Steroidogenic acute regulatory-related lipid transfer domain protein 5 localization and regulation...

Steroidogenic acute regulatory-related lipid transfer domain protein 5localization and regulation in renal tubules

Yu-Chyu Chen,1 Renate K. Meier,1 Shirong Zheng,2 Syed J. Khundmiri,3 Michael T. Tseng,4

Eleanor D. Lederer,3,5,6 Paul N. Epstein,2,5 and Barbara J. Clark1,5

Departments of 1Biochemistry and Molecular Biology, 2Pediatrics, 3Medicine, and 4Anatomical Sciences and Neurobiology,5Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville; and 6Veterans AffairsMedical Center, Louisville, Kentucky

Submitted 22 July 2008; accepted in final form 25 May 2009

Chen Y-C, Meier RK, Zheng S, Khundmiri SJ, Tseng MT,Lederer ED, Epstein PN, Clark BJ. Steroidogenic acute regulatory-related lipid transfer domain protein 5 localization and regulation inrenal tubules. Am J Physiol Renal Physiol 297: F380–F388, 2009.First published May 27, 2009; doi:10.1152/ajprenal.90433.2008.—STARD5 is a cytosolic sterol transport protein that is predominantlyexpressed in liver and kidney. This study provides the first report onSTARD5 protein expression and distribution in mouse kidney. Im-munohistochemical analysis of C57BL/6J mouse kidney sectionsrevealed that STARD5 is expressed in tubular cells within the renalcortex and medullar regions with no detectable staining within theglomeruli. Within the epithelial cells of proximal renal tubules,STARD5 is present in the cytoplasm with high staining intensityalong the apical brush-border membrane. Transmission electronmi-croscopy of a renal proximal tubule revealed STARD5 is abundant atthe basal domain of the microvilli and localizes mainly in the roughendoplasmic reticulum (ER) with undetectable staining in the Golgiapparatus and mitochondria. Confocal microscopy of STARD5 dis-tribution in HK-2 human proximal tubule cells showed a diffusepunctuate pattern that is distinct from the early endosome markerEEA1 but similar to the ER membrane marker GRP78. Treatment ofHK-2 cells with inducers of ER stress increased STARD5 mRNAexpression and resulted in redistribution of STARD5 protein to theperinuclear and cell periphery regions. Since recent reports showelevated ER stress response gene expression and increased lipid levelsin kidneys from diabetic rodent models, we tested STARD5 andcholesterol levels in kidneys from the OVE26 type I diabetic mousemodel. Stard5 mRNA and protein levels are increased 2.8- and1.5-fold, respectively, in OVE26 diabetic kidneys relative to FVBcontrol kidneys. Renal free cholesterol levels are 44% elevated in theOVE26 mice. Together, our data support STARD5 functioning inkidney, specifically within proximal tubule cells, and suggest a role inER-associated cholesterol transport.

START; cholesterol transport; diabetic nephropathy; ER stress; prox-imal tubules

STARD5 BELONGS to the StAR-related lipid-transfer (START)domain protein superfamily that is composed of known orpredicted lipid-binding proteins. This family is defined by thepresence of a START domain of �200 amino acids that sharesa three-dimensional helix-grip fold structure that is conservedfrom plants to humans (11, 22). STARD5 is a member of theSTARD4 subfamily and analysis of STARD4 and STARD5deduced amino acid sequences predicts �22-kDa soluble pro-teins composed entirely of the START domain and lacking anymembrane or organellar targeting sequence (25, 29). The liver

and kidney are the major mouse tissues that express Stard4 andStard5 mRNA (29).

Recent reports on STARD5 expression and regulation inliver showed that STARD5 is localized to the Kupffer cells,i.e., macrophages within the reticuloendothelial system of theliver, and not hepatocytes (24). STARD5 is also expressed incell lines derived from human macrophages and monocytes aswell as mast cells, lymphoblasts, and promyeloblasts (24).Double immunofluorescence studies in human THP-1 macro-phages revealed STARD5 was localized to the perinuclearregions of the cell and colocalized with Golgi but not endo-some markers (24). In addition, overlapping the immunofluo-rescent images of STARD5 with filipin, a polyene antibioticforming fluorescent complex with cholesterol, demonstratedcolocalization of STARD5 with membranes enriched in freecholesterol. STARD5 binds cholesterol and 25-hydroxycholes-terol (25, 29). Agents that promote endoplasmic reticulum(ER) stress, such as thapsigargin-treated NIH-3T3 cells orcholesterol-loaded mouse macrophages, promote an increase inStard5 mRNA expression (28). These data suggest a role forSTARD5 as a cytosolic sterol transporter that shuttles choles-terol between intracellular membranes; e.g., from the cyto-plasm to the ER and/or from the ER to Golgi (24, 30).

