ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

and summary Mendelian randomization analyses were performed according to previous publication (2,3).

Human kidney single nuclear ATAC-sequencing
Adult human kidney single nuclear ATAC seq-data was used from previous publications (2,3).

Animal studies
The mice were fed ad libitum with water and rodent standard chow diet.6-to 7-week-old male and female C57BL/6J mice were used in the study.Mice were randomly assigned to experimental groups for all experiments including drug efficacy studies.To induce kidney injury, we employed two widely used kidney disease models including unilateral ureteral obstruction (UUO) and folic acid nephropathy (FAN).UUO surgery experiments were conducted on male and female mice.
Briefly, UUO was performed by ligating the right kidney ureter, and the left kidney served as a sham operated kidney.Post-surgical procedures were followed according to the IACUC guidelines.For FAN models, 300mM sodium bicarbonate (NaHCO 3 ) solution was first made to solubilize folic acid (FA) and injected into male mice (FA 250 mg/kg i.p at single dose).Mice were sacrificed 7 days following injection or surgery.For the drug injection studies, mice were first injected one day before the UUO surgery.

Adenine induced chronic kidney disease model
Adenine (#A11500) was purchased from RPI (Saint Louis, MO, USA).Adenine was dissolved in water at a concentration 2.5 mg/ml.Male mice were given adenine by oral gavage at a dose of 50mg/kg body weight daily for four weeks (5).Control male mice received 0.2ml of vehicle every day for four weeks.Animals were sacrificed on day 30.BUN and serum creatinine was analyzed by BUN (#B7552-150, Horiba Pointe Scientific) and creatinine kits (#DZ072B-KY1, Diazyme).
Daily body weights were recorded.

Crispr SNP deletion experiments
Two Crispr guides were generated for the open chromatin of the prioritized SNP region.Guide RNAs were designed using Crispor software (6) and cloned into pLKO5.SgRNA.EFS.GFP plasmid (#57822, Addgene) using Esp3I restriction enzyme (#FD0454, Thermo).Guide RNA sequences and human primers were listed in Supplemental table 3. Bacterial transformation was performed using OneShot-Stbl3 competent cells (#C737303, Thermo) and isolated plasmids were verified by Sanger sequencing.The guide RNA containing plasmids were transfected into HEK293 cells stably expressing Cas9 (gift from Dr. Liling Wan, University of Pennsylvania) using Lipofectamine 3000 (#L300015, Thermo).After 72h of transfection, puromycin (4µg/ml) was added for an additional 3 days.The cells were harvested, and RNA, and DNA were isolated.The DNA was cloned into TOPO-TA vector and TOP10 chemical competent cells (#K4500J10, Thermo).
Genomic region deletion was further verified by Sanger sequencing.The isolated RNA was used to measure gene expression by quantitative real time PCR.

Gene expression analysis
A total of 15mg of kidney tissue was homogenized in 1ml of Trizol (Ambion) with Qia Tissue Lyzer for 1min 15sec at 4 o C.After homogenization in Trizol, 200ul of chloroform was directly dispensed into the Trizol lysate and vortexed for 15sec at RT. Lysates were then spun down at 12000rpm for 15min at 4oC, and the upper aqueous layer was collected into new, clean tubes.Next, 500ul of isopropanol (100%) was slowly added through the wall of the tubes and mixed gently.The tubes were then spun down at 12500rpm at 4 O C for 15min, and the pellet was washed with 70% ethanol (made from clean 100% ethanol) at 10000rpm for 10min at 4oC.Finally, the RNA pellet was dried at RT for 15min, resuspended in clean RNase free and Dnase free water.RNA was pretreated with DNase before proceeding to cDNA conversion.A total of 2,000 ng RNA was converted into cDNA using the High-capacity cDNA Reverse Transcription Kit (#4368813, Applied Biosystems).Realtime quantitative PCR analysis was performed to measure the relative gene expression by normalizing the CT values of gene of interest with endogenous control gene (Gapdh was used).The data was calculated and presented as fold change by (2 -ΔΔCT method).
Primer sequence listed in Supplemental table 4.

