NEFA FATP2 staining was detected in Slc27a2 / kidneys (not synthesis15,16 and diminished b-oxidation17 couldleadto shown).Figure3Econfirms FATP2 mRNA expression in

82 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 81–91, 2018 www.jasn.org BASIC RESEARCH

membrane domains.38,39 To screen for FATP2 membrane expression, crude membrane fractions from cultured proximal tubules were probed by immunoblotting. Figure 4A reveals FATP2 expression in LLC-PK1 and HK-2 proximal tubule cell membranes. Immunocytochemistry experiments 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 fi tectable by RT-PCR in LLC-PK1 cells (Figure 3E), perhaps con rm 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 described 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 2 2 for regulation of proximal tubule NEFA reabsorption. wild-type and Slc27a2 / 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- 2 2 be a membrane-associated transporter containing two trans- nal perfusion of wild-type compared with Slc27a2 / tubules. In contrast, basolateral NEFA was diminished compared with apical uptake, and the magnitude and kinetics of basolateral NEFA transport were identical in micro- 2 2 perfused wild-type and Slc27a2 / proximal 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

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Proximal Tubule Luminal FATP2- Mediated NEFA Uptake Is Cytotoxic Accumulation of NEFAs and NEFA metabolites 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 sion.12,14 To directly test whether FATP2 membrane preparations were immunoblotted with anti-FATP2 antibodies (upper 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 2 2 tubule FATP2, wild-type and Slc27a2 / 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 2 2 areas were significantly different between wild-type and blunted in Slc27a2 / mouse proximal tubule cells (Figure 2 2 Slc27a2 / 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. 2 2 wild-type versus Slc27a2 / mice after induction of albuminuria 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 2 2 was significantly attenuated in Slc27a2 / 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 pathophysiologyof 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 albumin molecule that leaks across the damaged glomerular filtration barrier has the capacity to bind up to seven NEFA molecules, it has been suggested that proximal tubule reabsorption and intracellular accumulation of NEFAs are cytotoxic and contribute to tubular atrophy pathogen- 12,14 Figure 5. FATP2 is expressed in proximal tubule apical membranes. Formalin-fixed esis. Using combined in vitro and ex 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

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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 NEFAwas largely mediated by membrane-localized FATP2. Apical NEFA uptake capacity at equilib- riumwassignificantlygreater 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 normal circumstances, NEFA uptake across the basolateral membrane might be targeted to a subcellularcompartmentassociatedwithme- tabolism, whereas damaged glomeruli filter albumin-bound NEFAs, which are transpor- ted by apical FATP2, and then may traffictoa compartment that ultimately initiates apo- Figure 6. Proximal tubule FATP2-regulated NEFA uptake is time- and concentration- ptosis.Thus,disparatecytotoxicityafterapical dependent. (A) BODIPY-NEFA (500 nM NEFA complexed with 0.2% albumin in Hepes versus basolateral NEFA uptake could be ex- 2 2 [10-mM]–buffered saline) uptake after luminal perfusion in wild-type versus Slc27a2 / 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 2 2 albumin in Hepes [10-mM]–buffered saline) uptake after luminal perfusion in wild-type Slc27a2 / compared with wild-type proxi- 2 2 versus Slc27a2 / 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- 2 2 take. Plasma membrane FATPs are coupled type and Slc27a2 / 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 fl (mean from n=4). which accelerate NEFA ux 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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2 2 Figure 7. FATP2 mediates tubulointerstitial injury. Wild-type and Slc27a2 / 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 treatment of hepatosteatosis.49,50 Some candidates have dictate whether albuminuric renal diseases progress or AKI emerged, although enthusiasm has been dampened, because develops after a relapse. With this caveat in mind, our in many of the compounds with the best inhibition profiles (e.g., vivo and in vitro experiments were performed using albumin anesthetics and tricyclic antidepressants) could cause nonspe- that was saturated with palmitate, the most toxic long-chain cific membrane disruption rather than specificinhibitionof 2/2 fatty acid. Slc27a2 mice developed less reduction in tubular FATP2 and at high IC50 values (midmicromolar), which may epithelial cell number and interstitial fibrosis in the albumin be unattainable in blood. However, pharmacodynamics stud- overload model of inducible proteinuria, in which mice ies have not been conducted, and if these drugs are filtered received daily intraperitoneal injections of palmitate-loaded and/or secreted by the kidneys, low doses could result in suf- 2 2 albumin. Furthermore, Slc27a2 / and shRNA-treated prox- ficiently high luminal concentrations to be effective inhibitors imal tubule cell lines were almost completely protected from of proximal tubule FATP2 and lipoapoptosis-dependent tubu- palmitate-induced apoptosis, suggesting that FATP2 plays a lar atrophy. major pathophysiologic role in the apical uptake of filtered There are six FATP isoforms, all of which contain a signature NEFAs that result from glomerular injury. Y/FIY/FTSGTTG AMP binding motif within a cytoplasmic Proximal tubule lipotoxicity is likely to result from a com- loop, and they are predicted to contain two extracellular do- bination of increased uptake, increased synthesis, and de- mains, which form an NEFA binding region after dimeriza- 25 creased catabolism of NEFA. The intracellular pathways are tion. As a group, FATPs are efficient transporters, with Km complex and may not be accessible to small molecule pharma- ranging from 0.3 to 20 mM,30,51,52 which is consistent with our cologic inhibitors, and therefore, they represent less rational observations for proximal tubule NEFA transport. However, therapeutic targets compared with apical FATP2. High- the low- to midmicromolar concentrations used in our exper- throughput screens of small molecule libraries for FATP2 in- iments represent total concentration, whereas circulating free hibitors have been conducted to identify lead molecules for the NEFA concentration is in the low nanomolar range.53 There

