LRNA9884, a Novel Smad3-Dependent Long NoncodingRNA, Promotes Diabetic Kidney Injury in db/db Mice viaEnhancing MCP-1–Dependent Renal InflammationYing-ying Zhang,1,2 Patrick Ming-Kuen Tang,1,3 Philip Chiu-Tsun Tang,1 Jun Xiao,1 Xiao-Ru Huang,1

Chen Yu,2 Ronald C.W. Ma,1 and Hui-Yao Lan1

Diabetes 2019;68:1485–1498 | https://doi.org/10.2337/db18-1075

Transforming growth factor-b/Smad3 signaling plays animportant role in diabetic nephropathy, but its underlyingworking mechanism remains largely unexplored. Thecurrent study uncovered the pathogenic role and un-derlying mechanism of a novel Smad3-dependent longnoncoding RNA (lncRNA) (LRNA9884) in type 2 diabeticnephropathy (T2DN). We found that LRNA9884 was sig-nificantly upregulated in the diabetic kidney of db/dbmice at the age of 8 weeks preceding the onset ofmicroalbuminuria and was associated with the progres-sion of diabetic renal injury. LRNA9884 was induced byadvanced glycation end products and tightly regulatedby Smad3, and its levels were significantly blunted indb/db mice and cells lacking Smad3. More importantly,kidney-specific silencing of LRNA9884 effectively atten-uated diabetic kidney injury in db/db mice, as shown bythe reduction of histological injury, albuminuria excre-tion, and serum creatinine. Mechanistically, we identifiedthat LRNA9884 promoted renal inflammation-drivenT2DN by triggering MCP-1 production at the transcrip-tional level, and its direct binding significantly enhancedthepromoter activity ofMCP-1. Thus, LRNA9884 isa novelSmad3-dependent lncRNA that is highly expressed indb/db mice associated with T2DN development. Target-ing of LRNA9884 effectively blocked MCP-1–dependentrenal inflammation, therefore suppressing the progres-sive diabetic renal injury in db/db mice. This study revealsthat LRNA9884 may be a novel and precision therapeutictarget for T2DN in the future.

Type 2 diabetes is a major metabolic disorder that causesa profound medical and socioeconomic burden worldwide(1). Type 2 diabetes is associated with a series of compli-cations on multiple organ systems, where diabetic ne-phropathy (DN) is the most severe complication ofdiabetes and is a leading cause of end-stage kidney disease(2). The pathological features of DN include diffuse glo-merular basement membrane thickening, mesangial ex-pansion, podocyte reduction, and renal fibrosis (3).Increasing evidence demonstrates that low-grade renalinflammation is a key mechanism leading to diabetesand diabetic complications (4).

Emerging evidence shows that long noncoding RNAs(lncRNAs), defined as RNAs.200 nucleotides that do notencode any protein, play an important role in the de-velopment of type 2 diabetic nephropathy (T2DN) (5,6). Ithas been shown that an lncRNA transcript (lncMGC) isincreased in the glomeruli of mouse models of DN inresponse to endoplasmic reticulum stress and that target-ing this lncRNA can protect against early DN (7). Inaddition, lncRNA CYP4B1-PS1-001 protects against theprogression of T2DN via inhibiting proliferation andfibrosis of mesangial cells during the early stage of DN,and lncRNA metastasis associated lung adenocarcinomatranscript 1 (MALAT1) is dysregulated in DN (8,9). A recentstudy also found that an lncRNA taurine-upregulated gene1 (Tug1) is reduced in diabetic kidney and that overex-pression of Tug1 largely improves mitochondrial bioener-getics in the podocytes of diabetic mice via a peroxisome

1Department of Medicine & Therapeutics, Li Ka Shing Institute of Health Sciences,and Lui Che Woo Institute of Innovative Medicine, Shenzhen Research Institute,The Chinese University of Hong Kong, Hong Kong2Department of Nephrology, Tongji Hospital, Tongji University School of Medicine,Shanghai, China3Department of Anatomical and Cellular Pathology, State Key Laboratory ofTranslational Oncology, Prince of Wales Hospital, The Chinese University of HongKong, Hong Kong

Corresponding author: Hui-Yao Lan, hylan@cuhk.edu.hk

Received 4 October 2018 and accepted 14 April 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1075/-/DC1.

Y.-y.-Z. and P.M.-K.T. contributed equally to this work.

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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proliferator–activated receptor-g coactivator-1a–dependentmechanism (10). Nevertheless, the potential roles andworking mechanisms of lncRNAs in DN are still largelyunknown (11).

By using high-throughput RNA sequencing, we uncov-ered the involvement of lncRNAs in renal diseases on bothanti- glomerular basement membrane (GBM) and unilat-eral ureteric obstruction (UUO)-induced mouse kidneydisease models and identified 21 novel Smad3-dependentlncRNAs participating in the renal pathogenesis (12).Among these lncRNAs, a novel Smad3-dependent lncRNA,Erbb4-IR, is highly upregulated in the UUO kidney andT2DN with progressive renal fibrosis, and knockdown thislncRNA can protect kidneys from both UUO and diabeticinjury (13,14). In the current study, we also found thata potential lncRNA transcript LRNA9884 was the highestexpressed Smad3-dependent lncRNAs in the kidneys ofdb/db mice compared with the db/m mice as well as theErbb4-IR level. We therefore further elucidated its poten-tial pathogenic role and underlying mechanism in T2DN.