Stard5 was originally detected at the mRNA level in mousekidney (29), but no studies to date have characterized STARD5protein expression, regulation, or function in this tissue. There-fore, one focus of this study was to examine STARD5 proteindistribution and regulation in mouse kidney. Second, recentreports showed that kidneys of type I diabetic rodent modelshave increased ER stress, increased expression levels of renalfatty acid and cholesterol biosynthetic enzymes, and elevatedcholesterol metabolite levels (18, 23, 36). Therefore, we rea-soned that STARD5 may be elevated in type I diabetic kidney,a tissue with potential for elevated cholesterol and ER stressconditions. We tested this hypothesis using the OVE26 trans-genic mouse that we previously established as model for earlyonset type I diabetes mellitus (5). OVE26 mice develop dia-betes within the first weeks of birth due to pancreatic �-celldestruction as a result of cell-specific overexpression of cal-modulin. Specifically, compared with FVB parental mouseline, the OVE26 mice display hallmarks of diabetic nephrop-athy (DN) including hyperglycemia, hypoinsulinemia, hyper-triglycerolemia, and macroalbuminuria, along with increasedkidney weight, enlarged glomeruli, and expanded mesangialmatrix with thickening of the basement membrane (5, 33, 40).By day 20, serum glucose levels are twofold greater in OVE26than FVB control counterparts (170 � 10 mg/dl for FVB vs.373 � 40 mg/dl for OVE26) with a threefold increase in serum

Address for reprint requests and other correspondence: B. J. Clark, Dept. ofBiochemistry and Molecular Biology, Univ. of Louisville, Louisville, KY40202 (e-mail: bjclark@louisville.edu).

Am J Physiol Renal Physiol 297: F380–F388, 2009.First published May 27, 2009; doi:10.1152/ajprenal.90433.2008.

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triglyceride levels by 3 mo of age. OVE26 mice also displayurine albumin levels greater than 15 mg/day, which is at least10-fold higher than any other diabetic mouse model (40). Thismakes them the closest mouse model to human DN withrespect to this critical feature of DN.

We demonstrate herein that STARD5 has a cytosolic distri-bution in normal kidney with enhanced association with the ERand apical membranes of renal proximal tubules. Induction ofER stress in a human proximal tubule cell line (HK-2) in-creases STARD5 mRNA expression and promotes cellularredistribution of the protein. Using OVE26 diabetic mousekidneys as model for renal hyperlipidemia, we show STARD5expression levels are elevated relative to the kidneys of FVBcontrol mice. We add new information to the diabetic profile ofthe OVE26 mouse model of early onset type I diabetes mellitusby reporting that OVE26 kidneys have elevated free choles-terol levels. Together, our data support a role for STARD5 inrenal proximal tubules and suggest STARD5 is part of thecellular stress response, possibly to help manage increasedlipid load, such as may occur in diabetic mouse kidney.

MATERIALS AND METHODS

Materials. Avidin/Biotin Blocking kit, Vectastain ABC Elite re-agent, Vector M.O.M Immunodetection kit (PK-2200), diaminoben-zidine tetrahydrochloride (DAB), and hematoxylin eosin were pur-chased from Vector Laboratories (Burlingame, CA). Permount resinand glass slides were from Fisher Scientific (Hanover Park, IL) andthe Lysing matrix D was from Q-BIOgene (Carlsbad, CA). RNeasykits were purchased from Qiagen (Valencia, CA). M-MLV RT,TaqMan probe, and SYBR Green PCR Core kit were fromPerkinElmer Applied Biosystems (Foster City, CA). Araldite 502 kitand propylene oxide were purchased from Ted Pella (Ridding, CA).Glutaraldehyde, cacodylate, and osmium tetroxide were obtainedfrom Electron Microscopy Science (Hatfield, PA). Amplex Red Cho-lesterol Assay Kit, biotinylated goat anti-rabbit IgG, and nonimmunegoat serum were purchased from Invitrogen (Carlsbad, CA). STARD5antibodies and a polyclonal rabbit anti-human were generous giftsfrom Dr. J. A. Breslow (Rockefeller University, New York, NY) (10,28). GST-STARD5 antigen was from ProteinTech (Chicago, IL).Horseradish peroxidase (HRP)-donkey anti-rabbit secondary antibodywas purchased from Amersham Biosciences (Piscataway, NJ). Mono-clonal mouse anti-rabbit GAPDH antibody was obtained from RDI(Concord, MA). NHERF-1 antibody (rabbit anti-mouse NHERF-1)was a gift from Dr. E. Weinman (Department of Medicine, Universityof Maryland) (14). Cholesteryl-linoleate, -oleate, -palmitate, -arachid-onate, and -sterate were purchased from Sigma (St. Louis, MO).

Animal management. Adult male C57BL/6J kidney sections were agenerous gift from Dr. K. Ramos (Dept. of Biochemistry and Molec-ular Biology, University of Louisville). Diabetic OVE26 transgenicmice were created as previously described (5). Their ability to produceand secrete insulin is impaired due to pancreatic beta cell-specificoverexpression of calmodulin. OVE26 mice were created on the FVBbackground and the line is maintained in the Animal Facility ofUniversity of Louisville by breeding OVE26-positive males to FVBfemales. This eliminates potential effects of a diabetic pregnancy.

