BASIC RESEARCH www.jasn.org

Protein O-GlcNAcylation Is Essential for the Maintenance of Renal Energy Homeostasis and Function via Lipolysis during Fasting and Diabetes

Sho Sugahara,1 Shinji Kume,1 Masami Chin-Kanasaki,1,2 Issei Tomita,1 Mako Yasuda-Yamahara,1 Kosuke Yamahara,1 Naoko Takeda,1 Norihisa Osawa,1 Motoko Yanagita,3 Shin-ichi Araki,1,2 and Hiroshi Maegawa1

1Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan; 2Division of Blood Purification, Shiga University of Medical Science Hospital, Otsu, Shiga, Japan; and 3Department of Nephrology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

ABSTRACT Background Energy metabolism in proximal tubular epithelial cells (PTECs) is unique, because ATP pro- duction largely depends on lipolysis in both the fed and fasting states. Furthermore, disruption of renal lipolysis is involved in the pathogenesis of diabetic tubulopathy. Emerging evidence suggests that protein O-GlcNAcylation, an intracellular nutrient-sensing system, may regulate a number of metabolic pathways according to changes in nutritional status. Although O-GlcNAcylation in PTECs has been demonstrated experimentally, its precise role in lipolysis in PTECs is unclear. Methods To investigate the mechanism of renal lipolysis in PTECs—specifically, the role played by protein O-GlcNAcylation—we generated mice with PTECs deficient in O-GlcNAc transferase (Ogt). We analyzed their renal phenotypes during ad libitum feeding, after prolonged fasting, and after mice were fed a high- fat diet for 16 weeks to induce obesity and diabetes. Results Although PTEC-specific Ogt-deficient mice lacked a marked renal phenotype during ad libitum feeding, after fasting 48 hours, they developed Fanconi syndrome–like abnormalities, PTEC apoptosis, and lower rates of renal lipolysis and ATP production. Proteomic analysis suggested that farnesoid X receptor–dependent upregulation of carboxylesterase-1 is involved in O-GlcNAcylation’s regulation of lipolysis in fasted PTECs. PTEC-specificOgt-deficient mice with diabetes induced by a high-fat diet de- veloped severe tubular cell damage and enhanced lipotoxicity. Conclusions Protein O-GlcNAcylation is essential for renal lipolysis during prolonged fasting and offers PTECs significant protection against lipotoxicity in diabetes.

JASN 30: 962–978, 2019. doi: https://doi.org/10.1681/ASN.2018090950

Mammalian cells use glucose, fatty acids (FAs), and involved in the pathogenesis of tubulopathy in ketone bodies to produce ATP, which is essential for kidney diseases, including diabetic kidney disease their survival and function. The identity of the nu- (DKD).527 Therefore, renal lipolysis has become trient used for ATP production depends on feeding status and cell type. In most cells, glucose is the principal source of ATP in the fed state, but FAs Received September 25, 2018. Accepted March 9, 2019. or ketone bodies become the main source during Published online ahead of print. Publication date available at the fasting state. Energy metabolism in kidney www.jasn.org. proximal tubular epithelial cells (PTECs) is Correspondence: Dr. Shinji Kume or Dr. Hiroshi Maegawa, De- unique, because ATP production here is thought partment of Medicine, Shiga University of Medical Science, Tsukinowa- to largely depend on lipolysis and subsequent cho, Seta, Otsu, Shiga 520-2192, Japan. Email: skume@belle. b-oxidation, regardless of feeding status.12 4 shiga-med.ac.jp or [email protected] Furthermore, a disruption of renal lipolysis is Copyright © 2019 by the American Society of Nephrology

962 ISSN : 1046-6673/3006-962 JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH the focus of an emerging research field, and better under- Significance Statement standing of renal lipolysis may contribute to the develop- ment of novel therapeutic approaches for DKD. Lipolysis is of particular importance for energy homeostasis in In general, the mechanisms by which cells take up and use proximal tubular epithelial cells (PTECs), and it is dysregulated FAs differ between the fed and fasting states. In the fed state, during the pathogenesis of diabetic kidney disease. In knockout mice lacking O-GlcNAc transferase specifically in PTECs, the authors cells obtain FAs from circulating triglycerides after lipoprotein demonstrated that protein O-GlcNAcylation, an intracellular nutri- lipase (LPL)–dependent lipolysis at the endothelial surface ent sensing system, is essential for renal lipolysis andATP production and directly transfer FAs to mitochondria for ATP produc- during prolonged fasting. They also found evidence that this novel tion (Supplemental Figure 1A).8 In contrast, during the regulatory mechanism of renal lipolysis involves farnesoid X re- – fasting state, cells take up free FAs that are released from ceptor dependent upregulation of carboxylesterase-1 and that deficiency of renal protein O-GlcNAcylation exacerbates tubulop- adipocytes and circulate bound to plasma albumin. After athy in diabetic kidney disease. These findings suggest that ma- they are transported into cells, these FAs can be stored in nipulation of the renal lipolytic mechanism to overcome the effects intracellular lipid droplets and are used for ATP production of prolonged fasting might represent a novel therapeutic approach after being once again liberated by intracellular lipolysis for diabetic kidney disease. (Supplemental Figure 1B).9 GiventhatPTECsrequirecon- tinuous lipolysis for ATP production, there must be intra- (RCALS) at Shiga University of Medical Science. All experi- cellular mechanisms that sense changes in nutritional status mental protocols were approved by the Gene Recombination and regulate lipolytic processes in the fed and fasting states. Experiment Safety Committee (approval number 28–12) and However, little is known about the physiologic mechanism the RCALS at Shiga University of Medical Science (approval underpinning lipolysis-associated energy metabolism in number 2016–8-10). PTECs. Cells have evolved a nutrient-sensing system that involves Generation of Tamoxifen-Inducible PTEC-Specific Ogt O-GlcNAcylation, a post-translational modification. Knockout Mice O-GlcNAcylation involves the addition of UDP-O–linked The Ogt gene resides on the X chromosome.20 Ogtf/f mice were n-acetylglucosamine to proteins by O-GlcNAc transferase obtained from the Jackson Laboratory (Bar Harbor, ME). This (Ogt).10212 Because UDP-O–linked n-acetylglucosamine strain originated in a B6;129 background and has been is derived from metabolites involved in FA, amino backcrossed to C57BL/6 for at least ten generations. Proximal acid, glucose, and nucleotide metabolism, the extent of tubular epithelial cell–specific O-GlcNAc transferase knock- O-GlcNAcylation is indicative of overall intracellular nutri- 2 out (PTEC-Ogty/ ) mice were generated by breeding female ent status. Accumulating evidence from experimental stud- Ogtf/f mice with male N-myc downstream-regulated gene-1 ies conducted using tissue-specificOgt-deficient mouse (NDRG1) promoter–derived tamoxifen (TM)-inducible CreERT2 models indicates that O-GlcNAcylation is required for he- mice with a C57BL/6 background21 (Figure 1A). Eight-week-old patic gluconeogenesis and lipolysis-dependent thermogen- male Ogty/f and Ogty/f mice carrying Ndrg1CreERT2 were admin- esis in brown adipose tissue during fasting.13,14 Thus, in istered 150 mg/kg per day TM for 5 consecutive days.21 Twelve manycells, O-GlcNAcylation may regulate a number of met- weeks after this induction, urine samples were collected from abolic pathways according to changes in nutritional status. 2 20-week-old Ogty/f and PTEC-Ogty/ mice using a metabolic O-GlcNAcylation in PTECs has been demonstrated experi- cage under both fed and 48-hour fasting conditions. Then, mentally15219; however, the precise role of this modification mice were euthanized, and renal cortical samples were col- in the physiology of energy homeostasis, including in lipol- lected (n=5–6). ysis in PTECs, has not been fully elucidated. We hypothesized that O-GlcNAcylation is involved in the mechanism of the continuous lipolytic activ- Mouse Models of Diabetes and Atherosclerosis ityinPTECsandthatitsdysregulationisinvolvedinthe Eighteen-week-old male db/db mice purchased from CLEA pathogenesis of diabetic tubulopathy. To address these Japan Co. (Osaka, Japan) were used as a model of type 2 di- hypotheses, we evaluated renal lipolysis and the renal phe- abetes, which is characterized by overt proteinuria in the ab- 2 2 notype of PTEC-specificOgt-deficient mice exposed to a sence of severe tubulopathy. ApoE / mice were generated by 2 prolongedfastorwithhigh-fatdiet(HFD)–induced crossbreeding male and female ApoE+/ mice.22 Their ApoE diabetes. +/+ littermates were used as controls. Eight-week-old ApoE+/+ mice were fed either a normal diet or an HFD for 24 weeks to induce obesity-related microalbuminuria without severe 2 2 METHODS tubulopathy. HFD-fed ApoE / mice were used as a model of diabetes- and atherosclerosis-associated severe tubulopathy. Ethics The normal diet (10% of total calories from fat) and HFD Animal experimentationwas conducted in accordancewith the (60% of total calories from fat) were purchased from Research guidelines of the Research Center for Animal Life Science Diets (New Brunswick, NJ).

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 963 BASIC RESEARCH www.jasn.org

A B Cre

( ) Ogtf/f mice ( ) NDRG1-CreERT2 mice Ogt

Ogt gene on X chromosome RL2 X LoxP Exon X LoxP NDRG1 promoter Cre ERT2 X LoxP Exon X LoxP (O-GlcNAcylation)

( ) Ogty/f β ( ) Ogty/f actin NDRG1-CreERT2 mice

y/f PTEC y/f PTEC

y/f Ogt Ogt y/- -Ogty/- -Ogty/- Ogt PTEC -Ogt Isolated PTECs Isolated non-PTECs Renal cortex (LTL-positive) (LTL-negative) X LoxPExon X LoxP Y X LoxP Exon X LoxP + C RL2 (O-GlcNAcylation) Y NDRG1 promoter Cre ERT2 y/f

Tamoxifen (TM) Tamoxifen (TM) Ogt

( ) PTEC-Ogty/- ( ) Ogty/f y/-

X LoxP LoxP X LoxP Exon X LoxP PTEC-Ogt Y Y

DE F 50 TM injection 200 TM injection 5 NS

40 4 P<0.05 150 30 NS NS 3 NS 100 NS NS NS 20 NS 2

y/f 50 y/f Body weight (g) 10 Ogt Ogt 1 Food intake (g/day) PTEC-Ogty/- Blood glucose (mg/dl) PTEC-Ogty/- 0 0 0

8 12 16 20 8 12 16 20 y/f y/-

(Week-old) (Week-old) Ogt PTEC -Ogt

Figure 1. Proximal tubular epithelial cell–specific O-GlcNAc transferase knockout (PTEC-Ogty/-) mice show little developmental defect. (A) The O-GlcNAc transferase (Ogt) gene resides on the X chromosome. Female Ogtf/f and male Ogty/f mice have loxP sites inserted into exon 10 of the Ogt gene. N-myc downstream-regulated gene-1 (NDRG1) promoter–derived tamoxifen (TM)-inducible CreERT2- expressing mice were used for proximal tubular epithelial cell (PTEC)–specific Cre expression. Male Ogty/f mice carrying CreERT2 and 2 Ogty/f mice were injected with TM for 5 consecutive days to induce Cre expression. The generated male PTEC-Ogt y/ and Ogty/f mice were used for the study. (B) Cre recombinase expression, Ogt protein expression, and protein O-GlcNAcylation, detected using an RL2 2 antibody, were lower in both renal cortical samples and isolated Lotus tetragolonobus lectin (LTL)–positive PTECs from PTEC-Ogty/ mice. PTECs were isolated using an anti-LTL antibody. (C) Immunostaining for renal protein O-GlcNAcylation in 20-week-old Ogty/f and 2 2 PTEC-Ogty/ mice. Protein O-GlcNAcylation was lower mainly in the renal cortex of PTEC-Ogty/ mice. Original magnification, 340 in 2 left panels; 3200 in center panels; 3400 in right panel. (D) Change in mean body mass. PTEC-Ogty/ mice showed lower body mass gain at 20 weeks of age than control Ogty/f mice (n=5 per group). (E) Change in mean casual blood glucose concentration. There was no difference in casual blood glucose concentration between two genotypes. (F) Food intake at 20 weeks old was similar between the two genotypes. Horizontal bars indicate the median values for each group. P,0.05 was considered statistical significance.