RESEARCH DESIGN AND METHODS

Animal ModelMale db/db and db/m mice were purchased from theChinese University of Hong Kong Laboratory AnimalServices Centre. All mice were fed in a standard animalhouse with 12-h/12-h light/dark cycle and were sacrificedby intraperitoneal injection of ketamine/xylene. Smad3knockout (KO) db/db mice were generated by crossingheterozygous db/m with heterozygous Smad3+/2

(C57BL/6 background), as previously described (14). Allstudies and experimental procedures were approved by theChinese University of Hong Kong Animal ExperimentationEthics Committee, and the experimental methods wereperformed in accordance with the approved guidelines.

Real-Time PCR AnalysisTotal RNA was isolated from the cultured cells and renalcortex of kidney tissues using Trizol (Invitrogen, Carlsbad,CA) according to the manufacturer’s instructions. Real-time PCR was performed by SYBR Green Supermix us-ing the CFX96 PCR System (Bio-Rad, Hercules, CA). Theprimers used in this study, including mouse collagen I,fibronectin, MCP-1, and b-actin, have been previouslydescribed (15–18). Other primers include LRNA9884 for-ward 59- ACCACAGCAGATACCCAAGC-39 and reverse 59-CAGCAAGCTCCTTTTTCCAC-39. The relative level of thedetected gene was normalized with the internal controlb-actin. Relative changes in target mRNA expression weredetermined by using the 22DDCT method.

Rapid Amplification of cDNA EndsThe full length sequence of LRNA9884 was obtained byconducting rapid amplification of cDNA 59 and 39 ends(RACE) with SMARTer RACE 59/39 Kit (Clontech, Moun-tain View, CA), as previously described (13). In brief, thecDNA generated from the total RNA of diabetic injured

kidney from the 20-week-old db/db mice was used as thetemplate for RACE PCR according to the user’s manual. Thefull length of LRNA9884 cDNA was synthesized by PCR withthe forward primer 59- AAAACAGGAGCAGGAAGAATTGGG-39 and reverse primer 59- TTGAACTCTGGTCTTCAGAC-39.

Bioinformatics AnalysisGenomic location of the LRNA9884 was identified by BasicLocal Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi and http://www.genome.ucsc.edu/). Smad3 binding site on the LRNA9884 genomicsequence was identified by the Evolutionary ConservedRegions (ECR) browser (https://ecrbrowser.dcode.org/),as reported in our previous study (12). The protein-coding potential of the LRNA9884 sequence was evalu-ated by two widely used computational programs: codingpotential calculator (CPC) (http://cpc.cbi.pku.edu.cn/programs/run_cpc.jsp) and the coding potential assess-ment tool (CPAT) (http://lilab.research.bcm.edu/cpat/index.php) (19,20). For evaluation in CPC, transcriptswith scores higher than 1 are predicted to be “coding,”lower than21 are “noncoding,” and between21 and 1 areclassified as “weak noncoding” (21, 0) or “weak coding”(0, 1). For identifying the direct binding site of LRNA9884on the MCP-1 promoter, the sequence of LRNA9884from RACE and the promoter region of MCP-1 from theEukaryotic Promoter Database (https://epd.vital-it.ch/index.php) were analyzed by IntaRNA tool on the FreiburgRNA Tools website using the default settings (http://rna.informatik.uni-freiburg.de/IntaRNA/Input.jsp) (21),followed by dual-luciferase reporter assay for validatingtheir potential RNA-DNA interaction as in our previouswork (13,14).

In Situ HybridizationThe expression level of LRNA9884 in the kidney wasdetected by using in situ hybridization (ISH), as previouslydescribed (14). The kidney sections (4 mm) were fixed in4% (w/v) paraformaldehyde with 1% (v/v) DMSO. Afterrehydration, we use a permeabilization step by usingproteinase K. Probe for ISH is carried with a locked nucleicacid–digoxigenin labeled LRNA9884 probe (59-ACTT-GAAGGGTCCAGAAGAGAT-39) (Exiqon, Vedbaek, Denmark)or negative control scramble probe (59-GTGTAACACGTCTA-TACGCCCA-39) (Exiqon) in a humid chamber at 54°C for2 h. The sections were washed and incubated with anti-digoxigenin antibody conjugated to alkaline phosphatase(Roche Diagnostics, Indianapolis, IN) overnight at 4°C anddeveloped with phosphate/nitroblue tetrazolium (Sigma-Aldrich, St. Louis, MO) (22).

Fluorescence ISH and Immunofluorescence AssayFluorescence ISH (FISH) with immunofluorescence assaywas performed with a Cy3-conjugated LRAN9884 probe59-ACTTGAAGGGTCCAGAAGAGAT-39 and scramble con-trol 59-GTGTAACACGTCTATACGCCCA-39 (GenePharma,Shanghai, P.R. China) by using a FISH kit (C10910; RiboBio,

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Guangzhou, P.R. China) according to the manufacturer’sinstruction. Immunofluorescence assay was followedby FISH, and FITC-conjugated Pan Cytokeratin (53-9003-82; eBioscience, San Diego, CA) and nephrin(ab58968; Abcam, Cambridge, U.K.) were the primaryantibodies. All stained samples were imaged under a fluo-rescence microscope (Axio Observer.Z1; Carl Zeiss, Oberko-chen, Germany) (14).