All animals were housed in groups under a 12:12-h light-dark cyclewith constant access to laboratory chow and tap water. Kidneys wereisolated from 120-day-old male mice and processed according tomethods as below. All animal procedures were approved by theInstitutional Animal Care and Use Committee in agreement withNational Institutes of Health Assurance of Compliance with PublicHealth Service Policy on Humane Care and Use of LaboratoryAnimals.

Immunohistochemistry. C57BL/6J, FVB, and OVE26 mice werekilled by injection with pentobarbital sodium and their kidneys wereisolated and fixed in 10% neutral-buffered formalin at room temper-ature overnight and then embedded in paraffin. Immunohistochemicalstaining for STARD5 was performed by using 5-�m-thick sections ofkidney that were mounted on glass slides. The kidney sections weredeparaffinized, rehydrated, and the endogenous peroxidase activitywas blocked by incubation with 0.5% H2O2 in methanol for 30 min.The sections were then blocked by incubation with Avidin/BiotinBlocking kit for 30 min and followed by incubation with anti-STARD5 antibody (1:500 dilution) in PBS containing 0.3% TritonX-100 and 5% goat serum in a humidified chamber at 4°C overnight.The sections were rinsed in PBS containing 0.03% Tween 20, andincubated with a biotinylated goat anti-rabbit IgG (Molecular Probes;1:1,000 dilution) for 1 h, and then antibody complexes were detectedusing DAB as substrate with Vectastain ABC Elite reagent. To labelproximal tubules, we used peroxide-labeled Lotus tetragonolobusagglutinin (LTA; 1:500 dilution, Sigma), a lectin that specificallybinds to proximal tubules (17, 31). The sections were counterstainedwith hematoxylin eosin, dehydrated in graded concentration of alco-hol, and mounted with Permount resin. The slides were examinedusing a Zeiss HAL100 inverted microscope equipped with Zeissdigital camera and AxioVision Rel 4.3 software. Multiple kidneysections from three C57BL/6J and two animals each of FVB andOVE26 mice were examined.

Immunoelectronmicroscopy. C57BL/6J mice were anesthetized byinjection with pentobarbital sodium and tissues were fixed by wholebody perfusion with 2.5% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) injected through the left ventricle of the heart. Kidneys wereremoved, dissected, and immersed in the fixation solution overnight.Sections of 20 �m were cut in a vibratome and stained as describedabove for immunohistochemistry. In brief, sections were blocked byincubation with 5% nonimmune goat serum for 20 min at 25°C andthen incubated with anti-STARD5 antibody (diluted 1:500) at 4°Covernight followed by incubation with a biotinylated goat anti-rabbitIgG. STARD5 was visualized using DAB. With the aid of dissectingmicroscope, 3-mm3 cubes were dissected from the stained regions,dehydrated in graded ethanol, and embedded in Araldite 502 resin.Thin sections were cut with a diamond knife in an ultramicrotome andwere mounted on nickel grids. Nonstained sections were examined bya Philips CM10 electron microscope operated at 60 kV.

Confocal microscopy. The HK-2 human kidney proximal tubulecells were maintained at 37°C in a humidified atmosphere with 5%CO2 in DMEM/F12 medium supplemented with 10% (vol/vol) fetalbovine serum, 100 U/ml penicillin, and 100 �g/ml streptomycin. Thecells were seeded onto multichambered coverglass wells (Nunc,Naperville, CT) and grown to confluency before treatment withserum-free DMEM/F12 medium containing either 0.2% DMSO or 2.5�g/ml tunicamycin for 24 h. Confocal microscopy was performedfollowing our previously established protocol (12–14). In brief, thecells were washed with PBS, fixed by incubation in 4% paraformal-dehyde for 10 min, washed with PBS three times, and then incubatedin PBS containing 0.05% saponin for 15 min. To detect STARD5, thesaponin-permeabilized cells were incubated with rabbit �-STARD5(diluted 1:500 in PBS/saponin) for 1 h at room temperature, rinsedfive times with PBS-saponin, and incubated with either Alexa Fluor488 or Alexa Fluor 555 secondary antibody (goat �-rabbit, 1:1,000dilution in PBS-saponin) for 30 min at room temperature. The cellswere then washed three times with PBS-saponin, one time with PBS,and incubated with 300 nM DAPI in PBS for 5 min to stain the nuclei.For colocalization with GRP78 or EEA1, the cells were processed forSTARD5-Alexa Fluor 555 as outlined above except that following thesecond set of PBS-saponin rinses, the cells were incubated with eithermouse �-human GRP78 (sc1050, Santa Cruz Biotechnology) ormouse-�-human EEA1 antibodies (BD Biosciences) at 1:500 dilu-tions in PBS-saponin for 1 h at room temperature. The cells wererinsed with PBS-saponin and incubated with a goat-�-mouse Alexa

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Fluor 488 secondary antibody and processed for DAPI staining asoutlined above. Images were captured using a Zeiss Axiovert 100 Mconfocal microscope with a �63 water-immersion objective lens andanalysis was performed using Zeiss LSM510 software.