964 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

Mitochondrial Division Inhibitor Treatment Study images of complete membranes are shown in Supplemental 2 Eight-week-old male PTEC-Ogty/ mice were divided into Figure 2. two groups, vehicle and mitochondrial division inhibitor (Mdivi-1; Sigma-Aldrich, St. Louis, MO) treatment groups Quantitative Real-Time PCR (n=7 each), after 4 weeks of Cre induction. Mdivi-1 (50 mg/kg Real-time PCR was performed as described previously.23 The per day) or vehicle was intraperitoneally administered for 3 PCR primer sets are listed in Supplemental Table 2. 2 days in 12-week-old PTEC-Ogty/ mice commencing 24 hours before the fast and continuing for the duration of the fasting Proteomic Analyses period. After 48 hours of fasting, urine and kidney samples The proteomic analysis was conducted by Medical Proteoscope were collected. (Kanagawa, Japan). Whole data are shown in Supplemental Appendix and were deposited into jPOST (identification 2 HFD-Induced Diabetes in PTEC-Ogty/ Mice number JPST000549) and Proteome XChange (PXID identi- Eight-week-old male Ogty/f mice and Ogty/f mice carrying fication number PXD012456). Ndrg1CreERT2 (n=5pergroup)wereadministeredTMand Snap-frozen kidney cortices from 20-week-old Ogty/f and 2 started consuming the HFD. An intraperitoneal insulin toler- PTEC-Ogty/ mice after 24 hours of fasting were homoge- ance test was conducted as previously described5 before eu- nized using a pestle and sonicated in ice-cold lysis buffer thanasia. Kidney samples were collected after 16 weeks of the (20mMHEPES-NaOH,pH8.0,9Murea,25mMsodium dietary intervention. pyrophosphate, 10 mM b-glycerophosphate, and 1% vol/vol each of Phosphatase Inhibitor Cocktail solutions 2 and 3 Blood and Urine Analyses [Sigma-Aldrich]). The lysates generated were clarified by cen- Blood glucose concentrations were measured using a Glutest trifugation at 15,0003g for 10 minutes, and their protein con- sensor (Sanwa Kagaku, Nagoya, Japan). Urinary albumin, centrations were determined using a Bradford assay. Aliquots amino acids, and ions were measured using standard labora- were subjected to a cycle of dithiothreitol reduction. After re- tory methods. ductive alkylation, the protein solutions were diluted to achieve a urea concentration of #2 M with 20 mM HEPES- Histologic Analyses NaOH, and then, they were subjected to protein hydrolysis Three-micrometer-thick sections of frozen and paraffin- with bovine trypsin (tosyl phenylalanyl chloromethyl ketone embedded fixed samples were prepared. Oil-red O staining, treated; Sigma-Aldrich) at 37°C for 16 hours, with an enzyme- periodic acid–Schiff staining, hematoxylin and eosin staining, to-substrate ratio of 1:20 (wt/wt). Peptide samples were desalted and immunohistochemistry were performed as described pre- using C18 STAGE tips and dried at low pressure. The dried viously.5 The antibodies used for immunohistochemistry are samples were dissolved in a solvent consisting of water, aceto- listed in Supplemental Table 1. Terminal deoxynucleotidyl nitrile, and formic acid at a volume ratio of 98:2:0.1 and diluted transferase–mediated digoxigenin-deoxyuridine nick-end to 250 ng/ml. Of this, 2 ml (containing 500 ng protein) were labeling staining was performed using a TACS 2 TdT DAB used for liquid chromatography (LC)-mass spectrometry kit (Trevigen, Gaithersburg, MD). Quantitative analysis was (MS)/MS using the following specifications and settings. performed by three independent nephrologists in a blinded Briefly, peptide separation was performed with an Ultimate fashion.23 A tubulointerstitial damage score was assigned as 3000 RSLCnano (Thermo Fisher Scientific) containing a C18 described previously.23 capillary LC column (Nano HPLC Capillary Column; 75-mm internal diameter, 150-mm length, 3-mm particle size; Nikkyo Evaluation of Mitochondrial Morphology Using Technos, Tokyo, Japan). The mobile phases consisted of formic Transmission Electron Microscopy acid, acetonitrile, and water at volume ratios of 0.1:0:100 for Mitochondrial structure was assessed by transmission elec- mobile phase A and 0.1:90:10 for mobile phase B. The peptides tron microscopy examination of at least 100 mitochondria were continuously eluted at a rate of 350 nl/min in gradient per sample at a magnification of 8000 as previously reported mode: the initial proportion of 5% mobile phase B was in- with some minor modifications.24 Mitochondrial size and creased to 40% B over 120 minutes, and it was subsequently number of mitochondria per PTEC were measured, and mi- increased to 95% B over the next 10 minutes. Protonated pep- tochondrial density was calculated as the number of mito- tides in the gas phase were analyzed sequentially by MS/MS chondria per square micrometer PTEC area. For aspect ratio in positive ion mode consisting of a full-range scan in the mass- measurements, the ratio between the major and minor axes of to-charge range of 300–1500 and subsequent product ion scans the ellipse equivalent to each mitochondrion was determined. for each of the ten most intense ions in the full-scan mass spectrum. Western Blotting Western blot analysis of proteins from the renal cortex or Label-Free Quantification and Peptide Identification isolated PTECs was performed as previously described.25 Label-free relative peptide quantitation was performed by di- The antibodies are listed in Supplemental Table 1. Scanned rect comparison of each MS scan profile using the Progenesis

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 965 BASIC RESEARCH www.jasn.org

QI for proteomics software (version 2.0; Nonlinear Dynamics, Burlingame, CA). Isolated PTECs were resuspended in culture Durham, NC). The normalized abundances obtained for each medium (BulletKit; Clonetics) and seeded into culture dishes. 2 peptide were subjected to statistical analysis using one-way Isolated PTECs from 20-week-old Ogty/f and PTEC-Ogty/ ANOVA. In this study, the detected feature was taken to be mice were treated with an farnesoid X receptor (FXR) agonist statistically significant when the P value for a given peptide was (GW4064; ChemScene, Monmouth Junction, NJ) or DMSO ,0.05. To identify the peptide sequence, peak lists were cre- as a treatment control for 24 hours after 12 hours of starvation. ated using Progenesis LC-MS. The MS and MS/MS data Collected samples were used for detecting carboxylesterase 1 obtained were searched against mouse protein sequences (CES1) mRNA expression. (16,930 entries) in the Swiss-Prot database (January 2013) and the amino acid sequences of protein contaminants (116 CES1 Overexpression in PTECs entries) in The Proteome Machine Organization using A pcDNA3 plasmid for the expression of CES1 was obtained MASCOTsoftware, version 2.5 (Matrix Science, London, United from OriGene (Rockville, MD), and a pcDNA3-null plasmid Kingdom). The search parameters were enzyme, semitrypsin; was used as a control. Transfection in isolated PTECs of 2 maximum missed cleavage, two; peptide tolerance, 65 20-week-old Ogty/f and PTEC-Ogty/ mice was performed ppm; MS/MS tolerance, 6 0.02 D; mass, monoisotopic using Lipofectamine 3000 Reagent (Invitrogen). mass; fixed modification, carbamidomethyl (C, +57.021 D); and variable modifications, oxidation (M, +15.099 D). The Fluorescence Microscopy of Isolated PTECs and false discovery rate was estimated on a decoy database using Intracellular Lipid Droplets the MASCOTsoftware. We used a 1% false discovery rate as a Lipid droplets in fixed cells were visualized using boron-dipyr- cutoff for the export of results from the analysis. romethene (BODIPY) 493/503 (Invitrogen).27,28 Isolated PTECs were incubated in culture medium containing 150 Bioinformatic Analyses of Protein Expression Data mM oleic acid for 2 hours, then washed twice with PBS(2), Pathway analysis of the list of data from the proteomic analysis and incubated in Krebs–Ringer buffer without glucose and was performed using KeyMolnet (KM Data, Tokyo, Japan). oleate for 3 hours. To label lipid droplets, 200 ng/ml BODIPY KeyMolnet is a bioinformatics integration platform that en- 493/503 was added to the cells before imaging. Nuclei were ables the analysis of specific pathways on the basis of data visualized using 49,6-diamidino-2-phenylindole. collected from recent papers. By importing the list of Entrez Fluorescence analysis of CES1 protein distribution was gene identifications, KeyMolnet automatically provides the performed as previously described29 using Alexa Fluor 594 corresponding molecules in the form of nodes in a network. anti-rabbit IgG as the secondary antibody. After immunos- In the various network-searching algorithms, the “interaction” taining, cells were counterstained using BODIPY 493/503 search identifies molecular networks containing a group of and 49,6-diamidino-2-phenylindole. Immunofluorescence molecules that showed differential regulation in this study. was detected using a fluorescence microscope (BX61; Olym- The significance was scored using following formula, in which pus, Tokyo, Japan). O = the number of overlapping molecular relationships be- tween the extracted network and the canonical pathway, Measurement of ATP Content and LPL Activity V = the number of molecules displayed in the search result, ATP concentration in the kidney cortex and cultured cells was C = the number of molecules belonging to specific pathways, measured using a firefly bioluminescence kit (AMERIC-ATP T = the total number of molecules recorded in KeyMolnet, and Kit; Wako Pure Chemical Industries) and an intracellular ATP X =thes-variable that defines incidental agreements: assay kit (Toyo Ink Group, Tokyo, Japan). Renal LPL activity was measured using an LPL activity assay kit (Cell Biolabs, Inc., ð Þ¼∑MinðC;VÞ ð Þ Score P x¼0 f X San Diego, CA). f ðXÞ¼ C CX z T 2 CCV 2 X=T CV ; Measurement of Renal Triglyceride and Cholesterol with Score ¼ 2 log ðScoreðPÞÞ; ScoreðVÞ¼O=V; and ScoreðCÞ 2 Content ¼ O=C: Total lipid was extracted from renal cortices using the method of Bligh and Dyer.30 Triglyceride and cholesterol contents were Isolation of Primary PTECs and Farnesoid X Receptor analyzed using the TG Assay Kit (Wako Pure Chemical Indus- Agonist Treatment tries). Total cholesterol and free cholesterol were measured Primary PTECs were isolated as described previously.26 Briefly, using the total cholesterol assay kit (Cell Biolabs, Inc.). kidney cortices were digested in HBSS containing collagenase Cholesteryl ester was calculated by subtracting free cholesterol type II (200 U/ml; Life Technologies, Grand Island, NY) and from total cholesterol. hyaluronidase (0.2%; Wako Pure Chemical Industries, Osaka, Japan). Selection of PTECs was performed using a CELLection Immunoprecipitation Study Biotin Binder Kit (Invitrogen, Carlsbad, CA) and biotinylated Immunoprecipitation in renal cortical samples from 20-week- Lotus tetragonolobus agglutinin lectin (Vector Laboratories, old wild-type C57/BLK6J mice was conducted as previously