Histone extraction and western blotting
To extract histones from kidneys, we first isolated nuclei and proceeded with acid-histone extraction method (8).Briefly, we washed nearly 40mg of kidney tissue with ice-cold PBS and minced it in the nuclei isolation buffer (NIB-250 is 15mM Tris-HCl at pH 7.5,60mM KCl,15mM NaCl, 5mM MgCl2, 1mM CaCl2, and 250mM sucrose, to which 0.1% Nonidet P-40, 1x protease inhibitor cocktail, 1mM DTT, and 10mM sodium butyrate).We collected the kidney pieces into glass Dounce homogenizer on ice.After 5 min of incubation on ice, we spun down the homogenates, collected the nuclei pellet, washed it twice with NIB-250 without Nonidet P-40 and proceeded with histone extraction.
We incubated the nuclear pellet with 0.4 N H 2 SO4 at a 5:1 ratio for 2h at 4 O C. We then spun down the acidified nuclei at 11000rcf for 10min at 4 O C and collected the soluble fraction containing histones into a new tube and precipitated with 20% trichloroacetic acid at final concentration overnight at 4 O C. We spun down the samples at 11000rcf for 10min at 4 O C to sediment the histone pellet at the bottom of the tube.We then washed the histone pellets with 1ml of ice-cold acetone containing 0.1% 12N HCl, followed by two washes with ice-cold 100% acetone.We air-dried the pellet and dissolved them in RIPA buffer.Two micrograms of histone lysates were loaded onto 15% SDS-PAGE gels.Western blotting was performed as described above and probed with anti-H3K27ac (#ab177178, abcam) and anti-total H3 (#4620, CST) antibodies.Imaged in Li-COR imager (Odyssey® XF).

H&E and Sirius Red staining
The tissues were fixed in formalin, dehydrated by an ethanol gradient (30%, 50%, 75% and 95%) and then submitted to the histology Core in 100% ethanol.Once the tissue was sectioned, H&E and Sirius red staining was performed.Images were acquired in Olympus 5000 microscope with Cell Sense software.Percentage of relative fibrosis was quantified in image J.

Primary tubular epithelial cell isolation and in vitro experiments
Primary kidney tubular epithelial cells (TECs) were isolated from young pups (2.5-3wk old) of WT, Acss2 -/-, Fasnf/-and Scapf/f mice.Kidneys were collected on a petri dish on ice, minced in RPMI media (Corning), and then then digested with 200ug/ml collagenase IV (1mg/ml, calbiochem) at 37 O C for 30min.Collagenase was inactivated by adding 100μl of fetal bovine serum (100% FBS), and cells were passed through 100μm, 70μm and finally 40μm strainers.Cells were then centrifuged at 1000rpm for 5min at 4 O C. The cell pellet was resuspended in 1 ml of sterile icecold RBC lysis buffer (Hy-Clone) and incubated for 2min on ice.Lysis was inhibited by adding icecold PBS and then centrifuged at 1000rpm for 10min at 4 O C. Finally, the cell pellet was resuspended in RPMI complete media (10% FBS with antibiotics 1X ITS and 50ng/ml human EGF) and plated in 10cm dishes.Cells were grown in the incubator at 5% CO2 at 37 O C, and the medium was changed every other day.
Cells were serum restricted in 0.5% FBS for 24h.Cells were then treated with 20ng/ml TGF-β1 for 48h in the presence or absence of FASNall (4µM/ml) or TVB-3664 (10nM/ml) in 0.5% serum media for 48h.Fasnf/-or Scapf/f cells were treated with adenovirus Ad5CMV-eGFP (Ad-GFP) or Ad5CMVCre-eGFP (Ad-Cre-GFP) (University of Iowa Gene Transfer Vector Core, Iowa City, IA) at a concentration of 0.5µl/ml for 24h in serum free media, and the infection efficiency was assessed by observing GFP signal under a fluorescence microscope before every experiment.
For, siRNA transfection experiments, a smart pool of non-target control siRNA and mouse siFasn were purchased from Dharmacon (#L-040091-00-0005, Horizon Biosciences).Cells were seeded in 6-well plates, grown overnight at 80-90% confluent, and then transfected with 20pM siFasn in RNAimax in OptiMEM for 48h.After transfection, cells were treated with TGF-β1 (20ng/ml) for 48h in 0.5% serum containing media.RNA or protein was isolated from these cells, and knockdown efficiency was determined by quantifying the relative Fasn expression using real time qPCR.

Cholesterol measurement
Total kidney cholesterol was quantitatively estimated using established methods (#K603-100, BioVision).Approximately 10-15mg of kidney tissue was homogenized in 300 µl of chloroform: isopropanol: NP-40 (7:11:0.1) in a microcentrifuge.The homogenate was centrifuged at 15000g for 10min, and the liquid layer was collected into a new microtube.The supernatant was air dried at 50 O C to remove chloroform and the samples kept under vacuum pressure (SpeedVac, Thermo Scientific) for 30 min to remove trace organic solvent.The dried lipids were dissolved in 200 µl of assay buffer and performed cholesterol measurements as per the manufacturer protocol.