Quantitative real-time PCR. Total RNA was isolated from wholekidney tissue by homogenization using FastPrep 110 (Savant, Hol-brook, NY) with Lysing matrix D with RNeasy kit. Reverse transcrip-tion (RT) was performed by incubating 1 �g of total RNA withrandom hexamers, dNTPs, and M-MLV reverse transcriptase at 37°Cfor 1 h. Thirty nanograms of the resulting cDNA were mixed with 300nM primers, which were designed for mouse Stard5 gene (sense5�-CTCAAAGGACACGGAAGAGC-3� and anti-sense AAGAC-CCACACAGGGACAAG) and SYBR GREEN PCR Core Reagentaccording to the manufacturer’s protocol. TaqMan human 18 S rRNAprobe and primers were included in the reaction as a normalizationcontrol. Reactions were performed at the condition of 94°C for 30 s,60°C for 20 s, 72°C for 30 s for a total of 45 cycles using an ABIPrism 7700 Sequence Detector (PerkinElmer Applied Biosystems).Reactions were performed in duplicate, and the average thresholdcycle (CT) was used in subsequent calculations to determine theStard5 mRNA levels in OVE26 kidney (diabetic) relative to FVB(control) using the ��CT method (Applied Biosystem). In brief, a�CT value was determined using 18S and ��CT values were deter-mined by selecting a single �CT value from the control group to useas the calibrator for all samples. Thus, the error bars in the controlgroup represent the interanimal variation in target gene expression.

RNA was isolated from HK-2 cells by TRIzol extraction andethanol precipitation. RT was performed as described above and 30 ngcDNA were used for qPCR using Assay-on-Demand TaqMan GeneSystem (ABI Biosystems) for human STARD5 and 18S rRNA, re-spectively. Treatments were in duplicate and qPCR in triplicate foreach experiment and ��CT method was used with to determine therelative expression of target gene expression as described for thekidney samples. The data were calibrated to the DMSO samples foreach experiment and mean values � SE were determined.

Western blot analysis. Kidney tissues (30 mg) from 120-day-oldFVB (n 4) and OVE26 (n 4) mice were homogenized in RIPAlysis buffer using a FastPrep 110 with Lysing matrix D (Qbiogene).The resultant homogenate was centrifuged at 12,000 g for 5 min at4°C and the supernatant was recovered as the lysate. Equivalentprotein (30 �g) of the lysate was resolved by electrophoresis on a 12%SDS-PAGE gel and transferred to Bio-Rad PVDF membrane. Mem-branes were incubated with a primary antibody against humanSTARD5 (1:2,000) followed by incubation with a HRP-donkey anti-rabbit secondary antibody (1:3,000). Specific signals were visualizedby chemiluminescence (Western Lighting, Amersham Biosciences) orSuperSignal West Pico (Pierce Biotechnology, Rockland, IL) andKodak BioMax Light film exposure. The membranes were strippedand reprobed with anti-GAPDH (1:2,000; Fig. 1) or reprobed forNHERF-1 (1:3,000) followed by mouse-anti-�-actin (1:5,000). Thespecific immunoreactive protein bands for STARD5, NHERF-1, and�-actin were digitized and quantified by UN-SCAN-IT gels software(Silk Science, Orem, UT). Multiple exposures were used to capturesignals within the linear range of the film and STARD5 and NHERF-1expression levels were normalized to �-actin within each film.

Cholesterol measurement. Kidney tissues (30 mg) from 120-day-old FVB (n 4) and OVE26 (n 4) mice were homogenized in 10mM Tris �HCl (pH 7.4), 250 mM sucrose, 0.1 mM EDTA usingFastPrep 110 with Lysing matrix D. Total lipid was extracted from thekidney lysate using chloroform:methanol:water (1:2:1 vol:vol:vol)following the method of Bligh and Dyer (1). The organic extract wasdried under nitrogen and the lipids were resuspended in the reactionbuffer and total and free cholesterol levels were measured simulta-neously using Amplex Red Cholesterol Assay Kit following manu-facturer’s instructions. In brief, the lipid extract was incubated withthe reaction buffer in the absence or presence of cholesterol esteraseto determine free and total cholesterol levels, respectively. Cholesterol

ester levels were calculated by subtraction of free cholesterol fromtotal cholesterol values. The assay was standardized by using li-noleate-, oleate-, palmitate-, arachidonate-, and sterate-esters of cho-lesterol as substrates to validate that the cholesterol values werenondetectable in the absence of cholesterol esterase. All data wereexpressed as micrograms of cholesterol per milligram of lysate.

Statistical analyses. Statistical tests were performed using Graph-Pad Prism software (GraphPad Software, San Diego, CA) and astatistically significant difference between the control and treatedsamples was determined using an unpaired Student’s t-test and Mann-Whitney post hoc test. P 0.05 was considered significant.