966 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

2 described.31 The antibodies used in this assay are listed in significantly lower in the fasted PTEC-Ogty/ mice than con- Supplemental Table 1. trols (Figure 3B), suggesting that the Fanconi syndrome–like abnormalities induced by the Ogt deficiency were mediated by Statistical Analyses ATP depletion rather than the generalized downregulation of The Wilcoxon rank sum test and the Kruskal–Wallis test fol- transporters. lowed by the Dunn–Bonferroni post hoc test were used for Mitochondria are of great importance for ATP production. statistical comparisons of two groups and multiple groups, Although the mitochondrial area and numbers in PTECs were 2 respectively. P,0.05 was considered to represent statistical similar in Ogty/f and PTEC-Ogty/ mice, regardless of feeding significance. status, there was a lower aspect ratio in mitochondrial mor- 2 phology in the fasted PTEC-Ogty/ mice (Figure 3, C and D), suggesting an increase in mitochondrial fission. To determine RESULTS whether there was a causal relationship between the renal 2 phenotype and this mitochondrial fission, PTEC-Ogty/ 2 Generation of PTEC-Ogty/ Mice mice were treated with a Drp-1 inhibitor (Mdivi-1) that blocks 2 The Ogt gene resides on the X chromosome.20 PTEC-Ogty/ mitochondrial fission32 and then, fasted for 48 hours. The mice were generated by crossbreeding Ogtf/f mice with administration of Mdivi-1 promoted the elongation of the NDRG1 promoter–derived TM-inducible CreERT2 mice.21 mitochondria (Figure 3E), but it did not ameliorate the Fan- 2 Eight-week-old male Ogty/f mice and Ogty/f mice carrying coni syndrome–like abnormalities in the fasted PTEC-Ogty/ Ndrg1CreERT2 were treated with TM (Figure 1A). Effective mice (Figure 3F), suggesting that an increase in mitochondrial Cre recombinase expression, Ogt deletion, and deficient pro- fission was not the primary event leading to impaired renal tein O-GlcNAcylation in the renal cortical samples and the function in the mice. 2 isolated PTECs of PTEC-Ogty/ mice were confirmed Next, FA metabolism was evaluated. LPL is essential for FA by Western blot analysis (Figure 1B) and immunostaining uptake into cells during the fed state.8 The expression and 2 (Figure 1C). Low levels of Cre recombination were found in lipase activity of LPL were similar in Ogty/f and PTEC-Ogty/ 2 the non-PTECs isolated from the renal cortex of PTEC-Ogty/ mice during the fed state (Figure 4, A–C). Furthermore, LPL mice (Figure 1B), suggesting that changes mediated by defi- was not O-GlcNAcylated (Figure 4D), and the expression of cient O-GlcNAcylation in renal cortical samples should glycosylphosphatidylinositol-anchored HDL-binding protein mainly reflect the modification of PTECs. 1, an adaptor protein for LPL, was also similar in the two ge- Twelve weeks after the induction with TM, body mass gain notypes (Figure 4A). Thus, the LPL system was not affected by 2 was slightly but significantly lower in PTEC-Ogty/ mice, al- the Ogt deficiency. These findings are consistent with the find- 2 though their casual blood glucose concentration and daily ing of no marked renal phenotype in the fed PTEC-Ogty/ food intake were similar to those of control Ogty/f mice mice. throughout the experimental period (Figure 1, D–F). In contrast, 48 hours of fasting was associated with greater intracellular lipid droplet formation and higher renal triglyc- Functional and Histologic Abnormalities in PTEC- eride content in the PTECs of Ogty/f mice, and the difference 2 2 Ogty/ Mice were greater in the PTEC-Ogty/ mice (Figure 4, E and F). This One of the principal functions of PTECs is the reabsorption of finding provoked the hypothesis that Ogt deficiency impairs macro- and micromolecules from the urinary space. There lipid droplet breakdown and subsequent utilization of FAs for were no significant differences in the urinary excretion of al- ATP production during fasting. To investigate this bumin, glucose, phosphate, uric acid, amino acids, and ions possibility, a primary cell culture study was conducted (Figure between the two groups during the fed state (Figure 2A). In 4G). When isolated PTECs were treated with oleate, an un- 2 contrast, PTEC-Ogty/ mice excreted higher concentrations saturated FA, in complete medium, lipid droplets were formed of these substances in urine after 48 hours of fasting (Figure in the cells, but ATP concentration did not differ between 2B). In addition to this Fanconi syndrome–like phenotype, two genotypes (Figure 4, H and I). After 3 hours of culture significantly larger numbers of terminal deoxynucleotidyl in Krebs–Ringer buffer without oleate and glucose, the lipid transferase–mediated digoxigenin-deoxyuridine nick-end la- droplets disappeared from the Ogty/f cells, and apoptosis did beling–positive apoptotic cells and more cleavage of caspase 3 not occur (Figure 4, H–J). In contrast, the droplets remained, 2 2 were identified in the fasted PTEC-Ogty/ mice (Figure 2, and there was more apoptosis in cells from PTEC-Ogty/ mice C–F) accompanied by larger numbers of vesicle-like structures accompanied by a lower ATP concentration (Figure 4, H–J). in hematoxylin and eosin–stained sections (Figure 2C). These observations are consistent with the above hypothesis.

2 Impaired Renal Lipolysis in Fasted PTEC-Ogty/ Mice Altered Expression of Lipolytic Enzymes in Fasted 2 The expression of several transporters in PTECs did not differ PTEC-Ogty/ Mice 2 between fasting Ogty/f and PTEC-Ogty/ mice (Figure 3A). In After 48 hours of fasting, there were no differences in contrast, the ATP concentration in renal cortical samples was the renal phosphorylation of hormone-sensitive lipase

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 967 BASIC RESEARCH www.jasn.org

A Ad libitum-fed condition NS NS NS 1.5 NS 100 4.0 0.20 NS 150 60 NS 3.0 0.15 1.0 100 40 50 2.0 0.10 0.5 50 20 1.0 0.05 U-IP (mg/day) U-Alb (µg/day) U-Glu (mg/day) U-UA (mg/day) U-Cys (µmol/day) 0 0 0 0 U-Asp (µmol/day) 0 0

NS 500 0 0.04 NS 0.20 NS 0.8 NS NS 400 0.03 0.15 0.6 40 300 Ogty/f 0.02 0.10 0.4 200 y/- 20 PTEC-Ogt 100 0.01 0.05 0.2 U-Ca (mg/day) U-K (mmol/day) U-Na (mmol/day) U-Gln (µmol/day) 0 U-Ser (µmol/day) 0 0 0 0

B 48-h fasting condition P<0.05 100 P<0.01 0.6 5.0 0.10 P<0.01 75 60 P<0.01 P<0.05 P<0.05 75 4.0 0.4 50 40 3.0 50 0.05 2.0 0.2 25 20 25 1.0 U-IP (mg/day) U-Alb (µg/day) U-UA (mg/day) U-Glu (mg/day) U-Cys (µmol/day) 0 0 0 0 U-Asp (µmol/day) 0 0

P<0.01 200 80 0.02 0.10 0.2 P<0.05 P<0.01 P<0.01 150 60 P<0.01 Ogty/f 100 40 0.01 0.05 0.1 PTEC-Ogty/- 50 20 U-Ca (mg/day) U-K (mmol/day) U-Na (mmol/day) U-Gln (µmol/day) 0 U-Ser (µmol/day) 0 0 00

C y/f y/- D Ogt PTEC-Ogt P<0.01

Fed Fast Fed Fast P<0.01

P<0.05 10

8

6 P<0.05 TUNEL stain 4 NS NS number / field 2 TUNEL-positive cell

0 y/f y/f y/- y/- Ogt Ogt Ogt Ogt PTEC- PTEC- HE stain Fed Fast

EF3.0 P<0.05

Cleaved 2.0 caspase 3

/ β actin 1.0 β actin Cleaved caspase 3 0 y/f y/- Ogty/f PTEC-Ogty/- Ogt Ogt Fast PTEC- Fast

Figure 2. Fasted proximal tubular epithelial cell–specific O-GlcNAc transferase knockout (PTEC-Ogty/-) mice develop Fanconi syndrome-like phenotype and cell apoptosis. (A) There were no significant differences in the 24-hour urinary excretion (U) of albumin (Alb); glucose (Glu); phospate (IP); uric acid (UA); some amino acids, including asparagine (Asp), cystine (Cys), glutamine (Gln), and