H labeled FAO measurements in mice kidneys
FAO measurements were performed by tracing tritium labeled water ( 3 H 2 O) (9).Frozen whole kidney extracts prepared in Krebs-Ringer bicarbonate buffer containing HEPES (#K4002, Sigma).
Nearly 500µg of protein was used for FAO measurements.Briefly, the kidney homogenates were incubated with master cocktail (Krebs-Ringer bicarbonate buffer containing 100mg/ml fatty acid free BSA, 2.5mM palmitic acid, 10mM carnitine, and 4μCi of 9,10-3H-palmitoyl-CoA) for 2h at 37OC and 600rpm in dark.The homogenates were then subjected to Folch's lipid extraction protocol (2:1 chloroform and methanol) and further precipitated with 10% trichloro acetic acid (#T6399, Sigma).After high-speed centrifugation at 4C, 1ml of supernatants were passed through activated AG 1-X8 resin formate columns (#7316221, BioRad) and eluted in roughly 1ml volume ( 3 H2O) into glass vials.Nearly 500ul of elutes were mixed into 3ml of scintillation cocktail and read in a liquid scintillation counter (Beckman Coulter).The radioactive counts were subtracted from the sample containing no protein and from sample with cold palmitic acid (#P5585, Sigma).
The final counts were normalized to the control samples and presented as relative FAO rate.

In vitro palmitic acid oxidation test by Seahorse analyzer
Real-time fatty acid oxidation analysis was performed in renal tubule cells using an XF-96 Extracellular Flux Analyzer with the Palmitate Oxidation Stress Test Kit and FAO Substrate (#102720-100, Agilent Seahorse Bioscience).Briefly, primary tubule cells were isolated from WT and Acss2 -/-mice and were cultured in a Seahorse 96-well plate at a density of 5x10^3 cells per well.The day before performing the OCR analysis, the cell culture medium was replaced with substrate-limited medium (DMEM (#A14430-01), 0.5 mM glucose, 1 mM Glutamax, 0.5 mM carnitine, and 1% FBS) and maintained up to 18 hours.An hour before performing the OCR analysis, the substrate-limited medium was exchanged with FAO assay medium (1x potassium bicarbonate buffer with 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES).Etomoxir (40 µM) was added 15 minutes before the start of the OCR analysis to the specified wells.Control cells were supplemented with BSA (0.17 mM) while test cells were supplemented with 1 mM palmitic acid conjugated BSA (0.17 mM).The OCR analysis was performed by treating cells with 2 µM oligomycin, 1 µM fluoro-carbonyl cyanide phenylhydrazone (FCCP), and 0.5 µM rotenone plus 1 µM antimycin A at final concentration.

In vivo DNL tracing with deuterated water
DNL tracing was performed at the GC-MS core at the University of Pennsylvania (10).To assess total lipogenesis, mice were subjected to UUO for seven days.On the 6th day, mice were fasted overnight at ~7 pm.On the 7th day, the mice were refed for three hours and then injected with 400ul of deuterated water (#151882, Sigma) prepared in 0.9% saline via i.p injection and continued feeding for three more hours.Systemic blood was collected by cardiac puncture, and livers and kidneys were harvested using clamps pre-cooled in liquid nitrogen.The blood was allowed to coagulate on ice for 15 min, and spun down at 10,000g for 5 min at 4C to collect serum.
The frozen liver and kidney samples were ground at liquid nitrogen temperature with a Cryomill (Qiagen).Saponification of lipids and gas chromatography-mass spectrometry (GC-MS) analysis were performed at the GC-MS core.Briefly, 5μl of serum, and 100 mg of liver or kidney powder was saponified, and fatty acids were extracted by adding 0.5 ml of hexane, vortexing, and transferring the top hexane layer to a new glass vial.Separation was performed by reversedphase ion-pairing chromatography on a C8 column coupled to negative-ion mode, full-scan GC-MS at 1-Hz scan time and 100,000 resolving power (Agilent 7890A Gas Chromatograph; 5975 Mass Spectrometer; Thermo Fischer Scientific).Palmitate was analyzed using GC-MS, and the absolute amount of newly made palmitate was assumed equivalent to the rate of DNL.Data analysis with MAVEN software and natural isotope correction were performed by the GC-MS core.

Kidney Triglycerides quantification
Kidney triglycerides were measured using Triglyceride Calorimetric Assay kit (#10010303, Cayman).Approximately 20mg of kidney tissue was homogenized in NP40-substitute assay buffer containing protease and phosphatase inhibitors.Homogenates were collected after centrifuging at 10,000g for 10min at 4 O C.