RESULTS

Localization of STARD5 in mouse kidney. A 22-kDa STARD5-immunospecific band was detected in whole cell lysates isolatedfrom mouse kidneys, demonstrating for the first time STARD5protein expression in kidney (Fig. 1A). The STARD5-specificsignal was present in cell lysate from mouse bone marrow-derived macrophages as expected. Antibody specificity wasverified by the absence of a signal then the STARD5 antibodywas preincubated with GST-STARD5 antigen (Fig. 1B). Im-munohistochemical staining of C57BL/6J mouse kidney sec-tions demonstrated that STARD5 protein was expressed in therenal tubules within the cortex and outer medulla while it wasundetectable in the glomeruli (Fig. 2A). STARD5 was alsodetected along the transitional epithelium lining the lumen ofrenal pelvis (Fig. 2E).

Prominent staining for STARD5 was observed at the cor-tico-medullary region, and higher magnification of this regionrevealed a diffuse cytoplasmic distribution with intense stain-ing for STARD5 along the apical membrane of the proximaltubules (Fig. 2, A and B). STARD5 staining pattern was similar

Fig. 1. STARD5 protein is detected in mouse kidney. A: STARD5 expressionin equivalent amount of protein lysate isolated from mouse bone marrow-derived macrophages (M) and C57BL/6J mouse kidney (K) tissue was detectedby Western blot analysis. The membrane was stripped and reprobed forGAPDH expression (bottom). Shown is a Western blot representative of dataobtained using 3 independent samples. B: kidney lysate samples were run induplicate and one membrane was incubated with STARD5 antibody (top) andthe second with STARD5 antibody preincubated with GST-STARD5 antigen[blocking peptide (BP)].

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to that for LTA (Fig. 2, C and D), a lectin that binds specifi-cally to proximal tubules (17, 31).

Immunoelectronmicroscopy analysis of STARD5 subcellu-lar distribution in proximal renal tubular epithelium showedSTARD5-positive staining within the cytoplasm from the api-cal membrane to the basolateral domain (Fig. 3A). Closerexamination of membrane structures indicated STARD5 wasabundant at the brush-border membrane, associated mainlywith the basal region of the microvilli and associated withinvaginating cell surface regions of the brush-border mem-brane (Fig. 3B), regions that are associated with endocytosis (8,9, 19). These data are consistent with the strong apical stainingof the proximal tubules in mouse kidney observed by immu-nohistochemisty (Fig. 2). In the cell interior, the electron denseparticles for STARD5 protein were mainly localized on therough ER with undetectable staining in the mitochondria orGolgi apparatus (Fig. 3, C and D, respectively).

Effect of ER stress on STARD5 expression and localizationin a human proximal tubule cell line. Two agents that arecommonly used to induce ER stress and trigger the unfoldedprotein response (UPR) are tunicamycin and thapsigargin.Tunicamycin inhibits N-glycosylation of proteins thereby pro-

moting protein retention in the ER while thapsigargin inhibitsthe ER Ca2�-ATPase resulting in increased cytosolic calcium.Both tunicamycin and thapsigargin activate GRP78 expressionand the UPR (27). GRP78 mRNA levels were increased 15-and 14-fold in tunicamycin- and thapsigargin-treated HK-2cells, respectively, indicating an intact UPR (Fig. 4). STARD5mRNA levels were also significantly increased (�3.5-fold)after tunicamycin or thapsigargin treatment.

STARD5 localization in DMSO- (control) and tunicamycin-treated HK-2 human proximal tubule cells was assessed byimmunofluorescence confocal microscopy. STARD5 was de-tected using an Alexa 488-conjugated secondary antibody andnuclei stained with DAPI (Fig. 5A). In control cells a punctatepattern of fluorescence was detected throughout the cell, sug-gesting potential STARD5 association with membrane-boundorganelles (Fig. 5A, Cont). In tunicamycin-treated cellsSTARD5 had a prominent perinuclear distribution and en-hanced staining at the cell periphery, indicating a cellularredistribution of STARD5 (Fig. 5A, Tuni).

Next, double immunofluorescence confocal microscopy wasused to test the potential association of STARD5 with endo-somal vesicles and ER membrane. As shown in Fig. 5B, the

Fig. 2. Immunohistochemical detection of STARD5 distribu-tion in mouse kidney. Kidneys from 3 C57BL/6J mice wereprepared and processed for immunohistochemical analyses forSTARD5 (A, B, E) and Lotus tetragonolobus agglutinin (LTA; C,D), as described in MATERIALS AND METHODS. A and C: represen-tative images for each target as indicated from a minimum of 3separate sections and 3 fields per section. B and D: magnifiedimages of the boxed regions indicated in the corresponding toppanel. The arrows denote STARD5 and LTA staining is similarfor proximal tubules. E: representative image of STARD5 immu-nodetection in the renal pelvis. F: background control for goatserum. *, Glomerulus.