968 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

(HSL) or the renal expression of HSL, adipose triglyceride triglyceride lipase, perilipin, CES1, and PPARa were not lipase, or perilipin, key regulators of intracellular lipid droplet (Figure 7, C and D). Furthermore, FXR agonist (GW4064) breakdown,33235 between the two genotypes (Figure 5, A and treatment significantly increased CES1 mRNA expression in B). Therefore, to identify the proteins and pathways mediat- isolated PTECs from Ogty/f mice, but it failed to increase 2 2 ing the impairment in lipolysis in the fasted PTEC-Ogty/ expression in cells from PTEC-Ogty/ mice (Figure 7E). These mice, a proteomic analysis of renal cortical samples from data suggest that the low CES1 expression is dependent on an 2 fasted Ogty/f and PTEC-Ogty/ mice was conducted (Figure interaction between FXR and O-GlcNAcylation. 5C). Proteomic analysis and subsequent pathway analysis 2 revealed that PTEC-Ogty/ mice had significant differences Low-Protein O-GlcNAcylation and CES1 Expression in in renal lipid metabolism from controls (Figure 5D). There an Animal Model of Atherosclerogenic Diabetes were several lipolytic enzymes listed in the output of the In addition to fasting, insulin resistance in adipose tissue under proteomic analysis (Supplemental Table 3), including much obese and/or diabetic conditions is associated with high plasma lower protein expression of CES1 family proteins, which can free FAs and a greater influxof free FAs into the kidneys, leading act as triglyceride hydrolases as well as cholesterol esterases36238 to renal lipotoxicity.5,23,41 Therefore, we next evaluated the (Figures 5E and 6A). role of O-GlcNAcylation in lipotoxic kidney injury. We first The finding of a large difference in CES1 protein level was measured protein O-GlcNAcylation in the kidneys of dia- corroborated by immunohistochemistry and Western blot betic db/db mice, HFD-fed obese ApoE+/+ mice, and HFD- 2 2 analysis (Figure 6, B–D). Furthermore, the concentration of fed ApoE / mice. Consistent with the results of previous cholesterol ester and the ratio of cholesterol ester to total cho- studies,23,42,43 the interstitial lesions in diabetic db/db mice lesterol concentrations in renal cortical samples from PTEC- andHFD-inducedobesemicewerenotsevere,butthose 2 2 2 Ogty/ mice were significantly higher than in control mice in HFD-fed ApoE / mice were more evident (Figure 8A). (Figure 6E), suggesting that renal CES1 activity is lower in Protein O-GlcNAcylation was more extensive in the kidneys of 2 PTEC-Ogty/ mice. In the isolated PTECs, the impairment the first two models, but it was much lower in the kidneys of 2 2 in lipid droplet breakdown was accompanied by lower CES1 HFD-fed ApoE / mice (Figure 8A). 2 expression in the PTEC-Ogty/ mice (Figure 6F, center panel). However, Ogt-deficient PTECs that overexpressed CES1 after Exacerbation of HFD-Induced Kidney Pathology in 2 transfection with a CES1-overexpression vector, which appear PTEC-Ogty/ Mice red in the right panel of Figure 6F, demonstrated a restored The above findings suggest that deficient protein O-GlcNAcyla- capacity for lipid droplet breakdown. Furthermore, CES1 tion in PTECs is involved in the exacerbation of tubulointerstitial 2 2 overexpression significantly ameliorated the low ATP lesions in HFD-fed ApoE / mice. To evaluate this 2 production and inhibited apoptosis in nutrient-deprived possibility, PTEC-Ogty/ mice were fed an HFD for 16 weeks primary Ogt-deficient PTECs after an oleic acid load (Figure to induce obesity and diabetes (Figure 8B). Although the body 2 6, G and H). mass of HFD-fed PTEC-Ogty/ mice was significantly lower In addition to the difference in protein expression level, and glucose tolerance was slightly better than in HFD-fed Ogt renal mRNA expression of CES1 was also lower in the kidneys y/f mice (Figure 8, C and D), tubular dilation, larger numbers 2 of PTEC-Ogty/ mice (Figure 6I), suggesting that the activity of vesicle-like structures, and fibronectin accumulation were 2 of a transcription factor regulating CES1 expression might observed in kidney sections of PTEC-Ogty/ mice (Figure 8, E have been inhibited by Ogt deficiency. Previous studies have and F) accompanied by lower CES1 expression and a massive demonstrated that peroxisome proliferator-activated recep- accumulation of lipid droplets (Figure 8F). tor-a (PPARa) and FXR regulate CES1 expression at the tran- scriptional level.39,40 The proteomic data showed that the expression of several proteins regulated by FXR was lower in DISCUSSION 2 the fasted PTEC-Ogty/ mice (Figure 7A), but the differences in the expression of proteins regulated by PPARa varied Because prolonged fasting is a life-threatening event, the pro- (Figure 7B). A series of immunoprecipitation studies revealed cess of evolution has caused organisms to develop a variety of that FXR was O-GlcNAcylated but that HSL, adipose cellular mechanisms for the maintenance of whole-body

2 serine (Ser); sodium (Na), calcium (Ca), and potassium (K) between 20-week-old control Ogty/f mice (n=5) and PTEC-Ogty/ mice (n=6) 2 under ad libitum–fed condition. (B) Urinary excretions of the indicated substances in PTEC-Ogty/ mice were significantly higher than those in Ogty/f mice (n=6 per group) after a 48-hour fast. (C and D) Apoptotic cell number determined by terminal deoxynucleotidyl 2 transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) staining was significantly higher in the fasted PTEC-Ogty/ mice (n=5 per group). Vacuolar changes in hematoxylin and eosin (HE)–stained proximal tubular epithelial cells were obvious in fasted 2 2 PTEC-Ogty/ mice. Original magnification, 3400. (E and F) Cleavage of caspase 3 was higher in the fasted PTEC-Ogty/ mice (n=5 per group). Horizontal bars in the graphs of A, B, D, and F indicate the median values. P,0.05 was considered statistical significance.

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 969 BASIC RESEARCH www.jasn.org

A B Renal ATP contents (µmol / g tissue)

NS NS NS P<0.05 NS NS NS 3.0 3.0 3.0 3.0 3.0 3.0 0.3 0.3 P<0.05

2.0 2.0 2.0 2.0 2.0 2.0 0.2 0.2

1.0 1.0 1.0 1.0 1.0 1.0 0.1 0.1 OAT1 / β actin NHE3 / β actin URAT / β actin SGLT2 / β actin NaPi-IIa / β actin ATP1b1 / β actin 0 0 0 0 0 0 0 0 y/f y/f y/f y/f y/f y/f y/f y/f y/- y/- y/- y/- y/- y/- y/- y/- Ogt Ogt Ogt Ogt Ogt Ogt Ogt Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt Fast Fast Fast Fast Fast Fast Fed Fast

CDOgty/f PTEC-Ogty/- Mitochondrial area Mitochondria number (pixel × 1,000)(Number/µm2) Aspect ratio

6 NS 6 NS 0.4 NS 0.4 NS 5 P<0.01 5 4 4 P<0.01

Fed 0.3 0.3 4 4 3 3 0.2 0.2 2 2 2 2 0.1 0.1 1 1 0 0 0 0 0 0 y/f y/f y/f y/f y/f y/f y/- y/- y/- y/- y/- y/- Transmission EM Fast Ogt Ogt Ogt Ogt Ogt Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt Fed Fast Fed Fast Fed Fast

EF Fasted P<0.01 NS NS NS NS PTEC-Ogty/- 10 0.8 4.0 0.10 0.08 8 0.6 3.0 0.06 6 0.4 2.0 0.05 0.04 4 0.2 1.0 0.02 Aspect ratio Mdivi-1(-) 2 U-IP (mg/day) U-UA (mg/day) U-Glu (mg/day) 0 0 0 0 U-Na (mmol/day) 0 Transmission EM Mdivi-1(-) Mdivi-1(-) Mdivi-1(-) Mdivi-1(-) Mdivi-1(-) Mdivi-1(+) Mdivi-1(+) Mdivi-1(+) Mdivi-1(+) Mdivi-1(+)

Mdivi-1(+) Fasted Fasted Fasted Fasted Fasted PTEC-Ogty/- PTEC-Ogty/- PTEC-Ogty/- PTEC-Ogty/- PTEC-Ogty/-

Figure 3. Transporters expression and mitochondrial fission are not involved in the renal phenotype of fasted proximal tubular epi- thelial cell–specific O-GlcNAc transferase knockout (PTEC-Ogty/-) mice. (A) There were no significant differences in renal mRNA 2 expression levels of the indicated transporters other than SGLT2 between 20-week-old control Ogty/f mice and PTEC-Ogty/ mice after 2 48 hours of fasting (n=5 each). SGLT2 expression in the fasted PTEC-Ogty/ mice was higher than in the fasted Ogty/f mice. (B) There was no significant difference in renal ATP levels between the two genotypes under the fed condition, whereas the level was signifi- 2 cantly lower in PTEC-Ogty/ mice after 48 hours of fasting (n=5 each). (C and D) Aspect ratio of mitochondria in proximal tubular 2 epithelial cells of PTEC-Ogty/ mice was smaller than that in Ogty/f mice, although there were no differences in mitochondria area and number. Original magnification, 38000. (E and F) Mitochondrial division inhibitor (Mdivi-1) treatment enhanced mitochondrial fusion, 2 but it did not improve excess urinary excretion of the indicated molecules in the fasted PTEC-Ogty/ mice at 12 weeks old (n=7 each). Original magnification, 38000. Horizontal bars in the graphs of A, B, and D–F indicate the median values. P,0.05 was considered statistical significance. EM, electron microscopy.

ATP production, regardless of feeding status. In mammals, the Therefore, this modification likely represents one of the abil- substrate used to generate ATP depends on the cell type. PTECs ities that PTECs have acquired to cope with prolonged fasting metabolize FAsto provide ATP during both feeding and fasting. during the course of evolution. Here, we show that protein O-GlcNAcylation is involved in the Although one previous report has suggested that maintenance of lipolysis during prolonged fasting (Figure 9A). O-GlcNAcylation in PTECs is important for the maintenance

970 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

ABCD 3.0 NS 3.0 NS 3 NS IB: LPL

2.0 2.0 LPL 2

β 1.0 / β actin 1.0 actin 1 LPL activity (unit / g tissue) y/f y/-

mRNA of GPIHBP1 Ogt PTEC-Ogt IP: RL-2-Ab mRNA of LPL / β actin

0 0 0 Pre-immune y/f y/f y/f y/- y/- y/- IP: Control IgG Ogt Ogt Ogt (O-GlcNAcylation) PTEC-Ogt PTEC-Ogt PTEC-Ogt

Fed Fed Fed EFG Fed Fast Renal triglyceride Ogty/f PTEC-Ogty/- content (µmol / g tissue)

30 P<0.01 y/f 20 P<0.05 Oleate (+) Ogt NS 10

0 ATP measurement (Basal) y/- y/f y/f y/- y/-

Oil-red O stain Oleate (-)

Ogt Ogt in KRB buffer PTEC-Ogt PTEC-Ogt PTEC-Ogt Fed Fast ATP measurement Lipid droplet (BODIPY) Apoptosis HIJ Oleate (+) Oleate (-) ATP contents in isolated At basal 1-hour 3-hour PTECs (pmol / µg protein) 10 NS 4 NS 8 Cleaved casp3 y/f 3

Ogt 6 2 Full length PARP 4 Cleaved PARP 2 1 β actin y/- 0 0 y/f y/f y/- y/- Ogty/f PTEC-Ogty/- Ogt Ogt

PTEC-Ogt Oleat (-) 3-hour PTEC-Ogt PTEC-Ogt

At basal Oleat (-) 3-hour

Figure 4. Lipid droplet breakdown is impaired in proximal tubular epithelial cell–specific O-GlcNAc transferase knock- out (PTEC-Ogty/-) mice. (A) There were no significant differences in the renal mRNA expression of lipoprotein lipase (LPL) and glycosylphosphatidylinositol-anchored HDL-binding protein 1 (GPIHBP1) between 20-week-old control Ogty/f mice and PTEC- 2 Ogty/ mice under ad libitum–fed condition (n=5 per group). (B and C) There were no significant differences in renal protein expression and activity of LPL between the two genotypes under the fed condition. (D) Immunoprecipitation (IP) study using an RL2 antibody that recognizes protein O-GlcNAcylation followed by immunoblotting (IB) with an LPL antibody. LPL was not O-GlcNAcylated. (E and F) Renal neutral lipid accumulation identified using (E) Oil-red O staining and (F) triglyceride content measurement. The fasting- 2 induced increase in renal triglyceride was significantly greater in PTEC-Ogty/ mice. Original magnification, 3200 in E. (G) Protocol for the cell culture study using isolated proximal tubular epithelial cells (PTECs). (H) Oleate treatment led to the formation of boron- 2 dipyrromethene (BODIPY)–stained lipid droplets in the isolated PTECs of 20-week-old Ogty/f mice and PTEC-Ogty/ mice. Lipid droplet 2 degradation after oleate removal did not occur in the PTECs of PTEC-Ogty/ mice. Original magnification, 3400. (I) After oleate removal, 2 the ATP concentration in the PTECs of PTEC-Ogty/ mice was lower than in cells from control Ogty/f mice (n=6 per group), although the basal ATP concentration before oleate removal did not differ between the two genotypes (n=5 per group). (J) After oleate removal, 2 cleavage of caspase 3 was higher in cultured PTECs from PTEC-Ogty/ mice. Horizontal bars in the graphs of A, C, F, and I indicate the median values. P,0.05 was considered a statistical significance. KRB, Krebs–Ringer buffer; PARP, poly(ADP-ribose) polymerase.