Oil Red O staining
For Oil Red O staining, we cut 5µm thin frozen sections.Briefly, the sections were dried at RT for 15min and fixed in prechilled 10% formalin buffered PBS for 10min.Slides were washed three times with water for 5min, and finally rinsed in 60% isopropanol for 5min.Lipids were stained by incubating slides in fresh Oil Red 'O' working solution for 30-60min at RT and rinsed in 60% isopropanol for five seconds.Slides were washed three times with water and counterstained with hematoxylin for 3min.Finally, slides were washed in 70% ethanol and mounted with 90% glycerol, and immediately proceeded for microscopic analysis.Images were acquired in EVOS FL inverted fluorescence microscope Invitrogen).

NADPH/NADP+ ratio measurements
The NADPH/NADP+ ratio was calculated according to the manufacturer protocol (#G1009, Promega).Briefly, an equal number of cells were cultured in a 96-well format and starved overnight for TGF-β1 treatments.Next, the cells were lysed in 20% DTAB containing Basic solution for 10min at RT.Samples were processed according to the protocol for measuring NADPH and NADP+ from the same well simultaneously.The absolute and relative NADPH/NADP+ values were calculated according to the manufacturer's formula.

GSH/GSSH ratio
Reduced and oxidized glutathione levels were measured according to the Glutathione Colorimetric Detection Kit (#EIAGSHC, Thermo Scientific) from the same well after completion of all treatments.

Mitochondrial Quality assessments and Mitophagy
Primary cells were cultured on microscopic cover glass overnight until they reached 70% confluency.Cells were incubated in a 5µM MitoSox (#M36008, Thermo Scientific) solution for 10min in incubator and processed for imaging and quantifications.Cells were also incubated in 10µM JC-1 (#T3168, Thermo Scientific) for 10min and then proceeded with imaging and quantifications.
Mitophagy was assessed as described in our earlier paper (11).Primary cells were transfected with Mito 'Q' mCherry-eGFP COX8 plasmid (#78520, Addgene) for 48h.Cells were processed for various treatments and proceeded with imaging.Cells cultured as above and pretreated with 100µM MitoTempo (#SML0737, Sigma) for 2h, followed by TGF-β1 treatments while MitoTempo was still present.The images of JC-1, mitosox stainings, Mito 'Q' mCherry-eGFP COX8, and MitoTempo experiments were all acquired in Olympus 5000 microscope with Cell Sense software.

In situ hybridization
In situ hybridization was performed on formalin-fixed paraffin-embedded tissue sections using the RNAscope 2.5 HD Duplex Detection Kit (#322436, ACD Bio) according to the manufacture protocol.Freshly cut kidney tissue sections were used in all in situ experiments.For the Gsdmd in situ hybridization, the antigen retrieval was performed for 30min in heated water bath.The following probes were used for the RNAscope in situ assay: Mm Acss2, Hs-ACSS2, Mm-Gsdmd, Mm-Lrp2, Hs-LRP2 and Mm-Hnf4a.In situ hybridization quantification was performed manually according to the ACD Bio RNAscope 2.5 HD Duplex user manual.
Human kidney bulk RNA-seq, and single nuclear RNA sequencing data (previously generated) can be viewed at the http://www.susztaklab.com/hk_genemap/scRNAwebsite.
Relative Fasn expression