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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 accumulation 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 basolateralNEFAuptake,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 that apical FATP2-directed NEFA uptake in shRNA- or scrambled oligonucleotide sequence (control)–transfected HRPT cells and 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 2 2 control (n=3). (G) Proximal tubule cell lines derived from wild-type and Slc27a2 / disease, such as diabetic nephropathy. Using mice were incubated with 100 mM palmitate complexed with 0.2% fatty acid–free in vivo, ex vivo,andin 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 2 2 Apical NEFA uptake in Slc27a2 / 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 2 2 functional NEFA transporters in the proximal tubule. Consid- and 129S-Slc27a2tm1Kds/J (Slc27a2 / )micewerepurchasedfrom 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 2 2 Slc27a2 / cells (Figure 8) and previous observations that 2 2 Human Samples Slc27a2 / 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: 81–91, 2018 Proximal Tubule FATP2 87 BASIC RESEARCH www.jasn.org involving human tissues were performed with approval of the insti- SimplePCI imaging software (Compix Inc., Cranberry Township, tutional review board of the MetroHealth System, Case Western Re- PA).Theuptakeratewasdeﬁned as the maximum slope within serve University. the ﬁrst10seconds,whichwasdeterminedusingSigmaPlot2000 software (SPSS, Chicago, IL). Cell Lines LLC-PK1 and HK-2 cell lines were purchased from ATCC (Manassas, RT-PCR VA); HRPT cells were a gift from Lorraine Racusen (Johns Hopkins Total RNA was extracted from cell lines or mouse kidney cortex using