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 971 BASIC RESEARCH www.jasn.org

ABpHSLser563 HSL ATGL Perilipin

pHSLser563 y/f HSL Ogt

ATGL Fast y/- Perilipin

β actin PTEC-Ogt Ogty/f PTEC-Ogty/- Fast C D Rank Pathway Score Score(p) Score(v) Score(c) y/- y/f PTEC-Ogt Ogt 1 Lipoprotein metabolism 122.575 1.262E-037 0.018 0.301 2 MMP signaling pathway 67.509 4.761E-021 0.018 0.092 Granzyme signaling 3 46.941 7.403E-015 0.007 0.228 pathway Transcriptional 4 46.587 9.459E-015 0.007 0.224 regulation by Nrf 48h fast Alternative complement 5 36.447 1.067E-011 0.004 0.538 pathway Intermediate filament Isolation of renal cortex 6 36.356 1.137E-011 0.010 0.085 signaling pathway

Fold E High Low Protein Function Protein sample change preparation 10.3 Control Knockout Carboxylesterase 1C (Ces1c) TG and CE hydrase 1.9 Control Knockout Carboxylesterase 1D (Ces1d) TG and CE hydrase 1.8 Control Knockout Carboxylesterase 1F (Ces1f) TG and CE hydrase Proteomic analysis 1.7 Control Knockout Carboxylesterase 1E (Ces1e) TG and CE hydrase

Figure 5. Carboxylesterase 1 (CES1) expression is decreased in the kidney of fasted proximal tubular epithelial cell–specific O-GlcNAc transferase knockout (PTEC-Ogty/-) mice. (A) Immunostaining and (B) Western blot analysis of phosphorylated hormone-sensitive lipase (pHSL), hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and perilipin. During fasting, there were no significant differences in renal phosphorylation or the expression levels of the indicated proteins between 20-week-old control Ogty/f mice and 2 PTEC-Ogty/ mice (n=6 per group). Original magnification, 3400. (C) Brief protocol for the proteomic analysis of renal cortical samples 2 from 20-week-old control Ogty/f and PTEC-Ogty/ mice after 48 hours of fasting. (D) Pathway analysis of data from the proteomic analysis. The detailed meaning of each calculated Score, Score(P), Score(V), are Score(C) is given in Supplemental Appendix. (E) In 2 the proteomic analysis, the expression levels of carboxylesterase 1 (CES1) family proteins were lower in fasted PTEC-Ogty/ mice. CE cholesterol ester; TG, triglyceride. of sodium-glucose transport protein function during hyp- diabetes-associated hyper O-GlcNAcylation is associated with oxia,18 the precise role of O-GlcNAcylation in PTECs has not less phosphorylation of endothelial nitric oxide synthase and been fully characterized to date, in part because appropriate Akt16 as well as changes in the microvilli of PTECs mediated by genetically modified mouse models have not previously been higher expression of a-actinin 4.17 In addition, hypertension available. This study, which used genetic deletion of protein is associated with greater renal O-GlcNAcylation, which leads O-GlcNAcylation in PTECs, has demonstrated that this mod- to the downregulation of megalin in PTECs and subsequent ification is essential for normal renal energy homeostasis and proteinuria.15 In contrast, we have demonstrated that defi- the function and survival of PTECs. In the light of our recent cient O-GlcNAcylation also causes Fanconi syndrome–like report showing that O-GlcNAcylation is also critical for abnormalities in fasting and exacerbates tubulopathy in podocyte function,44 this modification may be important for mice with diabetic atherosclerosis (Figure 9B). These appar- many types of kidney cells in addition to PTECs and podocytes, ently conflicting results may imply that both deficiency and because it can be identified in most parts of the nephron. excess of O-GlcNAcylation are harmful to PTECs, and the The high levels of renal O-GlcNAcylation present maintenance of O-GlcNAcylation within a narrow effective in disease have been thought to be pathogenic. For instance, range is important for the normal function of PTECs.

972 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

ABFed Fast D y/f y/- y/f y/- NS Triglycerides Ogt PTEC-Ogt Ogt PTEC-Ogt FA NS FA FA 2.0 P<0.01 P<0.01 Triglyceride 1.5 CES1 hydrase IHC: Ces1 FA 1.0 FA Glycerol C 0.5 FA Ces1 CES1 / β actin 0 y/f y/f y/- y/- β actin Ogt Ogt

Ogty/f PTEC-Ogty/- Ogty/f PTEC-Ogty/- PTEC-Ogt PTEC-Ogt Fed Fast Fed Fast

E F G 2.0 30 P<0.01 8 P<0.05 NS P<0.01 1.5 P<0.05 6 P<0.05 20 1.0 4 10

CE content 0.5 2 ATP contents (µmol / g tissue) (pmol / µg protein)

0 CE / Total Cholesterol 0 0 CES1 / Bodipy DAPI y/f y/f y/f y/f y/- y/- y/- y/- Ogt Ogt Ogt Ogt y/f y/- y/- CES1(-)

Ogt PTEC-Ogt PTEC-Ogt CES1(+) CES1(-) CES1(-) CES1(+) +CES1(-) y/f PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC-Ogt PTEC y/-

Ogt -Ogt Fed Fast Fed Fast

HI 2.5 P<0.01 2.0 P<0.01 C-casp3 1.5 1.0 β actin β actin 0.5

mRNA of CES1 / 0 y/f y/- y/- y/f y/f Ogt PTEC-Ogt PTEC-Ogt y/- y/- CES1(-) CES1(-) CES1(+) Ogt Ogt PTEC-Ogt PTEC-Ogt

Fed Fast

Figure 6. Decreased Carboxylesterase 1 (CES1) expression is responsible for the impaired lipid droplet breakdown in proximal tubular epithelial cell–specific O-GlcNAc transferase knockout (PTEC-Ogty/-) mice. (A) CES1 is a triglyceride hydrolase that hydrolyzes tri- 2 glyceride to liberate glycerol and fatty acids (FAs). (B–D) Renal CES1 expression in 20-week-old control Ogty/f mice and PTEC-Ogty/ 2 mice, demonstrated using (B) immunostaining and (C and D) Western blotting, in the kidneys of fasted PTEC-Ogty/ mice was lower than in fasted Ogty/f mice, regardless of feeding status (n=5 each). Original magnification, 3400. (E) Renal cholesterol ester (CE) 2 concentration and the ratio of cholesterol ester to total cholesterol concentrations were higher in PTEC-Ogty/ mice (n=5 per group). (F) Overexpression of CES1 restored triglyceride degradation in the isolated proximal tubular epithelial cells (PTECs) of 20-week-old 2 PTEC-Ogty/ mice. Red, green, and blue colors indicate CES1 protein expression, boron-dipyrromethene (BODIPY)–stained lipid droplets, and 49,6-diamidino-2-phenylindole (DAPI)–stained nuclei, respectively. Original magnification, 3400. (G and H) Overexpression of CES1 (G) restored ATP production (n=5 per group) and (H) inhibited apoptosis, which is indicated by cleavage of 2 2 caspase 3 in isolated PTECs from the 20-week-old PTEC-Ogty/ mice. (I) Renal CES1 mRNA expression in the PTEC-Ogty/ mice was lower, regardless of feeding status (n=5 per group). Horizontal bars in the graphs of D, E, G, and I indicate the median values. P,0.05 was considered statistical significance.

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 973 BASIC RESEARCH www.jasn.org

A B Fold High Low Fold High Low FXR-regulated proteins PPARα-regulated proteins change condition condition change condition condition 7.9 Control Knockout Fibrinogen alpha chain 5.5 Control Knockout Apolipoprotein A-II 4.1 Control Knockout Apolipoprotein A-I Very long-chain acyl-CoA 1.6 Control Knockout Organic solute transporter synthetase 3.7 Control Knockout subunit beta 1.5 Control Knockout Peroxisomal bifunctional enzyme 3.6 Control Knockout Fibrinogen gamma chain 3.1 Control Knockout Complement C3 1.5 Control Knockout Platelet glycoprotein 4 Long-chain-fatty-acid--CoA 2.7 Control Knockout Fibrinogen beta chain 1.2 Control Knockout ligase 1 Canalicular multispecific 2.3 Control Knockout organic anion transporter 1 Medium-chain specific acyl-CoA 1.1 Knockout Control Intercellular adhesion molecule dehydrogenase, mitochondrial 1.4 Control Knockout 1 N(G),N(G)-dimethylarginine Carnitine O-palmitoyltransferase 1.4 Control Knockout 1.2 Knockout Control dimethylaminohydrolase 1 1, muscle isoform 1.1 Control Knockout Fatty acid synthase 0.500 1.000 2.000 Hydroxymethylglutaryl-CoA 1.5 Knockout Control Expression ratio: KO/Cont synthase, mitochondrial 0.750 1.500

C (O-GlcNAcylation) D E P<0.01 IB: HSL P<0.01 IB: RL2 40 IB: ATGL P<0.01 30 IB: Perilipin 20 IgG FXR

mRNA of NS IB: CES1 10 CES1 / β actin Pre-IP IP 0 IB: FXR IB: PPARα FXR agonist ––+ +

(GW4064) y/f y/- IgG RL2 IgG RL2 Ogt Ogt Pre-IP Pre-IP IP IP

Figure 7. O-GlcNAcylation to farnesoid X receptor (FXR) is essential for Carboxylesterase 1 (CES1) expression. (A and B) Fold dif- ferences in the renal expression of proteins regulated by (A) FXR and (B) peroxisome proliferator-activated receptor-a (PPARa)inthe 2 proteomic data. The expression of FXR-regulated proteins was lower in the kidneys of PTEC-Ogty/ mice, and the expression of PPARa-regulated proteins was inconsistent. (C) Immunoprecipitation studies using RL-2 and FXR antibodies in renal cortex samples from 20-week-old wild-type mice. FXR was O-GlcNAcylated. (D) Immunoprecipitation study using an RL-2 antibody to detect O-GlcNAcylated proteins. Hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin, carboxylesterase 1 (CES1), and PPARa were not O-GlcNAcylated. (E) FXR agonist (GW4064) treatment significantly increased mRNA expression of CES1 in isolated 2 proximal tubular epithelial cells from 20-week-old Ogty/f mice, but it failed to increase it in cells from PTEC-Ogty/ mice (n=5 per group). Horizontal bars in the graphs of E indicate the median values. P,0.05 was considered statistical significance. IB, immunoblot; IP, immunoprecipitation; KO, knockout.