Cell CultureImmortalized murine kidney proximal tubular epithelial cells(mTECs) (a gift fromDr. Jeffrey B. Kopp, National Institutes ofHealth) were cultured in DMEM/F-12 (Gibco, Carlsbad, CA),supplemented with 10% FBS (Gibco) and 1% antibiotic/antimycotic solution (Life Technologies, Grand Island, NY)(13,15,16). The murine SV40 transfected mesangial cell lineSV40 MES 13 (mMC) (ATCC, Manassas, VA) was maintainedin a 3:1mixture ofDMEMandHam’s F-12medium containing5% FBS, and 1% antibiotic/antimycotic solution (Life Tech-nologies) (14). Mouse embryonic fibroblasts (MEFs) lackingSmad3 or Smad2 were generated and characterized as de-scribed previously (23), and were cultured in DMEM/F-12medium (Gibco), as previously described (24). Advanced gly-cation end products (AGE), at a dose of 50 mg/mL (Cat#A8426; Sigma-Aldrich), was added to induce responses, whichwas previously dissolved in endotoxin-free 1 3 PBS. Thecomposition of AGE includes protein and 95% biuret, andspecific of AGE is bovine, their activity on Smad3 activationand MCP-1 expression were validated as position control.To inhibit Smad3 activity, cells were pretreated with theSmad3 inhibitor SIS3 (S0447; Sigma-Aldrich) at a dose-dependent pattern of 1–4 mmol/L for 2 h before AGEstimulation.

Chromatin Immunoprecipitation AnalysisChromatin immunoprecipitation (ChIP) was performedwith the SimpleChIP Enzymatic Chromatin IP Kit (magneticbeads) (#9003; Cell Signaling, Danvers, MA), as previouslydescribed (15,18,25). Immunoprecipitation was performedwith the antibody against Smad3 (#9513; Cell Signaling) ora normal IgG as a negative control. Precipitated DNA frag-mentswere detected byPCRusing a specific primer of promoterregion of LRNA9884: forward 59-TCGCCTCCAGTTGTCTTTCT-39, reverse 59-CCTGAAGGACAGCCACTCTC-39.

Ultrasound-Mediated Gene Transfer-MediatedLRNA9884 KnockdownGroups of eight male mice were used: db/db mice weretreated with pSuper.puro vector (Oligoengine, Seattle WA)empty vector (EV) control or plasmid overexpressingshRNA sequence targeting LRNA9884 (shRNA) (59-GATCC-GACCACAGCAGATACCCAAGCttcaagagaGCTTGGGTATCTGCT-GTGGTCTTTTTTGAATTCA-39) via ultrasound-mediated genetransfer method from the age of week 12 and sacrificed atweek 20. To maintain the transgene expression levels,LRNA9884 shRNA expressing plasmids were transfected atage 12, 15, and 18 weeks, as previously described (13,14). In

brief, mice received the mixed solution (400 mL/mouse)containing the LRNA9884 shRNA-pSuper.puro vector orpSuper.puro EV (200 mg/mouse) and lipid microbubbles(Sonovue; Bracco, Milan, Italy) at a ratio of 1:1 (v/v) viathe tail vein injection, then immediately using an ultra-sound transducer (Therasonic; Electro Medical Supplies,Wantage, U.K.) directly placed on the skin of the backagainst the left kidney with a pulse-wave output of 1 MHzat 2 W/cm2 for a total of 5 min on each side, as previouslydescribed (13–16).

Histology and ImmunohistochemistryKidneys sections were fixed in 4% paraformaldehyde,and stained with periodic acid-Schiff (PAS), peri-odic acid-silver methenamine (PASM), and Massontrichrome staining methods. Immunohistochemistrywas performed in 4-mm paraffin sections by using a mi-crowave-based antigen-retrieval technique. The antibodiesused in this study included F4/80 (MCA497; Serotec,Oxford, U.K.), interleukin-1b (IL-1b) (sc-7884; SantaCruz Biotechnology, Dallas, TX), MCP-1 (sc-1785; SantaCruz Biotechnology), and tumor necrosis factor-a (TNF-a)(sc-1351; Santa Cruz). After immunostaining, sectionswere counterstained with hematoxylin. The mesangialmatrix index was quantified using Image-Pro Plus software(Media Cybernetics, Bethesda, MD) by PAS staining in10 random fields (original magnification 3200), as pre-viously described (13). Glomerulosclerosis was quantifiedby Masson trichrome staining, where 20 glomeruli wererandomly selected from each section, and positive signalswithin the selected glomerulus were highlighted, mea-sured, and represented as the percentage positive areaof the entire glomerulus. The percentage of positive stain-ing areas of MCP-1, IL-1b, and TNF-a expression wasquantified using the same software in 10 consecutivefields, whereas positive cells for F4/80+ cells were countedunder the power field of microscope in 10 random areas ofkidney tissues using a 0.25-mm2 graticule fitted in theeyepiece of the microscope and expressed as cells/mm2

(13,14,26).

Renal Function MeasurementMice were placed in metabolic cages for collection of 24-hurinary samples every 4 weeks from the age of 8 weeks.Urinary microalbumin was measured by competitive ELISAaccording to the manufacturer’s instructions (Exocell,Philadelphia, PA). Urinary and serum creatinine was mea-sured by an enzymatic kit (Stanbio Laboratories, Boerne,TX). Urinary albumin excretion was expressed as totalurinary albumin–to–creatinine ratio (mg/mg), as previ-ously reported (26).