University). LLC-PK1 and HRPT cells were maintained in DMEM-F12 the RNeasy Mini kit (Qiagen, Hilden, Germany) in accordance with (Invitrogen Life Technologies, Carlsbad, CA) plus 10% FBS (HyClone) the protocol described by the manufacturer. RNAconcentrations were and 1% penicillin/streptomycin-fungizone (Sigma-Aldrich, St. Louis, MO) determined using the NanoDrop 2000 Spectrophotometer (Thermo as described previously.12 HK-2 cells were cultured in Keratinocyte- Fisher Scientific). Reverse transcription was performed using Super- SFMsupplementedwith5ng/mlEGFand40mg/ml bovine pituitary Script IV VILO Master-mix (Invitrogen Life Technologies) according extract (Gibco/Thermo Fisher Scientific, Waltham, MA). to the manufacturer’s instructions. Each amplification reaction was conducted in 25-ml volume using Choice Taq DNA Polymerase (Den- Microperfusion Experiments ville Scientific Inc., Holliston, MA) according to the recommended Microdissected proximal tubules were perfused with fluorescently protocol and PCR cycling conditions. PCR products underwent 1% labeled NEFA using established methods.26 Animals were anesthe- agarose gel electrophoresis, and bands were identified by ethidium tized with ketamine (100 mg/kg body wt intraperitoneally) and xy- bromide staining and photographed. Human FATP2 primer se- lazine (20 mg/kg body wt intraperitoneally). The abdominal cavity quences were 59-GGAGATACATTCCGGTGGAA-39 (forward) and was opened, and the left kidney was superfused twice with ice cold 59-TGATCTCAATGGTGTCCTGT-39 (reverse), yielding a 257-bp am- 150 mM sodium chloride; then, it was removed and placed in phys- plicon. Human CD36 primer sequences were 59-GGAACAGAGGCT- iologic saline at 4°C. Coronal slices were cut, and proximal tubule GACAACTT-39 (forward) and 59-TCGCAGTGACTTTCCCAATAG-39 segments were isolated from the kidney cortex using microforceps (reverse), yielding a 347-bp amplicon. Mouse GAPDH primer se- under a stereomicroscope at 4°C to 10°C. S2 segment tubules ranging quences were 59-CTGCCATTTGCAGTGGCAAAGTGG-39 (forward) from 0.7 to 1.0 mm were transferred to a temperature-regulated and 59-TTGTCATGGATGACCTTGGCCAGG-39 (reverse); human chamber and perfused using concentric glass pipettes at 37°C. GAPDH primer sequences were 59-GTCTTCACCACCATGGA- Tubules were perfused with BODIPY-conjugated 4,4-difuoro-5,7- GAAG-39 (forward) and 59-GCTTCACCACCTTCTTGATGT- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (final CATC-39 (reverse). concentration 2.5 mM; Invitrogen Life Technologies) complexed with 0.2% fatty acid–free albumin in buffer containing 130 mM NaCl, Immunohistochemistry 58 2.5 mM NaH2PO4,4mMKCl,1.2mMMgSO4,6mML-alanine, Methods have been described previously in detail. Mouse kidney 1 mM trisodium citrate, 5.5 mM glucose, 2 mM calcium dilactate, was frozen at 280°C. Samples were sectioned to 5-mm thickness by and 10 mM Hepes, pH 7.4 at 37°C. The fluorescence detection system cryostat, fixed in paraformaldehyde (4%; 10 minutes at room tem- was mounted on a Nikon Diaphot inverted microscope (Nikon, perature), rinsed in PBS, and then permeabilized with Triton X-100 Tokyo, Japan). The intracellular BODIPY dye was excited at 490 (Sigma-Aldrich; 0.2% in PBS, 10 minutes at room temperature). nm, and emitted fluorescence was measured using a 510-nm dichroic Sections were blocked with rabbit or goat serum (5% in PBS, 1 mirror. Images were recorded using a 403 immersion oil objective hour at room temperature) or Mouse on Mouse reagent (Vector, and a Coolsnap HQ digital camera (Photometrics, Tucson, AZ), and Burlingame, CA). Primary antibodies were rabbit anti-FATP2 IgG fluorescence measurements were recorded using Metafluor version 7 (GeneTex, Irvine, CA; 1:50, 16 hours, 4°C), rabbit polyclonal imaging software (Universal Imaging, Downingtown, PA). 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 + NEFA Uptake in Proximal Tubule Cell Lines (BD Pharmingen, San Jose, CA; 1:200, 16 hours, 4°C), mouse anti–Na Uptake studies were performed with minor modifications to pub- /K+-ATPase IgG (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100, 16 lished methods.57 Cells were cultured to confluence on permeable hours, 4°C), or goat anti-Glut5 IgG (Santa Cruz Biotechnology; 1:100, supports or 12-mm coverslips, which were mounted in a temperature- 16 hours, 4°C). Secondary Alexa Fluor 488 and 568 antibodies were used controlled perfusion chamber of a Leica DMIRE2 inverted microscope (Invitrogen Life Technologies; 1:200, 1 hour at room temperature). stage. Solutions containing BODIPY-conjugated dodecanoic acid Proximal tubules were labeled with Texas red–conjugated Tetragonolobus (QBT fatty acid uptake assay; Molecular Devices, Sunnyvale, CA; purpureus lectin as previously described.5 Sections were mounted in final concentrations of 4 nM, 20 nM, 100 nM, 500 nM, and 2.5 mM) Vectashield aqueous mounting media containing DAPI nuclear coun- complexed with 0.2% essential fatty acid–free albumin were perfused terstain (Vector) and viewed by confocal microscopy (Leica, Wetzlar, into the recording chamber. Excitation l=490-nm pulses were de- Germany). livered and emission l=510-nm fluorescence was recorded at 20-second intervals until steady-state uptake was achieved. Epifluorescence Immunoblot Analyses and Immunoprecipitation was recorded using a SPOT-RT camera (Diagnostic Instruments, Lysates were probed from whole cells or crude membrane fractions. Sterling Heights, MI), and images were acquired and analyzed using Crude membranes were harvested by incubation with hypotonic