The circumstances under which protein O-GlcNAcylation is shown to be key pathogenic factors in the progressive decline suppressed or enhanced remain to be established. In general, hy- in renal function that occurs in patients with diabetes who perglycemia has been thought to increase cellular O-GlcNAcylation do not show albuminuria.47 Thus, a deficiency in protein levels in various tissues.45,46 Actually, in this study, protein O-GlcNAcylation may be involved particularly in DKD with- O-GlcNAcylation in PTECs was greater in diabetic db/db out albuminuria rather than in typical progressive DKD show- mice and HFD-induced obese mice, but no tubulointerstitial ing albuminuria. lesions were evident in these models. Surprisingly, the addi- Our proteomic analysis has shown that renal CES1 expres- tional presence of atherosclerotic lesions in HFD-induced sion is downregulated by deficient O-GlcNAcylation. CES1 is obese condition was associated with much lower renal protein generally thought to be a cholesterol esterase.37 In fact, renal 2 O-GlcNAcylation and severe tubulopathy. These findings sug- cholesterol ester accumulated in PTEC-Ogty/ mice during gest that hypernutrition in diabetes is associated with greater both the fed and fasting states, suggesting that Ogt deficiency renal protein O-GlcNAcylation as previously reported,46 but suppressed CES1 activity. Furthermore, a recent study has proatherosclerotic stimuli, such as hypoxia, may have the op- demonstrated that this enzyme has a triglyceride hydrolase posite effect. Recently, atherosclerosis and hypoxia have been activity.38 Our data showing that CES1 overexpression is

974 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH

AB db/m db/db ND-ApoE+/+ HFD-ApoE+/+ HFD-ApoE-/- ( ) PTEC-Ogty/- ( ) Ogty/f HE

High-fat diet (16 weeks) y/f C Ogt PTEC-Ogty/- 50 HFD start 40 TM injection 30 20 NS P<0.05

10 P<0.05 Body weight (g) P<0.05 F4/80 P<0.05 0 8 12162024 D (Week-old) y/f 200 Ogt y/- NS RL2 PTEC-Ogt 150

(O-GlcNAcylation) Fibronectin 100 NS NS

50 NS NS NS 0 Blood glucose (mg/dl)

CES1 0 30 60 90 120 (min after insulin injection)

E HE stain F Oil-Red O Fibronectin Ces1 10 P<0.05 y/f y/f 8 6 4 HFD-fed Ogt HFD-fed Ogt

damage score 2 Tubulointerstitial

0 y/- y/- y/f y/- Ogt HFD-fed HFD-fed HFD-fed HFD-fed PTEC-Ogt PTEC-Ogt PTEC-Ogt

Figure 8. Deficient O-GlcNAcylation exacerbates lipotoxicity-related kidney injury in high-fat diet (HFD)-induced obese type 2 diabetic mice. (A) Renal tubulointerstitial lesions, identified by hematoxylin and eosin (HE) staining and immunostaining for fibronectin and F4/80, were not severe in 18-week-old db/db mice and 32-week-old HFD-fed ApoE+/+ mice, but they were more severe in 2 2 32-week-old HFD-fed ApoE / mice. Renal protein O-GlcNAcylation and carboxylesterase 1 (CES1) expressions were high in db/db 2 2 mice and HFD-fed ApoE+/+ mice, but they were lower in HFD-fed ApoE / mice. Original magnification, 3400. (B) HFD intervention 2 2 study in Ogty/f mice and PTEC-Ogty/ mice (n=5 per group). (C) Change in mean body mass. PTEC-Ogty/ mice showed lower body mass gain during the HFD-feeding period. (D) Change in mean blood glucose concentration during an intraperitoneal insulin tolerance 2 test. There was no significant difference in insulin sensitivity between 24-week-old Ogty/f and PTEC-Ogty/ mice. (E) Renal tubular cell 2 damage in HE-stained sections was significantly worse in PTEC-Ogty/ mice. Original magnification, 3400. (F) Oil-red O staining of 2 positive neutral lipid and fibronectin deposition was higher in the kidneys of PTEC-Ogty/ mice, and it was accompanied by lower CES1 expression. Original magnification, 3400. Horizontal bars in E indicate the median values for each group. P,0.05 was considered statistical significance. ND, normal diet; TM, tamoxifen.

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 975 BASIC RESEARCH www.jasn.org

ABO-GlcNAcylation-deficiency Feeding Fasting under fasting or diabetes Fatty acid (FA) Triglycerides Triglycerides Triglycerides FA FA FA FA FA FA FA FA FA Liver Adipocytes Lipolysis (Hormone- Adipocytes Lipolysis (Hormone- Endothelial cells sensitive lipase) sensitive lipase)

Free fatty acid Free fatty FA FA Albumin Albumin FA FA FA FA (FFA) acid (FFA) FA FA FA FA FFA FFA FFA FFA FA FA FFA FFA LPL LPL LPL LPL FFA FFA

Lipid droplets Fatty acid Lipid droplets Fatty acid (Triglyceride) transporter (Triglyceride) transporter FA FA FA FA FA FA FA FA FA FA FA FA FA FA FA Lipolysis Nuclei Nuclei Nuclei FA FA FA FA Mitochondria FA FA Mitochondria FA FA Mitochondria FA FA FA FA ATP ATP production Lipolysis production CES1 Lipolysis

Proximal tubular Fasting epithelial cells Ogt-mediated protein FXR CES1 ATP production O-GlcNAcylation Impaired lipolysis Lipotoxicity Diabetes

Figure 9. Proposed hypothesis. (A) Ogt-mediated protein O-GlcNAcylation plays a critical role in maintaining renal lipolysis– dependent ATP production in proximal tubular cells during the switch from the fed to the fasting state. (B) Deficient O-GlcNAcylation impairs intracellular lipid droplet breakdown, which leads to lower ATP production during fasting and an exacerbation of intrarenal lipotoxicity in diabetes. The farnesoid X receptor (FXR)-carboxylesterase 1 (CES1) pathway may be involved in the maintenance of renal lipid metabolism, and it is regulated by O-GlcNAcylation. FA, fatty acid; FFA, free fatty acid. able to restore lipid droplet breakdown in isolated PTECs from clarification of the pathogenesis of kidney diseases. However, 2 PTEC-Ogty/ mice provides additional evidence for the role of our findings have revealed only part of the physiology of CES1 in triglyceride metabolism. In this study, although renal lipolysis; additional work is required to elucidate the 2 PTEC-Ogty/ mice demonstrated low CES1 protein expression, mechanisms in more depth. In most mammalian cell types, regardless of feeding status, they developed renal abnormalities LPL-dependent lipolysis of circulating triglycerides at the en- only after fasting. This suggests that the use of triglyceride, rather dothelial surface and CD36-dependent uptake of albumin- than cholesterol ester, as a substrate for CES1 is more important bound free FAs have been thought to be the means by which for ATP production during fasting. cells obtain FAs from the bloodstream during feeding and Previous work has demonstrated that loss and gain of func- fasting, respectively.8,9 However, it has still not been proven tion of CES1 in mice worsens and improves hepatosteatosis, that LPL is critical for FA uptake by PTECs during feeding. respectively.40 These results are consistent with our findings Furthermore, a recent study showed that CD36 is not involved that low CES1 expression is associated with renal lipotoxicity in FA uptake by PTECs during fasting.9 Thus, the regulation of in HFD-fed mice and that high CES1 expression ameliorates FA uptake by PTECs seems to be different from that in other cell damage in primary Ogt-deficient PTECs, and they suggest cell types. If the unique mechanisms involved in renal lipid that CES1 gain of function may represent a promising thera- metabolism can be elucidated in greater depth, this should peutic target for lipotoxicity-associated kidney diseases. Our contribute to better understanding of the pathogenesis of data also suggest that the transcription factor FXR is a candi- CKD, including DKD. date for regulation by O-GlcNAcylation, although a direct re- There were a couple of limitations to this study. First, lationship between CES1 and FXR has not been fully shown in NDRG1 is expressed in parts of the nephron other than PTECs. this study. Given that a renoprotective effect of an FXR agonist Therefore, although the Fanconi syndrome–like phenomenon 2 has recently been reported48250 and that this agonist increased and changes in renal cortical samples in PTEC-Ogty/ mice renal CES1 expression in our study, CES1 overexpression may most likely reflect changes in PTEC function, not all of the be involved in the mechanism underpinning FXR-mediated changes that occurred in this mouse model may be explained renoprotection. by abnormalities in PTECs. Second, regulation of lipid Our findings are consistent with the notion that a novel metabolism during fasting is one of the roles of renal finding regarding the physiology of renal lipolysis can lead to O-GlcNAcylation. Actually, our proteomics analysis identified

976 JASN JASN 30: 962–978, 2019 www.jasn.org BASIC RESEARCH some proteins with changed expression levels in the kidneys of Supplemental Table 1. List of antibodies used in this study. 2 PTEC-Ogty/ mice. For example, Renin-2 protein levels were Supplemental Table 2. List of primer sets used in this study. 2 largely increased in the kidneys of the fasted PTEC-Ogty/ Supplemental Table 3. Fold changes in expression levels of proteins mice. We still do not know the exact cause and significance of associated with fatty acid metabolism in the proteomic analysis this change. However, given that active renin was found in proximal using renal cortex samples obtained from Ogty/f mice (control) and 2 convoluted tubules,51 intrarenal protein O-GlcNAcylation may PTEC-Ogty/ mice (knockout). be directly involved in the regulation of RAS. Thus, unraveling the remaining cell-specific effects of protein O-GlcNAcylation should provide additional insights into renal physiology. REFERENCES In conclusion, intracellular lipid metabolism is maintained in PTECs using protein O-GlcNAcylation, despite increases in 1. Schmidt U, Guder WG: Sites of enzyme activity along the nephron. – FA uptake in situations, such as starvation or obesity and di- Kidney Int 9: 233 242, 1976 2. Balaban RS, Mandel LJ: Metabolic substrate utilization by rabbit prox- abetes. The protein O-GlcNAcylation-FXR-CES1 axis may be imal tubule. An NADH fluorescence study. Am J Physiol 254: F407– the mechanism responsible. Our results provide novel insight F416, 1988 into proximal tubule physiology and should contribute to bet- 3. Gullans SR, Brazy PC, Mandel LJ, Dennis VW: Stimulation of phosphate ter understanding of the kidney diseases associated with ab- transport in the proximal tubule by metabolic substrates. Am J Physiol – normal lipid metabolism. 247: F582 F587, 1984 4. Uchida S, Endou H: Substrate specificity to maintain cellular ATP along the mouse nephron. Am J Physiol 255: F977–F983, 1988 5. Kume S, Uzu T, Araki S, Sugimoto T, Isshiki K, Chin-Kanasaki M, et al.: Role of altered renal lipid metabolism in the development of renal injury ACKNOWLEDGMENTS induced by a high-fat diet. J Am Soc Nephrol 18: 2715–2723, 2007 6. Kang HM, Ahn SH, Choi P, Ko YA, Han SH, Chinga F, et al.: Defective We thank Naoko Yamanaka and Keiko Kosaka (Shiga University of fatty acid oxidation in renal tubular epithelial cells has a key role in Medical Science) and the Central Research Laboratory of Shiga kidney fibrosis development. Nat Med 21: 37–46, 2015 7. JiangT,WangZ,ProctorG,MoskowitzS,LiebmanSE,RogersT,etal.:Diet- University of Medical Science for technical assistance. fi induced obesity in C57BL/6J mice causes increased renal lipid accumula- This study was supported by Grants-in-Aid for Scienti c Research tion and glomerulosclerosis via a sterol regulatory element-binding (KAKENHI) 25713033 (to Dr. Kume), 25461219 (to Dr. Chin- protein-1c-dependent pathway. J Biol Chem 280: 32317–32325, 2005 Kanasaki), and 18H02862 (to Dr. Maegawa) from the Japan Society 8. Goldberg IJ, Eckel RH, Abumrad NA: Regulation of fatty acid uptake for the Promotion of Science; grants from the Lilly Grant Office (to into tissues: Lipoprotein lipase- and CD36-mediated pathways. JLipid Res 50[Suppl]: S86–S90, 2009 Dr. Kume and Dr. Maegawa); Bayer Academic Support (Dr. Kume 9. Scerbo D, Son NH, Sirwi A, Zeng L, Sas KM, Cifarelli V, et al.: Kidney fi and Dr. Maegawa); a Sano Grant (to Dr. Kume); and an MSD Grant triglyceride accumulation in the fasted mouse is dependent upon se- (to Dr. Chin-Kanasaki). rum free fatty acids. J Lipid Res 58: 1132–1142, 2017 10. Hardivillé S, Hart GW: Nutrient regulation of signaling, transcription, and cell physiology by O-GlcNAcylation. Cell Metab 20: 208–213, 2014 DISCLOSURES 11. Hanover JA, Krause MW, Love DC: Bittersweet memories: Linking metabolism to epigenetics through O-GlcNAcylation. Nat Rev Mol Cell fi Dr. Kume reports grants from Lilly Grant Of ce, grants from Bayer Ac- Biol 13: 312–321, 2012 fi ademic Support, grants from Sano Grant,andgrantsfromtheJapanSociety 12. Janetzko J, Walker S: The making of a sweet modification: Structure and for the Promotion of Science during the conduct of the study. Dr. Chin- function of O-GlcNAc transferase. J Biol Chem 289: 34424–34432, 2014 Kanasaki reports grants from MSD Grant and grants from the Japan Society 13. MisraJ,KimDK,JungYS,KimHB,KimYH,YooEK,etal.:O-GlcNAcylation for the Promotion of Science during the conduct of the study. Dr. Maegawa of orphan nuclear receptor estrogen-related receptor g promotes hepatic fi reports grants from Lilly Grant Of ce, grants from Bayer Academic Support, gluconeogenesis. Diabetes 65: 2835–2848, 2016 and grants from the Japan Society for the Promotion of Science during the 14. Ohashi N, Morino K, Ida S, Sekine O, Lemecha M, Kume S, et al.: Pivotal conduct of the study. Dr. Sugahara, Dr. Tomita, Dr. Yasuda-Yamahara, role of O-GlcNAc modification in cold-induced thermogenesis by Dr.Yamahara,Dr.Takeda,Dr.Osawa,Dr.Yanagita,andDr.Arakihadnoth- brown adipose tissue through mitochondrial biogenesis. Diabetes 66: ing to disclose. 2351– 2362, 2017 15. Silva-Aguiar RP, Bezerra NCF, Lucena MC, Sirtoli GM, Sudo RT, Zapata- Sudo G, et al.: O-GlcNAcylation reduces proximal tubule protein re- SUPPLEMENTAL MATERIAL absorption and promotes proteinuria in spontaneously hypertensive rats. JBiolChem293: 12749–12758, 2018 16. Gellai R, Hodrea J, Lenart L, Hosszu A, Koszegi S, Balogh D, et al.: Role This article contains the following supplemental material online at of O-linked N-acetylglucosamine modification in diabetic nephropa- http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018090950/-/ thy. Am J Physiol Renal Physiol 311: F1172–F1181, 2016 DCSupplemental. 17. Akimoto Y, Miura Y, Toda T, Wolfert MA, Wells L, Boons GJ, et al.: Supplemental Appendix. Spreadsheet. Morphological changes in diabetic kidney are associated with in- Supplemental Figure 1. Fatty acid transport into cells during creased O-GlcNAcylation of cytoskeletal proteins including a-actinin 4. Clin Proteomics 8: 15, 2011 feeding and fasting 18. SuhHN,LeeYJ,KimMO,RyuJM,HanHJ:Glucosamine-inducedSp1O- Supplemental Figure 2. Full gelscan images for the immune blots of GlcNAcylation ameliorates hypoxia-induced SGLT dysfunction in primary the indicated figure number. cultured renal proximal tubule cells. JCellPhysiol229: 1557–1568, 2014