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early endosome marker EEA1 had a strong perinuclear stainingin control HK-2 cells and no significant overlap with STARD5was observed (EEA1 Cont merged images). GRP78 stainingfor ER membrane was dispersed throughout the cells andcolocalization with STARD5 was detected (Fig. 5B, GRP78Cont merged images). In tunicamycin-treated cells, EEA1staining was dispersed throughout the cells while retaining astrong perinuclear distribution. Colocalization with STARD5

was detected within the perinuclear region of the cell, consis-tent with redistribution of STARD5 to the perinuclear regionwith ER stress (Fig. 5B, EEA1 Tuni merged images). GRP78staining pattern was also changed after tunicamycin treatmentwith staining detected at cell edges and perinuclear regionsresulting in significant overlap between the GRP78 andSTARD5 (Fig. 5B, GRP78 Tuni merged images). These datademonstrate that STARD5 is colocalized with the ER mem-brane and that ER stress results in a redistribution of STARD5in the cell that is consistent with maintaining ER membraneassociation.

STARD5 expression is increased in OVE26 diabetic mousekidney. Stard5 steady-state mRNA levels were significantlyincreased (2.8-fold) in kidneys from OVE26 diabetic micecompared with FVB wild-type control mice (Fig. 6A). Westernblot analysis of mouse kidney lysates demonstrated thatSTARD5 protein was also significantly increased (1.5-fold) indiabetic OVE26 kidneys (Fig. 6, B and C). Expression of theNa-H exchanger regulatory protein (NHERF-1) was used as arenal tubule-specific marker to control for potential differencesin recovery of tubular protein in the kidney lysates (15, 35).There is no difference in NHERF-1 protein expression betweenthe FVB wild-type and OVE26 diabetic mouse kidney lysatesper milligram of total protein and assessment of the STARD5:NHERF-1 ratio (1.1 � 0.07 vs. 1.8 � 0.15 for FVB vs.OVE26, P 0.05) confirmed a significant increase inSTARD5 in the OVE26 kidney lysates. STARD5 localizationin kidneys from both FVB and OVE26 mice was assessedusing immunocytohistochemistry. The pattern of STARD5

Fig. 3. Transmission electron microscopy ofSTARD5 subcellular localization in mouseproximal tubules. Shown is a representativemicrograph of a renal proximal tubule withelectron opaque granules for STARD5 appar-ent from the apical to basolateral regions ofthe cytoplasm (A). Higher magnification ofthe apical membrane (B) and organelles(C-D) shows staining at the basal regions ofmicrovilli (Mv; B) but not the apical regions(Mv; A). Staining was detected along apicaltubules (arrow; B) and the endoplasmic retic-ulum (ER; C) but not with mitochondria (M; C)or Golgi apparatus (G; D). Asterisks indicateER. N, nucleus. Magnifications: A, �8,200;B, �18,000; C, �24,000; D, �27,000.

Fig. 4. STARD5 mRNA expression is increased by ER stress in HK-2 humanproximal tubule cells. HK-2 cells were treated with 0.5% DMSO, 2.5 �g/mltunicamycin (Tuni), or 0.5 �M thapsigargin (Tg) as indicated. After 24 h, totalRNA was isolated and STARD5 and GRP78 steady-state mRNA levels weredetermined by RT-qPCR as described in MATERIALS AND METHODS. Shown arethe mean relative values � SD from 3 independent experiments. *P 0.05compared with control based on the Student’s t-test.

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distribution in FVB and OVE26 was similar with intensestaining along the apical brush-border membrane of renaltubules, diffuse cytoplasmic staining, and no expression in theglomeruli (Fig. 1, Supplementary data; the online version of

this article contains supplemental data). Thus, despite theincrease in STARD5 protein, no distinctive change inSTARD5 cell-type expression was observed in OVE26 dia-betic mouse kidney.

Fig. 5. STARD5 localization in HK-2 humanproximal tubule cells by confocal microscopy.HK-2 cells were grown to confluence beforetreatment with either DMSO (Cont) or 2.5�g/ml Tuni for 24 h. The cells were fixed andprocessed following the protocol outlined inMATERIALS AND METHODS. A: STARD5 wasdetected using primary antibody followed byAlexa Fluor 488-conjugated secondary anti-body and then stained with DAPI. B: colocal-ization of STARD5 with EEA1 and GRP78.STARD5 was labeled using primary antibodyfollowed by Alexa Fluor 555. The cells werethen labeled with either EEA1 or GRP78 pri-mary antibody followed by Alexa Fluor 488.Fluorescent images were captured from mul-tiple fields from duplicate wells (n 8–10fields total) as outlined in MATERIALS AND

METHODS. Shown are representative fields forDMSO (Cont) and Tuni-treated cells with thesingle images (marker proteins EEA1 orGRP78 in green and STARD5 in red) andmerged images indicated. The boxed region ineach merged image is shown enlarged.