88 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 81–91, 2018 www.jasn.org BASIC RESEARCH buffer (1:10 dilution of PBS with protease inhibitor cocktail; Thermo previously described methods.12 Oligonucleotides were purchased Fisher Scientific). Lysed cells were homogenized, and large particles were fromtheMissionshRNAGeneSetandclonedintopLKO.1-puro removed by centrifugation at 5003g for 2 minutes. For immunoblots, plasmid (Sigma-Aldrich), and lentiviral particles were produced in the supernatant was centrifuged at 14,0003g for 30 minutes, and the HEK 293T packaging cells using the ViraPower Expression System resulting pellet was suspended in Laemmli buffer (125 mM Tris, pH 6.8, (Invitrogen Life Technologies). The viral supernatant was added to 2% SDS, and 5% glycerol), assayed for protein concentration by DC HRPT monolayers grown on 10-cm plates, and stably transfected protein assay (Bio-Rad, Hercules, CA), and frozen at 280°C. cells were identified for further studies. Three constructs were For biotinylation experiments, PBS-washed cells were incubated screened by immunoblotting for silencing of FATP2 expression: with sulfo-NHS-LC-Biotin (Thermo Fisher Scientific; 2 mM, 5 min- ccggCCATACTTCTTCCAGGACATACTCGAGTATGTCCTGGAA- utes, 4°C), washed again with PBS, and then incubated with glycine GAAGTATGGtttttg (shRNA1), ccggGCTGATTACCTACCTAGT- quenching buffer (0.1 M, 5 minutes, 4°C). Cells were lysed in 1 ml TATCTCGAGATAACTAGGTAGGTAATCAGCtttttg (shRNA2), lysis buffer (25 mM Tris, pH 7.4, 50 mM NaCl, 25 mM NaF, 10% and ccggCCTATGACTGAGGACATCTATCTCGAGATAGATGTCCT- glycerol, and 1% Triton X-100) and centrifuged (14,0003g,10min- CAGTCATAGGtttttg (shRNA3). utes, 4°C), and supernatants were assayed for protein concentration and stored at 280°C. Thawed samples of equal protein concentration In Vivo Induction of Proteinuria were mixed with streptavidin agarose beads (Thermo Fisher Scien- Proteinuria and tubulointerstitial disease were induced by adapted tific; 20 ml bead volume) with gentle rocking for 3 hours at 4°C and protein overload8,40 and LPS41,42 protocols. Both protocols used 6- to 2 2 then centrifuged (10003g, 30 seconds, 4°C). The pelleted beads were 8-week-old wild-type and Slc27a2 / mice on 129S genetic back- then washed with lysis buffer at 4°C. grounds. For the protein overload model, the right and left abdomen Whole-cell, crude membrane, and streptavidin-precipitated sam- were alternately cleansed with isopropanol on consecutive days for in- ples were lysed and denatured in boiling SDS-PAGE buffer (125 mM traperitoneal injections. Mice received daily (Monday through Friday) Tris,pH6.8,2%SDS,5%glycerol,1%b-mercaptoethanol, and sterile injections of endotoxin-free BSA (A-9430; Sigma-Aldrich; 10 mg/g 0.003% bromphenol blue) for 5 minutes. Immunoblot methods body wt) 5 d/wk for 3 weeks. For the LPS model, mice were injected with have been described previously.12 Briefly, samples (20 mgprotein endotoxin-free LPS (L-3137; Sigma-Aldrich; 10 mg per mouse) for 3 per lane) were resolved by SDS-PAGE and transferred to polyvinyli- consecutive days. All mice were euthanized the day after protocol dine difluoride membranes. Blots were blocked in 5% nonfat dried completion. Urine was collected by bladder puncture, and kidneys milk and probed with anti-FATP2 (GeneTex; 1:500, 16 hours, 4°C) or were harvested for quantitative histomorphometry analyses. 