Fasting Blood GlucoseBlood glucose levels were measured every 4 weeks by Accu-Chek glucose meter (Roche Diagnostics, Basel, Switzerland)after the mice were fasted for 6 h, as recommended by theAnimal Models of Diabetic Complications Consortium.

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Transfection of siRNA and PlasmidThe mTECs were transfected with 100 nmol/L LRNA9884siRNA (sense 59-GACCACAGCAGAUACCCAAGC-39, anti-sense 59-GCUUGGGUAUCUGCUGUGGUC-59) or nonsensecontrol (NC; sense 59-UUCUCCGAACGUGUCACGUTT-39,antisense 39-ACGUGACACGUUCGGAGAATT-59) usingLipofectamine RNAiMAX reagent (Invitrogen) accordingto the manufacturer’s instructions as previously reported(27), then stimulated with AGE. In addition, the mTECswere also transfected with 0.5 mg/mL pcDNA3.1 EV orplasmid containing LRNA9884 full-length RNA sequence,as shown in Supplementary Fig. 1 (LRNA9884; constructedby PharmaGene, Shanghai, China) using Lipofectamine2000 (Invitrogen) according to the manufacturer’s manual,and the culture.

Western Blot AnalysisProtein from renal cortex and cultured cells was extractedusing radio immunoprecipitation assay (RIPA) lysis buffer.Western blot analysis was performed as previously de-scribed (28,29). In brief, after nonspecific binding wasblocked with 5% BSA, nitrocellulose membranes wereincubated overnight at 4°C with primary antibodiesagainst phosphorylated-Smad3 (Cell Signaling Technology,Danvers, MA) and Smad3 (Cell Signaling Technology),collagens I (1310-01; Southern Tech, Birmingham, AL),fibronectin (sc-8422; Santa Cruz Biotechnology), andb-actin (sc-47778; Santa Cruz Biotechnology), followedby IRDye800-conjugated secondary antibodies (RocklandImmunochemicals, Gilbertsville, PA). Signals were detectedusing the LI-COR/Odyssey infrared image system (LI-CORBiosciences, Lincoln, NE), followed by quantitative anal-ysis using the Image J program (https://imagej.nih.gov/ij/?) (13,18). The ratio for the protein examinedwas normalized against b-actin and expressed as themean 6 SEM.

ELISAMedium from stimulated mTECs was collected to detectthe cytokine production using an ELISA kit. MCP-1 wasmeasured with Quantikine ELISA Kit (R&D Systems, Min-neapolis, MN) according to the product protocols.

Dual-Luciferase Reporter AssayThe pcDNA3.1+ plasmids containing LRNA9884 full-lengthsequence with (LRNA9884) or without (LRNA9884-mutant) predicted the binding site on the MCP-1 promoter;and pGL3-basic reporter plasmids containing MCP-1 pro-moter sequence with (pGL3–MCP-1) or without predictedLRNA9884 binding site (pGL3–MCP-1–mutant) were con-structed by Biowit Technologies Ltd., China (13,14). Theluciferase reporter assay was performed by Landbiology(Guangzhou, China) by using dual luciferase reporterassay kit (Promega, Madison, WI) as our previous studies(24). The empty vectors (pGL3-basic, pcDNA3.1+), orMCP-1 reporter plasmids (pGL3–MCP-1, pGL3–MCP-1–mutant), or LRNA9884-overexpressing pcDNA3.1+

plasmids (LRNA9884, LRNA9884-mutant) were cotrans-fected into mTECs. Luciferase activities were measuredat 48 h according to the manufacturer’s instructions byGloMax-Multi Detection System (Promega). The reporteractivity was represented by the ratio of the firefly lucif-erase activity (M1) to the Renilla luciferase activity (M2)as M1/M2, and shown as mean 6 SEM fold induction ofluciferase in three independent experiments.

Statistical AnalysisAll of the data are expressed as mean 6 SEM. Statisti-cal analyses were performed with one-way ANOVA,followed by Newman-Keuls multiple comparison fromGraphPad Prism 5.0 (GraphPad Software, La Jolla, CA).In addition, a repeated-analysis ANOVA was used foralbumin excretion, body weight, and fasting blood glucoseanalysis.

RESULTS

LRNA9884 Is a Novel Smad3-Dependent lncRNA and IsHighly Expressed and Associated With Progression ofRenal Injury in db/db MiceAmong all previously identified Smad3-depedent lncRNAs(11), real-time PCR analysis revealed that a potentiallncRNA transcript (na_9884) was highly expressed inthe diabetic kidney of db/db mice (Fig. 1A). More impor-tantly, expression of na_9884 progressively increased inthe diabetic kidneys of db/db mice compared with db/msince the age of 8 weeks (Fig. 1B). We then furthercharacterized this novel lncRNA by obtaining its full-length sequence via RACE (Supplementary Fig. 1). Ourresult identified that na_9884 is 508 nucleotides in fulllength, which locates on murine chromosome 5 (Chr5:52973035–52992344) and covers the whole genomic se-quence of an lncRNA transcript 5033403H07Rik (Fig. 1Cand D). By using the CPC and CPAT analysis, we confirmedthat na_9884 scores 21.01155 and 0, respectively, as anRNA without protein-coding capacity (Fig. 1E). Thus, wenamed this novel lncRNA as LRNA9884. By ISH, wefurther observed that LRNA9884 is highly expressed inthe nucleus of renal residential cells in the diabetic kidneyof 8-week-old db/db mice and further upregulated in20-week-old db/db mice (Fig. 1F).