344 Supplemental Figure 1 . 3 . 4 .Fasn
Prioritization of ACSS2 as a kidney disease risk gene.345 mapping regional plot showing single nucleotide variants associated with kidney eGFR GWAS dataset (N=1.5MEuropean population).X-axis chromosomal location and y-axis shows the strength of association (-log(p)).Variants shown in red color indicates correlation (extreme right r2) with underlying genes.Color key indicates linkage disequilibrium (r2).B. Human kidney ACSS2 gene expression in glomeruli (n=303) in microdissected samples.Y -axis shows normalized ACSS2 expression and X-axis shows genotype information.C. GWAS and eQTL effect sizes (upper plot tubule; lower plot glom) plotted for ACSS2 gene in 1.5M human samples.D. Transcript levels of CEP250 following risk regions deleted in HEK293T cells.The data was generated using samples used in Figure 1I.E. Transcript levels of SPAG4 following risk regions deleted in HEK293T cells.The data was generated using samples used in Figure 1I.F. Immunoblots of ACSS2, KIM-1 and GAPDH in whole kidney lysates of wild type (WT) (n=5) and Acss2 -/-mice (n=6).G. Transcript levels of Acss2 in kidneys of WT (n=5) and Acss2 -/-mice (n=6).H. Weekly body weights recorded in WT (n=5) and Acss2 -/-mice (n=6) at baseline.I. Blood urea nitrogen (BUN) in 10 weeks old WT (n=5) and Acss2 -/-(n=6) mice at baseline.J. Serum creatinine (sCr) in 10 weeks old WT (n=5) and Acss2 -/-(n=6) mice at baseline.K. Ki67 immunofluorescence in WT and Acss2 -/-mice at 10 weeks of age.Scale bars 10µm.L. Transcript levels of mKi67, Havcr1 and Lcn2 in 10 weeks old WT (n=5) and Acss2 -/-(n=6) mice at baseline.Data are represented as mean ± SEM.P values determined by one-way ANOVA for D, E, G, I, J, L after Tukey's multiple comparison.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Protein marker was cropped from all blots but was presented in full blots file.371 Supplemental Figure 2. Loss of ACSS2 protects from kidney disease.TGFβ1 induces de novo lipogenesis in primary tubule cells.Inhibition of de novo lipogenesis prevents kidney fibrosis.441 expression level of fatty acid synthase (Fasn) and perilipin 2 (Plin2) in TECs transfected with small interfering RNA against Fasn (siFasn) and treated with TGFβ1 for 48h.B. Gene expression levels of alpha smooth muscle actin (Acta2), Collagen 1a1 (Col1a1), collagen type 3a (Col3a) and fibronectin (Fn1) in TECs transfected with siFasn and treated with TGFβ1.C. Gene expression level of sterol regulatory element binding protein (SREBP) cleavage activating protein (Scap), Fasn, and Plin2 were measured in WT and Scapf/f TECs treated with Adeno-Cre virus (Ad-Cre) for 24h and treated with TGFβ1 for 48h.D. Gene expression level of Acta2, Col1a1, Col3a and Fn1 measured in WT and Scapf/f TECs treated with Ad-Cre and TGFβ1.WT and TGF β1 treated samples are the same as used in supplemental figure 2 panel O. E. Experimental design.F.Gene expression level of Fasn and Plin2 in kidneys of mice injected with FASNall or PBS followed by UUO injury.Supplemental Figure5.Suppression of mitochondrial ROS suppresses NLRP3inflammasome activation in primary tubular cells.
was assessed in primary tubular epithelial cells (TECs) transfected with mitoCox8 eGFP-mCherry plasmid and subjected to various mitophagy inducers for 2h.Scale bars 10µm.B. Mitolysosomes quantified in Image J. C. Immunoblots of LC3B and Parkin1 in primary TECs in fed, starve, and bafilomycin (BA).D. Quantification of immunoblots of LC3B in Image J. E. Relative gene expression of Nlrp3, IL1B, and caspase1 (Casp1) in TECs treated with TVB-3664 or TGFβ1 for 48hr.F. Relative gene expression of IL1B, IL18 and Casp1 in WT and Scapf/f TECs transfected with Adeno-Cre virus (Ad-Cre) and treated with TGFβ1.G. Relative gene expression of Fasn in WT and Fasnf/-TECs transfected with Ad-Cre and treated with TGFβ1.WT and TGF β1 samples are same used in the supplementary figure 4 panel C. H. Relative gene expression of IL1B, IL18 and Casp1 WT and Fasnf/-TECs transfected with Ad-Cre and treated with TGFβ1.I. Relative fluorescence of MitoSox quantified in WT and Acss2 -/-cells treated with vehicle or TGFβ1 or mitoTempo (MT).J. Relative fluorescence of MitoSox quantified in WT and Fasnf/-cells transfected with Ad Cre for 24h and treated with vehicle or TGFβ1 or MT.K. Relative gene expression of alpha smooth muscle actin (Acta2), Collagen 1a1 (Col1a1), collagen type 3a (Col3a) and fibronectin (Fn1) measured in primary TECs treated with TGFβ1 or MT.Data are represented as mean ± SEM.P values determined by one-way ANOVA after Tukey's multiple comparison.*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Vehicle, TGFβ1, MT, and MT+TGFβ1 data are the same for both panels I and J.The data was a representative of multiple experiments.Protein marker was cropped from all blots but was presented in full blots file.Supplemental Figure 6.Inhibition of de novo lipogenesis suppresses ROS-induced NLRP3 inflammasome activation.

Fasnf 7 .
Fatty acid synthesis correlated with fibrosis in CKD patients.