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- Urine Protein Electrophoresis tensity was detected by enhanced chemiluminescence. Blots were To determine albumin excretion, urine samples (10 mlperlane+2ml exposed to stripping buffer (Thermo Fisher Scientific; 10 minutes 63 SDS-PAGE buffer) were boiled for 10 minutes, loaded on 10%– at room temperature) and then reprobed with anti–a-tubulin (Santa 20% Tris-glycine gels, and electrophoresed at 150 V. Gels were then Cruz Biotechnology; 1:3000, 1 hour at room temperature), anti– stained overnight in solution containing 0.1% Coomassie Brilliant Na+/K+-ATPase (Santa Cruz Biotechnology; 1:1000, 1 hour at room Blue R-250, 50% methanol, and 70% glacial acetic acid. Gels were temperature), or anti-Glut5 (Santa Cruz Biotechnology; 1:500, 1 hour then destained in 50% methanol + 7% acetic acid solution, and digital at room temperature) IgG followed by HRP-conjugated IgG (1:10,000, images were obtained with a Canon Canoscan LiDE 120 scanner. 1 hour at room temperature). Quantitative Histomorphometry Immunocytochemistry Methods were conducted as previously described.12 Briefly, studies Methods have previously been described in detail.59 Cells were main- were conducted by two observers blinded to experimental conditions tained on sterile glass coverslips within six-well plates, blocked with on images viewed at 403 magnification, which were overlaid with a 5% low-IgG BSA and 0.2% Triton X-100 (Sigma-Aldrich) for 30 16322 grid within Adobe Photoshop (San Jose, CA) or ImageJ to minutes at room temperature, incubated with rabbit anti-FATP2 assess for tubular atrophy and interstitial fibrosis. Ten sections per IgG (1:50, 2 hours at room temperature), and then fixed in parafor- kidney were randomly selected for analysis. Coincidence of intersect- maldehyde (4%, 10 minutes at room temperature). Postfixed cells ing grid lines with tubule (nucleus, cytoplasm, or brush border), were incubated with Alexa Fluor 568 goat anti-rabbit IgG (1:200, 2 tubule lumen, or Masson trichrome–stained interstitium was coun- hours at room temperature). Coverslips were mounted in antifade, ted, whereas glomeruli and blood vessels were omitted from calcula- aqueous media containing DAPI (Vectashield; Vector) on standard tions. The total of the three compartments was defined as 1.0, and microscope slides. Images were viewed using a Leica confocal micro- mean values between the two observers for the proportion of the total scope with appropriate fluorescence filters. composed of tubule cells, tubule lumen, or interstitium were compared between experimental and control (saline-injected) mice. shRNA Transfection To achieve FATP2 knockdown, HRPT cells were transfected with Oil Red O Staining and Quantification lentiviral shRNA vectors containing a puromycin selection cassette After incubation with palmitic acid (100 mM, 24 hours) complexed and targeting human SLC27A2 (Ref-Seq NM_003645.3) according to with 0.2% albumin, cells were fixed with paraformaldehyde (4%, 10