LRNA9884 Is Markedly Induced in Renal TubularEpithelial and Mesangial Cells Under DiabeticConditionThe LRNA9884 expression was further confirmed by FISHassay with immunofluorescence costaining. Owing to lackof the specific marker in mouse mesangial cells, theepithelial cell marker keratin and podocyte marker nephrinwere detected in kidneys of 20-week-old db/db mice.Results demonstrated that LRNA9884 was largelyexpressed in the nucleus of epithelial cells and was lackingin podocytes compared with the db/m mice (Fig. 2A),suggesting that LRNA9884 was mainly expressed in epithe-lial cells and mesangial cells. Aberrant glucose metabolism

1488 Role of LRNA9884 in DN Diabetes Volume 68, July 2019

results in hyperglycemia, and accumulation of variousbyproducts (e.g., AGE) is suggested to promote renalinjury in diabetic kidney disease. Thus, we culturedmTECs and mMCs under high glucose or AGE conditionsin vitro. We found that LRNA9884 expression was

significantly upregulated in both mTECs and mMCs at 1 hby AGE (50 mg/mL) stimulation in a time- and dose-dependent manner (Fig. 2B–E), whereas its inductionwas significant at 3 h under high glucose (35 mmol/L)stimulation (Supplementary Fig. 2).

Figure 1—Characterization of LRNA9884 in kidney. A: Real-time PCR shows expression of 21 common lncRNAs in db/db and db/m mice(n = 8 in each group). B: Real-time PCR detects the upregulation of na_9884 in the kidney during progression of db/dbmice from the age of8 weeks. C: A full length of na_9884 (508 base pairs [bp], named as LRNA9884) amplified by PCR is detected by electrophoresis in 1%agarose gel. D: Gene location of LRNA9884 in chromosome 5 of the mouse genome. E: Scores calculated by using the CPC and CPATanalysis. UTR, untranslated region. F: ISH detects LRNA9884 expression (nuclear pattern, presumably by glomerular [g] and tubular cells) atthe age of 8 weeks and 20 weeks in the kidneys of db/db and db/mmice (original magnification3400). Each bar represents the mean6 SEMfor groups of eight mice. *P , 0.05, **P , 0.01, ***P , 0.001 vs. db/m mice.

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LRNA9884 Expression Is Tightly Regulated by Smad3Signaling In Vitro and In Vivo

In our previous study, induction of LRNA9884 was asso-ciated with chronic kidney diseasemousemodels in a Smad3-dependent manner as revealed by high-throughput RNA

sequencing (12), but its regulatory mechanism in dia-betic kidney injury is still unknown. Interestingly, weobserved the renal expression of LRNA9884 in 12-week-old db/dbmice was largely suppressed after Smad3 KO, asshown by ISH and real-time PCR (Fig. 3A and B). Thus, we

Figure 2—LRNA9884 expression is tightly regulated by AGE in mTECs and mMCs. A: FISH assay with immunofluorescence containingepithelial cell marker keratins (second panels from left) and podocyte marker nephrons (right-most panels) confirmed the cell-specificexpression of LRNA9884 (red) with the nucleus (DAPI, blue) in the diabetic injured kidney of 20-week-old db/dbmice. Real-time PCR showsthat AGE (50 mg/mL) induces LRNA9884 expression in mTECs (B and C) and mMCs (D and E ) in a time- and dose-dependent manner. Datarepresent themean6SEM for at least three independent experiments. *P, 0.05, **P, 0.01, ***P, 0.001 vs. time-matched control or controlgroup. #P , 0.05 vs. AGE (50 mg/mL) concentration.

1490 Role of LRNA9884 in DN Diabetes Volume 68, July 2019

further speculated that LRNA9884 may be regulatedby Smad3 under diabetic conditions. This was exam-ined in MEFs, in which AGE-induced upregulation ofLRNA9884 was also largely suppressed in MEFs, knock-ing out Smad3 but not Smad2 (Fig. 3C and D andSupplementary Fig. 3A and B). By using the ECR browser,we predicted a conversed Smad3 binding site on thepromoter region of LRNA9884 genomic sequence (Fig.4A). The physical binding of Smad3 protein on thepromoter region of the LRNA9884 genomic sequencewas dramatically enriched in mTECs by AGE stimula-tion as demonstrated by ChIP assay (Fig. 4B). Thusdeletion (Smad3 KO) or inhibition (SIS3) of Smad3effectively blocked the AGE-induced LRNA9884 ex-pression in MEFs and mTECs in vitro, (Fig. 4C and Dand Supplementary Fig. 3). Thus, our findings clearlydemonstrated that expression of LRNA9884 is tightlyregulated by Smad3 signaling and serves as a directSmad3 target gene under diabetic condition in vitroand in vivo.