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Cellular cholesterol levels are increased in OVE26 diabeticmouse kidney. Recent studies demonstrated an increased ex-pression of cholesterol biosynthetic enzymes and decreasedexpression of cholesterol export proteins in OVE26 diabeticmouse kidneys, suggesting that cholesterol levels might beelevated in these kidneys; however, no direct measurement ofrenal cholesterol levels was determined (23). Although thesignificance for increased cholesterol in diabetic kidneys is not

established, an increase in free cholesterol in the ER has thepotential to trigger ER stress. Therefore, we measured the renaltotal and free cholesterol levels in FVB and OVE26 mice. Asshown in Fig. 7, the kidneys of diabetic OVE26 mice have a44% increase in total cholesterol content compared with thekidneys from wild-type FVB mice. The increase in totalcholesterol is due to elevated free cholesterol, indicating thatfree cholesterol accumulation contributes to the overt increasein renal cholesterol levels in the diabetic OVE26 mice.

DISCUSSION

We report on the expression, distribution, and regulation ofSTARD5 in proximal tubules of normal mouse kidney anddemonstrate that STARD5 expression is elevated in the patho-logical state of DN. First, we show that STARD5 is expressedin mouse kidney with prominent staining detected at the apicalmembrane of proximal tubules at the cortico-medullary region.Our transmission electron microscopy (TEM) data provide thefirst high-resolution images of STARD5 subcellular localiza-tion and confirm the strong staining at the apical regions of theproximal tubule. The TEM data confirm STARD5 localizationwith the brush-border membrane and reveal association alongapical tubules, structures that are associated with membranerecycling from endocytic vesicles to the apical membrane (9).The potential significance of this distribution for STARD5 isthat proximal tubular epithelium has an asymmetrical distribu-tion of membrane lipids with the apical membrane beingenriched in cholesterol and sphingomylein that form uniquelipid raft microdomains (4, 20). These lipid rafts are located atthe basal region of the microvilli along the brush-bordermembrane, similar to STARD5 distribution, and are the site forendocytosis-mediated reabsorption of components from theglomerular filtrate (2, 3, 26). In cultured HK-2 human proximaltubule cells, we did not detect STARD5 colocalization with theearly endosome marker EEA1 under normal growth condi-tions. These data are consistent with an earlier study thatreported STARD5 distribution in THP-1 macrophages did notoverlap with a marker for the endosome recycling complex(24). Thus, future studies are necessary to define STARD5’s

Fig. 7. Increased renal cholesterol content in diabetic OVE26 mice. Total lipidwas extracted by from whole kidney lysates and total cholesterol, free choles-terol (Free CL), and cholesterol ester (CE) levels in the lipid extract weremeasured as detailed in MATERIALS AND METHODS. The data were expressed as�g cholesterol/mg protein and shown are the mean values � SE for valuesobtained from 4 FVB and 4 OVE26 mice. *P 0.05 based on Mann-Whitneytest.

Fig. 6. STARD5 expression is elevated in diabetic OVE26 mouse kidney.Total RNA or cell lysates were prepared from FVB (n 3) or OVE26 (n 3) whole kidney tissue. A: Stard5 mRNA levels in FVB and OVE26 mousekidney. RT-qPCR amplification for Stard5 and 18S rRNA was performed asdescribed in MATERIALS AND METHODS. The mRNA levels were expressedrelative to FVB mice using ��CT method and shown are the mean values �SE. B: Western blot analysis of STARD5 and NHERF-1 protein in FVB andOVE26 mouse kidney. Shown are the immunospecific bands for STARD5(top), �-actin (middle), and NHERF-1 (bottom) as indicated. C: immunoreac-tive bands were quantified as described in MATERIALS AND METHODS and theSTARD5 IOD was normalized to �-actin IOD. Shown are the mean STARD5/�-actin ratios � SE for each group. *P 0.05 based on Mann-Whitney test.

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function at the apical membrane and whether STARD5 isassociated with lipid rafts.

Our TEM data indicate STARD5 is associated with the ERmembrane in mouse renal proximal tubule cells. An ER asso-ciation is supported by our data that demonstrate colocalizationof STARD5 and the ER membrane marker GRP78 by doubleimmunofluorescence confocal microscopy in the HK-2 humanproximal tubule cell line. Previous work in THP-1 macrophagecells showed that STARD5 had a diffuse cytoplasmic distri-bution with enhanced perinuclear staining that colocalizes withthe Golgi apparatus (24). Chemical disruption of the Golgiapparatus resulted in dispersion of the STARD5 staining,suggesting STARD5 colocalized with membranes of high-cholesterol content (24). Filipin staining confirmed that theGolgi apparatus of THP-1 cells had high-cholesterol contentrelative to other cellular compartments (24). Although colocal-ization with the ER was not directly tested in THP-1 cells, theauthors proposed that STARD5 may function as an ER-Golgitransporter for cholesterol and 25-hydroxycholesterol to relieveER cholesterol load under conditions of cholesterol accumula-tion, e.g., during foam cell formation (24, 25). The apparentdifference in STARD5 membrane association in renal proximaltubule cells and THP-1 macrophages may reflect STARD5colocalization with cholesterol-rich membranes rather thanspecific membrane association in these cell types, such as theapical membrane for proximal tubule cells and Golgi formacrophages.