J Am Soc Nephrol 29: 81–91, 2018 Proximal Tubule FATP2 89 BASIC RESEARCH www.jasn.org minutes at room temperature), rinsed with PBS, and stained with the 6. Schelling JR, Nkemere N, Kopp JB, Cleveland RP: Fas-dependent Oil Red O (0.5%; Biovision; 5 minutes at room temperature) as pre- fratricidal apoptosis is a mechanism of tubular epithelial cell deletion in – viously described.12 Stained cells were rinsed with PBS and viewed chronic renal failure. Lab Invest 78: 813 824, 1998 fi 7. Thomas ME, Harris KPG, Walls J, Furness PN, Brunskill NJ: Fatty acids with a Leica Dmi8 inverted light microscope. For quanti cation, Oil exacerbate tubulointerstitial injury in protein-overload proteinuria. Am Red O was incubated for 30 minutes and washed with PBS and then J Physiol Renal Physiol 283: F640–F647, 2002 60% isopropanol. Oil Red O was eluted with isopropanol (100%, 10 8. Kamijo A, Kimura K, Sugaya T, Yamanouchi M, Hase H, Kaneko T, Hirata minutes at room temperature) followed by eluate absorbance mea- Y, Goto A, Fujita T, Omata M: Urinary free fatty acids bound to albumin – surement at 492 nm with a NanoDrop 2000 spectrophotometer aggravate tubulointerstitial damage. Kidney Int 62: 1628 1637, 2002 fi 9. van Timmeren MM, Bakker SJ, Stegeman CA, Gans RO, van Goor H: (Thermo Fisher Scienti c). Addition of oleic acid to delipidated bovine serum albumin aggravates renal damage in experimental protein-overload nephrosis. Nephrol Dial Transplant 20: 2349–2357, 2005 TUNEL Assays 10. Arici M, Chana R, Lewington A, Brown J, Brunskill NJ: Stimulation of Proximal tubule epithelial cell lines were cultured on coverslips and proximal tubular cell apoptosis by albumin-bound fatty acids mediated fl fi grown to near con uence. Ten to 12 random elds per coverslip were by peroxisome proliferator activated receptor-g. JAmSocNephrol14: assayed for apoptosis, which was assessed by TUNEL as previously 17–27, 2003 described.59 Nuclei of all cells were counterstained with DAPI, and 11. Arici M, Brown J, Williams M, Harris KP, Walls J, Brunskill NJ: Fatty acids fi data are presented as percentage of apoptotic cells. carried on albumin modulate proximal tubular cell bronectin pro- duction: A role for protein kinase C. Nephrol Dial Transplant 17: 1751– 1757, 2002 Statistical Analyses 12. Khan S, Abu Jawdeh BG, Goel M, Schilling WP, Parker MD, Puchowicz Unless otherwise noted, all results are expressed as means6SEM. MA, Yadav SP, Harris RC, El-Meanawy A, Hoshi M, Shinlapawittayatorn Two-tailed paired t tests were used for statistical analysis between K, Deschênes I, Ficker E, Schelling JR: Lipotoxic disruption of NHE1 two groups. Two-way ANOVA was used for comparisons between interaction with PI(4,5)P2 expedites proximal tubule apoptosis. JClin Invest 124: 1057–1068, 2014 more than two groups. Statistical significance is defined as P#0.05. 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: F896–F906, 2014 ACKNOWLEDGMENTS 14. Moorhead JF, Chan MK, El-Nahas M, Varghese Z: Lipid nephrotoxicity in chronic progressive glomerular and tubulo-interstitial disease. Lan- Fatty acid transporter-2 antibodies were a gift from Dr. Andreas Stahl cet 2: 1309–1311, 1982 (University of California at Berkeley). 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 This work was supported by National Institutes of Health grants glomerulosclerosis in diabetes mellitus. JBiolChem277: 18919– R01 HL128053 (to J.L.G.) and R01 DK067528 (to J.R.S.). 18927, 2002 16. Proctor G, Jiang T, Iwahashi M, Wang Z, Li J, Levi M: Regulation of renal fatty acid and cholesterol metabolism, inflammation, and fibrosis in Akita and OVE26 mice with type 1 diabetes. Diabetes 55: 2502–2509, DISCLOSURES 2006 None. 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 fibrosis development. Nat Med 21: 37–46, 2015 REFERENCES 18. Ruan XZ, Varghese Z, Moorhead JF: An update on the lipid nephrotoxicity hypothesis. Nat Rev Nephrol 5: 713–721, 2009 1. Coresh J, Selvin E, Stevens LA, Manzi J, Kusek JW, Eggers P, Van Lente 19. Nieth H, Schollmeyer P: Substrate-utilization of the human kidney. F, Levey AS: Prevalence of chronic kidney disease in the United States. Nature 209: 1244–1245, 1966 JAMA 298: 2038–2047, 2007 20. Guder WG, Wagner S, Wirthensohn G: Metabolic fuels along the 2. Tonelli M, Muntner P, Lloyd A, Manns BJ, James MT, Klarenbach S, nephron: Pathways and intracellular mechanisms of interaction. Kidney Quinn RR, Wiebe N, Hemmelgarn BR; Alberta Kidney Disease Network: Int 29: 41–45, 1986 Using proteinuria and estimated glomerular filtration rate to classify risk 21. Abumrad N, Harmon C, Ibrahimi A: Membrane transport of long-chain in patients with chronic kidney disease: A cohort study. Ann Intern Med fatty acids: Evidence for a facilitated process. J Lipid Res 39: 2309– 154: 12–21, 2011 2318, 1998 3. Risdon RA, Sloper JC, De Wardener HE: Relationship between renal 22. McArthur MJ, Atshaves BP, Frolov A, Foxworth WD, Kier AB, Schroeder function and histological changes found in renal-biopsy specimens F: Cellular uptake and intracellular trafficking of long chain fatty acids. from patients with persistent glomerular nephritis. Lancet 2: 363–366, J Lipid Res 40: 1371–1383, 1999 1968 23. 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Schelling JR: Tubular atrophy in the pathogenesis of chronic kidney This article contains supplemental material online at http://jasn.asnjournals. disease progression. Pediatr Nephrol 31: 693–706, 2016 org/lookup/suppl/doi:10.1681/ASN.2017030314/-/DCSupplemental.