Kidney-Specific Silencing of LRNA9884 ProtectsAgainst Diabetic Renal Injury in db/db Mice

Because LRNA9884 induction was highly associated withT2DN progression (Fig. 1B and F), we further investigatedits potential role in the pathogenesis of T2DN in db/dbmice. EVs or plasmids expressing shRNA specificallyagainst LRNA9884 were transfected into both kidneysof the db/db mice (100 mg/mouse per treatment) by usingan ultrasound-microbubble delivery system (9). Treatmentwith shRNA effectively suppressed expression of renalLRNA9884 in db/db mice compared with the EV-treated20-week-old control mice according to results of real-timePCR and ISH (Fig. 5A and B). More importantly, inhibitionof renal LRNA9884 significantly suppressed renal histo-logical injury, including glomerular matrix deposition andglomerulosclerosis (Fig. 5A, C, and D), and decreasedmicroalbuminuria excretion and serum creatinine (Fig.5E and F) as determined by PAS, PASM, and Massontrichrome stain and ELISA assays. However, blockade ofrenal LRNA9884 showed no significant therapeutic effects

Figure 3—LRNA9884 expression is positively regulated via Smad3-dependent mechanism in vitro and in vivo. A and B: ISH and real-timePCR detect LRNA9884 is highly expressed in the diabetic kidney (week 12) of S3WT db/dbmice. In contrast, expression of renal LRNA9884in S3KO db/db mice is comparable to normal db/m mice (3400). g, glomerulus. C and D: Real-time PCR analysis shows that AGE inducesLRNA9884 expression in Smad3WT (S3WT)MEF but not in Smad3 KO (S3KO)MEF cells. Each bar represents themean6SEM for groups ofsixmice or three independent experiments. *P, 0.05, **P, 0.01 vs. db/m or timematch control group. ###P, 0.001 vs. db/db-S3WTgroup.

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on the type 2 diabetes phenotype, including body weightand fasting blood glucose (Supplementary Fig. 4), suggest-ing LRNA9884 promotes type 2 diabetes complications viaa kidney-specific mechanism.

Silencing of LRNA9884 Attenuates Renal InflammationIn Vivo and In VitroImmunohistochemistry and real-time PCR detected thata significant reduction of inflammatory markers (i.e., MCP-1, TNF-a, and IL-1b) occurred in the kidney of 20-week-old db/db mice by shRNA-9884 treatment compared withthe EV-treated and db/db controls (Fig. 6). To elucidate thedetailed mechanism mediated by LRNA9884, we screenedSmad3-related downstream inflammatory biomarkers af-ter knockdown LRNA9884 by using real-time PCR. Resultsshowed that AGE-induced inflammation and proinflam-mation cytokines were downregulated after silencing ofLRNA9884 in vitro, among which inhibition of MCP-1expression by shRNA treatment was the most remarkableone, suggesting that LRNA9884 may trigger MCP-1–dependent renal injury under diabetic condition (Supple-mentary Fig. 5).

In addition, no significant changes in the AGE-inducedrenal fibrotic marker expression (i.e., collagen I, fibronec-tin, and phosphorylated Smad3) in mTECs after effectivesiRNA-mediated knockdown of LRNA9884 (siLRNA9884)compared with the nonsense-treated control as shownby real-time PCR and Western blot analysis (Fig. 7 andSupplementary Figs. 5 and 6). MCP-1 has a crucial rolein renal inflammation via promoting the infiltration ofinflammatory leukocytes into injured kidney; therefore,the underlying mechanism of how LRNA9884 regulatesMCP-1 production at the transcriptional level was furtherelucidated in this study, as shown below.

LRNA9884 Directly Triggers MCP-1 Production at theTranscriptional Level, Thereby Promoting RenalInflammation Under Diabetic ConditionsMechanistically, we found that LRNA9884 was continu-ously increased in the nucleus of mesangial and tubularepithelial cells during T2DN progression, which was asso-ciated with a marked upregulation ofMCP-1 in the diabeticinjured kidney (Figs. 1B and 6B). We also found thata potential binding site of LRNA9884 is predicted on

Figure 4—LRNA9884 expression is transcriptionally induced by AGE via a Smad3-dependent mechanism. A: The evolutionarily conservedregion of LRNA9884 in human (hg19) and mouse (mm10) genomes and the predicted binding site by Smad3 analyzed by the ECR Browser(underlined). B: ChIP assay shows that Smad3 physically binds the LRNA9884 promoter in response to AGE (50 mg/mL). C: Real-time PCRanalysis shows AGE induces LRNA9884 expression in Smad3 WT MEF but is absent in Smad3KO MEF cells. D: Real-time PCR shows thatthe addition of SIS3 inhibits AGE (50 mg/mL)-induced LRNA9884 expression in mTECs at 1 h. Each bar represents the mean6 SEM for threeindependent experiments. **P, 0.01 vs. control group. #P, 0.05, ##P, 0.01, ###P, 0.001 vs. SIS3 (0 mmol/L) concentration in responseto AGE (50 mg/mL) or db/db S3WT group.

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Figure 5—Kidney-specific silencing of LRNA9884 improves renal histology and function in db/dbmice.A: ISH, PAS staining, PASM staining,andMasson trichrome staining show changes in renal histology after knockdown of LRNA9884 in db/db kidney.B: Real-timePCR shows thatultrasound-microbubble-mediated LRNA9884 shRNA (shRNA) transfer effectively inhibits its expression in db/db kidney at the age of20 weeks compared with the EV-treated control. C and D: Quantification analysis of mesangial matrix index by PAS staining andglomerulosclerosis by Masson trichrome staining at the age of 20 weeks in kidneys. E and F: Urine microalbuminuria-to-creatinine ratio(UACR) and serum creatinine in db/dbmice after knockdown of LRNA9884. Data represent the mean6 SEM for eight mice per group. **P,0.01, ***P , 0.001 vs. db/m mice; #P , 0.05, ##P , 0.01 vs. db/db mice with EV treatment.