Next, we demonstrated that STARD5 mRNA expression isincreased by activation of ER stress in the HK-2 humanproximal tubule cells. These results are consistent with previ-ous studies that showed induction of Stard5 mRNA by induc-ers of ER stress in macrophages and NIH-3T3 cells (28). Wereport the novel finding that ER stress results in a redistributionof STARD5 in HK-2 cells from a diffuse, punctate pattern to anenhanced perinuclear and cell periphery pattern. The strong pe-rinuclear localization is similar to STARD5 distribution reportedin THP-1 macrophages (24). The finding that STARD5 iscolocalized with the ER membrane marker GRP78 in bothcontrol and ER-stressed HK-2 cells suggests STARD5 func-tions in cholesterol transport to and/or from the ER in renalproximal tubules.

Last, we extend our findings to the pathological condition ofDN. Our data demonstrate that Stard5 mRNA and protein areelevated in kidneys from OVE26 mice, a model of type Idiabetes. Recent studies showed that Grp78 mRNA and proteinexpression are significantly increased in kidneys from strepto-zotocin (STZ)-induced diabetic Wistar rats, a type I diabeticmodel, compared with nondiabetic controls (18). Immunohis-tochemical analysis of the kidneys from control and STZ-treated rats showed that GRP78 was increased in both glomer-uli and proximal tubules. ER stress response gene expression isalso increased in OVE26 mouse kidneys relative to FVBcontrol kidneys (personal communication, unpublished data,Dr. M. Barati, Dept. of Medicine, Kidney Disease Program,University of Louisville). Thus, the increase in STARD5 weobserve in OVE26 mouse kidney is most likely a consequenceof elevated ER stress.

Renal abnormalities are observed in early stages of DN andinclude an increase in glomerular filtration rate with an in-crease in kidney size and thickening of the basement mem-brane. Increased urine albumin levels and increased expression

of extracellular matrix proteins such as collagen and fibronec-tin in mesangial cells are indicative of progression to DN. TheOVE26 diabetic mouse displays all these characteristics of DN(5, 40) and our current data contribute to further characteriza-tion of the DN phenotype for these mice by demonstrating anincrease in renal free cholesterol content compared with FVBmice. Although lipid accumulation is known to occur withinthe kidney in diabetic animal models and humans, only re-cently have the mechanisms for lipid accumulation in diabetickidneys been investigated in detail. The potential for thekidney as the source for increased renal cellular triacylglyceroland cholesterol levels during diabetes is supported by demon-strated increased expression of enzymes for fatty acid andcholesterol synthesis in kidney from diabetic animal models(23, 32, 34, 36). For renal proximal tubules, cholesterol con-stitutes 20% of the total lipid content (37) and renal cholesterollevels are elevated in response to chemical insults or patholog-ical damage either directly to the tubules or to the glomeruli(38, 39). In fact, de novo cholesterol synthesis contributes to thecholesterol accumulation within the proximal renal tubules duringacute renal failure (39). Thus, it is likely that an increasedcholesterol load leads to morphological and functional changeswithin renal tubules that are associated with the development ofdiabetic kidney disease (7, 21). One possible consequence ischolesterol overload of the ER may promote the UPR to counterER stress (6). This outcome would be consistent with studiesthat demonstrated cholesterol-loaded macrophages have anactive UPR (16). Furthermore, cholesterol-loaded macro-phages were shown to have increased Stard5 mRNA expres-sion as a test for ER stress activating Stard5 mRNA expression(28). We report a similar correlative association between ele-vated STARD5 levels with elevated cholesterol levels in thekidneys of the OVE26 type 1 diabetic mouse model that havean active ER stress response. It remains to be determinedwhether the elevated cholesterol levels contribute to the ERstress in OVE26 diabetic kidney and thus indirectly regulateSTARD5 expression.

In summary, our data support STARD5 functioning in thekidney, specifically within the proximal tubule cells, and sug-gest a role in ER-associated cholesterol transport. STARD5function in proximal tubule cells may be similar to the pro-posed function in macrophages, i.e., to relieve ER cholesterolload under conditions of cholesterol accumulation (24), such asmay occur in diabetic kidney.

ACKNOWLEDGMENTS

We thank Dr. J. L. Breslow (Rockefeller University, New York, NY) for thegenerous gift of STARD5 antibody and Dr. J. Suttles (Dept. of Microbiology,University of Louisville), who provided the mouse bone marrow macrophagelysates. We thank Dr. A. Nanez (Dept. of Biochemistry and Mol. Biol.,University of Louisville) for providing the C57BL/6J kidney sections andexpertise and guidance for the immunohistochemistry studies. We appreciateDrs. C. Hu and C. Klinge (University of Louisville) for critical review of thiswork.

GRANTS

This work was supported by National Institutes of Health (NIH) GrantDK-51656 and Univ. of Louisville SOM Basic grant to B. J. Clark, NIH GrantDK-72032 to P. N. Epstein, and a Veteran Affairs Merit Review grant to E. D.Lederer.

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