J Am Soc Nephrol 29: 81–91, 2018 Proximal Tubule FATP2 91 CD36 Glut5 DAPI Merge

CD36 Glut5 DAPI

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Figure S1. CD36 expression in mouse kidney. Immunohistochemical CD36 co-localization with Glut5 and DAPI in wild-type mouse kidney. FFA1 Glut5 FFA1 Glut5 DAPI Merge DAPI

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Figure S2. FFA1 expression in mouse kidney. Immunohistochemical FFA1 co-localization with Glut5 and DAPI in wild-type mouse kidney. A B C D

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Figure S3. FATP2 is expressed in mouse proximal tubule apical membranes. Frozen sections from wild-type mouse kidneys were lightly fixed (4% paraformaldehyde, 2 mins, room temp) and probed with anti-Glut5 (B) or anti-Na+/K+-ATPase (F) and Alexa Fluor 488 secondary antibodies. Samples were probed with anti-FATP2 and Alexa Fluor 568-labeled secondary antibodies (A, E). Nuclei were labeled with DAPI (C) in the mounting medium. Images were merged and colocalization is depicted in yellow (D and G). A Time (hrs) 0 6 20 28 48 72 144 MW - 82 - 64 LPS dose 10 5 10 5 10 5 10 5 10 5 10 5 10 5 (mg/g wt)

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Figure S4. FATP2 regulates tubulointerstitial injury. Wild type and Slc27a2-/- mice were treated with three daily intraperitoneal endotoxin-free lipopolysaccharide (LPS) injections (10 mg/g) to induce tubular atrophy and interstitial fibrosis, as described in Methods. Coomassie blue-stained gel demonstrating albuminuria after administration of a single LPS dose (A). Mice were sacrificed and evaluated for tubulointerstitial injury, using previously described quantitative histomorphometry methods. Fraction of (B) tubular epithelial cells, (C) interstitium, and (D) tubular lumen within the tubulointerstitial compartment. Histogram data represent mean from N=2 per group. Palm + + BSA + + + + 15 * 10 *

% apoptosis

0 HK-2 LLC-PK1

Figure S5. HK-2 or LLC-PK1 proximal tubule cell lines were incubated with 100 µM palmitate complexed with 0.2% fatty acid-free albumin (Palm) or albumin only control (BSA) for 24 hrs and then assayed for apoptosis by TUNEL (N=3, *P <0.05 compared to BSA-treated control). 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,including microperfusedtubules,cellculture,and genetically manipulated mouse models, we localized fatty acid transporter-2 (FATP2) to the proximal tubule luminal membrane and showed that FATP2 mediates proximal tubule NEFA uptake and cytotoxicity. We conclude that FATP2 may, therefore, represent a potential therapeutic target for the prevention of CKD progression.