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the promoter region of the MCP-1 genomic sequence byusing the Freiburg RNA Tool (Fig. 8A and SupplementaryFig. 7). To investigate the functional role of LRNA9884, weperformed siRNA-mediated knockdown of LRNA9884 onmTECs and found that silencing of LRNA9884 largelyinhibited AGE-induced MCP-1 production in mTECs(Fig. 8B and C and Supplementary Fig. 8). Furthermore,overexpression of LRNA9884 could directly upregulate theexpression of MCP-1 without AGE stimulation (Fig. 8Dand Supplementary Fig. 8). More importantly, direct phys-ical interaction between LRNA9884 and the promoter

region of the MCP-1 genomic sequence was clearly dem-onstrated in vitro by dual-luciferase reporter assay, whereoverexpression of full-length LRNA9884 significantly en-hanced the transcription activity of MCP-1 via directbinding on its promoter region (pGL3–MCP-1–promoter),which was effectively prevented by deletion of the pre-dicted LRNA9884 binding site on the MCP-1 promoter(pGL3–MCP-1–mutant) or the LRNA9884 full-length se-quence (pcDNA3.1-LRNA9884-mutant) (Fig. 8E). There-fore, targeting of renal LRNA9884 effectively protectedagainst AGE-driven diabetic kidney injury by blocking

Figure 6—Knockdown of LRNA9884 inhibits inflammatory response in the db/db kidney. A: Immunohistochemistry (IHC) and quantitativeanalysis of MCP-1, IL-1b, and TNF-a expression (3400). B: Real-time PCR analysis of MCP-1, IL-1b, and TNF-amRNA expression in micekidneys. Data represent the mean 6 SEM for six mice per group. *P , 0.05, **P , 0.01 vs. db/m mice; #P , 0.05 vs. db/db mice with EVtreatment.

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renal inflammation via a MCP-1–dependent mechanism.This was further demonstrated by a significant reductionof the kidney-infiltrating macrophages in shRNA-9884–treated diabetic kidney compared with EV-treated anduntreated db/db controls (Supplementary Fig. 9). There-fore, LRNA9884 may represent as an effective noveltherapeutic target for AGE-driven T2DN.

DISCUSSION

It is well accepted that DN as a severe diabetic complicationis mediated by TGF-b1 via both Smad3-dependent and

non–Smad3-dependent signaling pathways (3,30,31). Inthis study, we uncovered the pathogenic role of a novelSmad3-dependent LRNA9884 in the disease progressionof T2DN and identified its molecular mechanism underdiabetic conditions in vivo and in vitro. We found thatLRNA9884 was a Smad3-dependent lncRNA highlyexpressed in the diabetic kidneys and by AGE-stimulatedmMCs/mTECs of Smad3 wild-type (WT) but not Smad3KO. We also found that the expression levels ofLRNA9884 were correlated with progressive diabetickidney disease in db/db mice. Further characterization

Figure 7—Knockdown of LRNA9884 has no effect on AGE-induced fibrotic responses in mTECs. A: Real-time PCR shows LRNA9884expression compared with nonsense-treated control (NC) after AGE-induced in mTECs. Real-time PCR shows fibronectin (Fn) (B) andcollagen I (Col-I) (C ) expression after knockdown of LRNA9884 in mTECs. D: Western blot (WB) analysis shows expression of Fn and Col-I inmTECs. Quantitative analysis of Fn (E) and Col-I (F ) in mTECs. Data represent the mean6 SEM for at least three independent experiments.*P , 0.05, **P , 0.01, ***P , 0.001 vs. time 0 h. ##P , 0.01 vs. time-matched control group.

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by obtaining its full-length sequence via RACE showedthat LRNA9884 is 508 nucleotides in length and locateswithin 5033403H07Rik at murine chromosome 5 (Chr5:52973035–52972344). Since LRNA9884 covered the wholegenomic sequence of its host gene, we speculated thatLRNA9884 is the completed sequence of 5033403H07Rik.Through the use of CPC and CPAT analysis, LRNA9884 wasconfirmed and classified as a novel noncoding RNA.

We found that expression of LRNA9884 was signifi-cantly induced by the metabolic byproduct and hypergly-cemia (AGE and high glucose) under diabetic conditionsbut not by the profibrotic factor TGF-b1 in the culturedrenal cells in vitro. Furthermore, it was markedly upregu-lated in the db/db mice kidneys but not in the UUOkidneys, as previously reported (13), which implied that

LRNA9884 is specifically induced under diabetic condi-tions. However, LRNA9884 has no regulatory role inglucose metabolism, as evidenced by blocking LRNA9884without altering the fasting blood glucose levels and bodyweight in db/dbmice. The high disease- and tissue-specificityof LRNA9884 encouraged us to further elucidate its ther-apeutic potential for T2DN.