JASN 30: 962–978, 2019 O-GlcNAcylation in Renal Lipolysis 977 BASIC RESEARCH www.jasn.org

19. Peruchetti DB, Silva-Aguiar RP, Siqueira GM, Dias WB, Caruso-Neves 35. Greenberg AS, Egan JJ, Wek SA, Garty NB, Blanchette-Mackie EJ, C: High glucose reduces megalin-mediated albumin endocytosis in Londos C: Perilipin, a major hormonally regulated adipocyte-specific renal proximal tubule cells through protein kinase B O-GlcNAcylation. phosphoprotein associated with the periphery of lipid storage drop- JBiolChem293: 11388–11400, 2018 lets. J Biol Chem 266: 11341–11346, 1991 20. Shafi R, Iyer SP, Ellies LG, O’Donnell N, Marek KW, Chui D, et al.: The O- 36. Alam M, Ho S, Vance DE, Lehner R: Heterologous expression, purifi- GlcNAc transferase gene resides on the X chromosome and is essential cation, and characterization of human triacylglycerol hydrolase. Protein for embryonic stem cell viability and mouse ontogeny. Proc Natl Acad Expr Purif 24: 33–42, 2002 Sci U S A 97: 5735–5739, 2000 37. Crow JA, Middleton BL, Borazjani A, Hatfield MJ, Potter PM, Ross MK: 21. Endo T, Nakamura J, Sato Y, Asada M, Yamada R, Takase M, et al.: Inhibition of carboxylesterase 1 is associated with cholesteryl ester re- Exploring the origin and limitations of kidney regeneration. J Pathol tention in human THP-1 monocyte/macrophages. Biochim Biophys 236: 251–263, 2015 Acta 1781: 643–654, 2008 22. Huang ZH, Reardon CA, Mazzone T: Endogenous ApoE expression 38. Dolinsky VW, Gilham D, Alam M, Vance DE, Lehner R: Triacylglycerol modulates adipocyte triglyceride content and turnover. Diabetes 55: hydrolase: Role in intracellular lipid metabolism. Cell Mol Life Sci 61: 3394–3402, 2006 1633–1651, 2004 23. Tanaka Y, Kume S, Araki S, Isshiki K, Chin-Kanasaki M, Sakaguchi M, 39. Ghosh S, Natarajan R: Cloning of the human cholesteryl ester hydrolase et al.: Fenofibrate, a PPARa agonist, has renoprotective effects in mice promoter: Identification of functional peroxisomal proliferator-acti- by enhancing renal lipolysis. Kidney Int 79: 871–882, 2011 vated receptor responsive elements. Biochem Biophys Res Commun 24. Ueda S, Ozawa S, Mori K, Asanuma K, Yanagita M, Uchida S, et al.: 284: 1065–1070, 2001 ENOS deficiency causes podocyte injury with mitochondrial abnor- 40. Xu J, Li Y, Chen WD, Xu Y, Yin L, Ge X, et al.: Hepatic carboxylesterase 1 mality. Free Radic Biol Med 87: 181–192, 2015 is essential for both normal and farnesoid X receptor-controlled lipid 25. Yamahara K, Kume S, Koya D, Tanaka Y, Morita Y, Chin-Kanasaki M, homeostasis. Hepatology 59: 1761–1771, 2014 et al.: Obesity-mediated autophagy insufficiency exacerbates 41. Herman-Edelstein M, Scherzer P, Tobar A, Levi M, Gafter U: Altered proteinuria-induced tubulointerstitial lesions. J Am Soc Nephrol renal lipid metabolism and renal lipid accumulation in human diabetic 24: 1769–1781, 2013 nephropathy. J Lipid Res 55: 561–572, 2014 26. Kimura T, Takabatake Y, Takahashi A, Kaimori JY, Matsui I, Namba T, 42. Chin M, Isono M, Isshiki K, Araki S, Sugimoto T, Guo B, et al.: Estrogen et al.: Autophagy protects the proximal tubule from degeneration and and raloxifene, a selective estrogen receptor modulator, ameliorate acute ischemic injury. JAmSocNephrol22: 902–913, 2011 renal damage in db/db mice. Am J Pathol 166: 1629–1636, 2005 27. Soumura M, Kume S, Isshiki K, Takeda N, Araki S, Tanaka Y, et al.: 43. Shibuya K, Kanasaki K, Isono M, Sato H, Omata M, Sugimoto T, et al.: Oleate and eicosapentaenoic acid attenuate palmitate-induced in- N-acetyl-seryl-aspartyl-lysyl-proline prevents renal insufficiency and flammation and apoptosis in renal proximal tubular cell. Biochem mesangial matrix expansion in diabetic db/db mice. Diabetes 54: 838– Biophys Res Commun 402: 265–271, 2010 845, 2005 28. Iwai T, Kume S, Chin-Kanasaki M, Kuwagata S, Araki H, Takeda N, et al.: 44. Ono S, Kume S, Yasuda-Yamahara M, Yamahara K, Takeda N, Chin- Stearoyl-CoA desaturase-1 protects cells against lipotoxicity-mediated Kanasaki M, et al.: O-linked b-N-acetylglucosamine modification of apoptosis in proximal tubular cells. Int J Mol Sci 17: pii: E1868, 2016 proteins is essential for foot process maturation and survival in podo- 29. Yasuda M, Tanaka Y, Kume S, Morita Y, Chin-Kanasaki M, Araki H, et al.: cytes. Nephrol Dial Transplant 32: 1477–1487, 2017 Fatty acids are novel nutrient factors to regulate mTORC1 lysosomal 45. Copeland RJ, Bullen JW, Hart GW: Cross-talk between GlcNAcylation localization and apoptosis in podocytes. Biochim Biophys Acta 1842: and phosphorylation: Roles in insulin resistance and glucose toxicity. 1097–1108, 2014 Am J Physiol Endocrinol Metab 295: E17–E28, 2008 30. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and puri- 46. Degrell P, Cseh J, Mohás M, Molnár GA, Pajor L, Chatham JC, et al.: fication. Can J Biochem Physiol 37: 911–917, 1959 Evidence of O-linked N-acetylglucosamine in diabetic nephropathy. 31. Kume S, Uzu T, Horiike K, Chin-Kanasaki M, Isshiki K, Araki S, et al.: Life Sci 84: 389–393, 2009 Calorie restriction enhances cell adaptation to hypoxia through Sirt1- 47. Bolignano D, Zoccali C: Non-proteinuric rather than proteinuric renal dependent mitochondrial autophagy in mouse aged kidney. JClin diseases are the leading cause of end-stage kidney disease. Nephrol Invest 120: 1043–1055, 2010 Dial Transplant 32[Suppl_2]: ii194–ii199, 2017 32. Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T, 48. Zhao K, He J, Zhang Y, Xu Z, Xiong H, Gong R, et al.: Activation of FXR et al.: Chemical inhibition of the mitochondrial division dynamin reveals protects against renal fibrosis via suppressing Smad3 expression. Sci its role in Bax/Bak-dependent mitochondrial outer membrane per- Rep 6: 37234, 2016 meabilization. Dev Cell 14: 193–204, 2008 49. Gai Z, Gui T, Hiller C, Kullak-Ublick GA, Farnesoid X: Farnesoid X re- 33. Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr., Londos C: ceptor protects against kidney injury in uninephrectomized obese Mechanism of hormone-stimulated lipolysis in adipocytes: Trans- mice. JBiolChem291: 2397–2411, 2016 location of hormone-sensitive lipase to the lipid storage droplet. Proc 50. Wang XX, Wang D, Luo Y, Myakala K, Dobrinskikh E, Rosenberg AZ, Natl Acad Sci U S A 89: 8537–8541, 1992 et al.: FXR/TGR5 dual agonist prevents progression of nephropathy in 34. Zechner R, Kienesberger PC, Haemmerle G, Zimmermann R, Lass A: diabetes and obesity. JAmSocNephrol29: 118–137, 2018 Adipose triglyceride lipase and the lipolytic catabolism of cellular fat 51. Chen M, Harris MP, Rose D, Smart A, He XR, Kretzler M, et al.: Renin and renin stores. J Lipid Res 50: 3–21, 2009 mRNA in proximal tubules of the rat kidney. J Clin Invest 94: 237–243, 1994

978 JASN JASN 30: 962–978, 2019 Online Data Supplement

Protein O-GlcNAcylation is essential for the maintenance of renal energy homeostasis and function via lipolysis during fasting and diabetes

Sho Sugahara,1 Shinji Kume,1 Masami Chin-Kanasaki,1,2 Isssei Tomita,1 Mako Yasuda-Yamahara,1 Kosuke Yamahara,1 Naoko Takeda,1 Norihisa Osawa,1 Motoko Yanagita,3 Shin-ichi Araki,1,2 Hiroshi Maegawa1

1Department of Medicine, Shiga University of Medical Science, Otsu, Shiga, Japan 2Division of Blood Purification, Shiga University of Medical Science Hospital, Otsu, Shiga, Japan 3Department of Nephrology, Graduate School of Medicine, Kyoto University Hospital, Kyoto, Japan

Contents

1. Supplemental Table 1. List of antibodies used in this study 2. Supplemental Table 2. List of primer sets used in this study 3. Supplemental Table 3. Fold changes in expression levels of proteins associated with fatty acid metabolism in the proteomic analysis using renal cortex samples obtained from Ogty/f mice (control) and PTEC-Ogty/- mice (Knockout) 4. Supplemental Figure 1. Fatty acid transport into cells during feeding and fasting 5. Supplemental Figure 2. Full gel scan images for the immune blots of the indicated figure number

Supplemental Table 1. List of antibodies used in this study.

Antibody Company Catalog No.

Cre recombinase Cell Signaling #12830

O-GlcNAc transferase (Ogt) Abcam #ab96718

RL2 (O-GlcNAcylation) Abcam #ab2739

Cleaved caspase 3 Cell Signaling #9661

Poly（ADP-ribose）polymerase (PARP) Cell Signaling #9542

β actin Sigma Aldrich #123M4876

Carboxylesterase 1 (CES1) Abcam #ab45957

Phosphorylated hormone sensitive lipase (pHSLser563) Cell Signaling #4139

Hormone sensitive lipase (HSL) Cell Signaling #4107

Adipose triglyceride lipase (ATGL) Cell Signaling #2138

Perilipin Cell Signaling #9349

Peroxisome proliferator-activated receptor-α (PPARα) Santa Cruz #sc-9000

Farnesoid X receptor (FXR) Abcam #ab28480

Fibronectin Millipore #AB2033

F4/80 BIO-RAD #MCA497GA

Lipoprotein lipase (LPL) Abcam #ab21356

Lotus Tetragonolobus lectin (LTL) Vector Laboratories #B-1325 Supplemental Table 2. List of primer sets used in this study.

mRNA Accession No. Forward Reverse

ATP1b1 NM_009721.6 5’-tgtgcaggttcaagcttgac-3’ 5’-ttcggtttgaagcccaacac-3’

NaPi-IIa NM_011392.2 5’-taactggctgtctgttctggtc-3’ 5’-tgaaggaagcaaccacaagc-3’

URAT NM_009203.3 5’-ttggacccgatgttcttctgg-3’ 5’-aagctgccattgaggttgtc-3’

SGLT2 NM_133254.4 5’-attgtctcgggctggtattgg-3’ 5’-acaagatgcacccagctttg-3’

NHE3 NM_001081060.1 5’-atcaccttttgcggcatctg-3’ 5’-actggccagcatcttcatagtg-3’

β actin NM_007393.5 5’-cgtgcgtgacatcaaagagaa-3’ 5’-tggatgccacaggattccat-3’

OAT1 NM_008766.3 5’-tggtttgccactagctttgc-3’ 5’-aggaagcacacaaacttggc-3’

LPL NM_008509.2 5’-gcccagcaacattatccagt-3’ 5’-ggtcagacttcctgctacgc-3’

GPIHBP1 NM_026730.2 5’-tgcaatcagacacagagctg-3’ 5’-acaagtgaagaagcggttcc-3’

CES1 NM_021456.4 5’-cttggatctctgaggtttgctc-3’ 5’-gggttttggtagcacaaagg-3’ Supplemental Table 3. Fold changes in expression levels of proteins associated with fatty acid metabolism in the proteomic analysis using renal cortex samples obtained from Ogty/f mice (control) and PTEC-Ogty/- mice (Knockout).

Fold Highest Lowest Description Function change condition condition 10.3 Control Knockout Carboxylesterase 1C Triglyceride esterase 3.6 Control Knockout Acylcarnitine hydrolase (Carboxylesterase 2) Carboxylic ester hydrolase 3.3 Knockout Control Acyl-coenzyme A thioesterase 11 Fatty acid synthase 2.6 Control Knockout Very long-chain specific acyl-CoA dehydrogenase, mitochondrial Beta oxidation 2.6 Knockout Control Perilipin-2 Lipid droplet 2.2 Control Knockout Acyl-coenzyme A thioesterase 1 Fatty acid synthase 2.1 Knockout Control Acyl-CoA synthetase family member 2, mitochondrial Fatty acid synthase 2.1 Control Knockout Very-long-chain enoyl-CoA reductase Fatty acid synthase 2.0 Control Knockout ATP synthase subunit epsilon, mitochondrial Mitochondria 2.0 Knockout Control Acyl-coenzyme A thioesterase 9, mitochondrial Fatty acid synthase 2.0 Control Knockout Enoyl-CoA hydratase domain-containing protein 3, mitochondrial Beta oxidation 2.0 Knockout Control Mitochondrial fission 1 protein Mitochondria fission 1.9 Knockout Control Glutamate dehydrogenase 1, mitochondrial Gluconeogenesis 1.9 Control Knockout Carboxylesterase 1D Triglyceride esterase 1.9 Control Knockout 3-ketoacyl-CoA thiolase B, peroxisomal Beta oxidation 1.8 Control Knockout Peroxisomal acyl-coenzyme A oxidase 1 Beta oxidation 1.8 Knockout Control Enoyl-[acyl-carrier-protein] reductase, mitochondrial Fatty acid synthase 1.8 Control Knockout Carboxylesterase 1F Triglyceride esterase 1.8 Control Knockout ATPase inhibitor, mitochondrial Mitochondria 1.7 Knockout Control 5'-AMP-activated protein kinase catalytic subunit alpha-1 AMPK 1.7 Knockout Control Elongation factor Tu, mitochondrial Mitochondria 1.7 Knockout Control Glycerol-3-phosphate dehydrogenase, mitochondrial Glycerol-3-phosphate 1.7 Knockout Control Hydroxymethylglutaryl-CoA lyase, mitochondrial Ketone metabolism 1.7 Knockout Control Acyl-coenzyme A synthetase ACSM1, mitochondrial Beta oxidation 1.7 Knockout Control Acyl-CoA dehydrogenase family member 10 Beta oxidation 1.7 Control Knockout Carboxylesterase 1E Triglyceride esterase 1.7 Knockout Control Glutaminase kidney isoform, mitochondrial Gluconeogenesis 1.6 Knockout Control Acyl-coenzyme A thioesterase 8 Fatty acid synthase 1.6 Knockout Control Acyl-coenzyme A thioesterase 13 Fatty acid synthase 1.6 Knockout Control Peroxisomal acyl-coenzyme A oxidase 2 Beta oxidation 1.6 Control Knockout Very long-chain acyl-CoA synthetase Beta oxidation 1.6 Knockout Control Acyl-CoA synthetase family member 3, mitochondrial Beta oxidation 1.6 Knockout Control Cytochrome c oxidase assembly protein COX15 homolog Mitochondria 1.6 Knockout Control Acyl-coenzyme A thioesterase Fatty acid synthase 1.6 Knockout Control Cytochrome c oxidase subunit 7A1, mitochondrial Mitochondria 1.6 Knockout Control Cytochrome c oxidase subunit 7A-related protein, mitochondrial Mitochondria 1.6 Knockout Control Glycerol-3-phosphate dehydrogenase [NAD(+)], cytoplasmic Glycerol-3-phosphate 1.6 Knockout Control Acyl-CoA dehydrogenase family member 9, mitochondrial Beta oxidation 1.5 Control Knockout Phosphoglycerate kinase 1 Gluconeogenesis/glycolysis 1.5 Knockout Control Peroxisomal acyl-coenzyme A oxidase 3 Fatty acid synthase 1.5 Control Knockout Fatty acid-binding protein, epidermal Fatty acid-binding protein 1.5 Knockout Control Enoyl-CoA hydratase, mitochondrial Beta oxidation 1.5 Control Knockout Enoyl-CoA delta isomerase 3, peroxisomall Beta oxidation 1.5 Knockout Control Succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial Ketolysis Supplemental Figure 1

(A) Feeding condition (B) Fasting condition

Triglycerides Triglycerides FA FA FA Fatty acid（FA） FA FA FA Liver Adipocytes Lipolysis (Hormone-sensitive lipase) Endothelial cells

FA FA Albumin Free fatty acid FA FFA FA FA FA (FFA) FA FA FA FA FFA FA FA FFA LPL LPL LPL LPL FFA

Lipid droplets Fatty acid transporter (Triglyceride) FA FA FA FA FA FA FA FA Lipolysis Nuclei FA Nuclei FA FA FA Mitochondria FA Mitochondria FA

ATP ATP production Lypolysis production

cells cells

Supplemental Figure 1. Fatty acid transport into cells during feeding and fasting. (A) During the fed state, most cells can utilize fatty acids in an lipoprotein lipase (LPL)-dependent manner. FAs are de-esterified by LPL on the endothelial surface, enter cells, and are metabolized to yield ATP in mitochondria. (B) During the fasting state, fatty acids released from adipocytes circulate bound to albumin, and are transported into cells. After this, FAs are esterified and stored in intracellular lipid droplets. Finally, stored FAs in the lipid droplet can be used to generate ATP following lipolysis. Supplemental Figure 2

Figure 1B. Cre Figure 1B. RL2 Figure 2E. Caspase 3

Figure 2E. β actin

Figure 1B. Ogt Figure 1B. β actin

Figure 4B. LPL (MW: 53 kd) Figure 4D. IP:RL2 IB:LPL Figure 4J. Caspase 3 Figure 4J. β actin

Figure 4J. PARP

Figure 4B. β actin

Figure 6C. CES1 Figure 6H. Caspase 3

Figure 5B. pHSL (Ser563) Figure 5B. ATGL

Figure 5B. HSL Figure 6C. β actin Figure 6H. β actin Figure 5B. Perilipin

Figure 5B. β actin

Figure 7C. Figure 7C. Figure 7D. Figure 7D. IP:RL2 IB:FXR IP:FXR IB:RL2 IP:RL2 IB:Perilipin IP:RL2 IB:CES1

Figure 7D. Figure 7D. Figure 7D. IP:RL2 IB:ATGL IP:RL2 IB:HSL IP:RL2 IB:PPARα Supplemental Figure 2. Full gel scan images for the immmune blots of the indicated figure number. The areas indicated by the red box were used in the representative figures of the manuscript.