First, we identified the biological function of LRNA9884in the development of T2DN and found that knockdown ofLRNA9884 effectively inhibited the AGE-induced inflam-mation markers, including MCP-1, IL-1b, and TNF-a, oncultured mTECs without influencing on the expression offibrosis markers such as collagen I and fibronectin in vitro.The inflammatory role for LRNA9884 was further con-firmed in vivo, where specifically silencing of LRNA9884 in

Figure 8—LRNA9884 directly interacts with MCP-1 in mTECs. A: Predicted binding site of LRNA9884 on the genomic sequence of MCP-1.B: ELISA detects MCP-1 protein expression in cell culture medium. C: Western blot shows MCP-1 protein expression after siRNA-mediatedknockdown of LRNA9884 (siLRNA9884) in mTECs. D: Western blot shows that transfection of LRNA9884-expressing pcDNA3.1+ plasmidincreased MCP-1 protein expression in mTECs compared with the EV (pcDNA3.1+) control. E: Dual luciferase assay shows the enhancedeffect of LRNA9884 on MCP-1 expression via its binding on the MCP-1 promoter region, which is prevented by deletion of the LRNA9884binding site in MCP-1 promoter (pGL3–mutant–MCP-1) or LRNA9884 sequences (LRNA9884-mutant), respectively. Data represent themean 6 SEM for at least three independent experiments. *P , 0.05, **P , 0.01, ***P , 0.001 vs. time 0 h or pGL3-basic group. #P , 0.05,##P , 0.01 vs. time-matched control or pGL3–MCP-1 group, ^^P , 0.01 vs. LRNA9884 group.

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the kidney of db/db mice was capable of inhibiting renalinflammation. Indeed, renal LRNA9884 was significantlyupregulated at the age of 8 weeks before the onset ofdetectable diabetic renal injury, such as microalbuminuria,suggesting that LRNA9884 may be pathogenic and re-sponsible for the early development of T2DN in db/dbmice. It is likely that the early upregulation of LRNA9884may trigger the early development of low-grade renalinflammation rather than renal fibrosis, because inhibitionof LRNA9884 expression suppressed renal inflammationin db/dbmice and in cultured mTECs without effect on theexpression of profibrotic cytokines that mainly exert a rolein renal fibrosis during the late progression of T2DN(3,32–34). Thus, LRNA9884 may function to trigger thedevelopment of low-grade renal inflammation in the earlydevelopment of T2DN.

Second, we revealed that LRNA9884 was tightly regu-lated by Smad3 under the diabetic condition. We previouslyreported that AGE is able to activate the Smad-signalingpathway via both TGF-b–dependent and –independentmanners (30,31). In this study, we found that the promoterregion of LRNA9884 contained a Smad3-binding site andthat deletion of Smad3 dramatically blocked the upregula-tion of LRNA9884 in the diabetic kidney of db/db micein vivo and in AGE-stimulated MEFs in vitro, suggestinga positive regulatory role of Smad3 in the induction ofLRNA9884 during renal inflammation under diabetic con-ditions. Interestingly, although LRNA9884 was upregulatedin the kidney of Smad3-WT db/dbmice, it did not participatein the AGE/Smad3-mediated renal fibrosis because knock-down of LRNA9884 did not influence expression of Col-Iand fibronectin in vitro. Thus, LRNA9884 is a downstreammediator of Smad3 specific for promoting renal inflamma-tion under diabetic conditions.

Importantly, we detected that the LRNA9884 maymediate renal inflammation in db/db mice via a MCP-1–dependent mechanism. This was supported by the findingsthat a physical interaction exists between LRNA9884and the MCP-1 promoter as identified by ChIP and dual-luciferase reporter assays. Moreover, we also demonstratedthat silencing of LRNA9884 dramatically suppressed MCP-1production in the diabetic kidney of db/db mice and inAGE-stimulated mTECs. The diabetic milieu is a low-gradeinflammatory disease, where the kidney-infiltrating macro-phages largely promote renal inflammation-driven DN (35).Silencing of LRNA9884 reduced the renal MCP-1 level,which largely suppressed the macrophage infiltration andtherefore significantly reduced the renal inflammation-driven kidney injured in db/db mice. All of these findingsrevealed that MCP-1 should be an important downstreamtarget gene of LRNA9884 responsible for the T2DN de-velopment. In this study, we identified the main function ofLRNA9884 is promoting tissue inflammation. Nevertheless,the biological function and working mechanism of this novelSmad3-dependent lncRNA is still largely unknown, and anunbiased approach to determine whether LRNA9884 alters

the gene expression of more than MCP-1 should be furtherinvestigated.

Finally, results from this study also provided evidencethat targeting LRNA9884 specifically on the diabetic kid-ney may represent a novel therapy for T2DN. Consis-tent with previous reports (7,14), by using a noninvasiveultrasound-microbubble-mediated technique, we were ableto effectively silence renal LRNA9884-mediated MCP-1production in db/dbmice, which largely blunted the kidneyinfiltration of inflammatory leukocytes (e.g., macro-phages), thereby suppressing renal inflammation andresulting in improved renal function in the db/db micewith diabetic kidney diseases. It should be noted thatalthough we tested the specificity of siLRNA9884 byBLAST analysis, the siLRNA9884 vector used in ourin vivo experiments may have had effects on other genesor transcripts than those targeted.

In conclusion, the current study identifies thatLRNA9884 is a Smad3-dependent lncRNA and plays aproinflammatory role in T2DN via an MCP-1–dependentmechanism. Our findings suggest that targeting renalLRNA9884 may represent a novel and precision therapyfor AGE-driven diabetic kidney diseases.

Funding. This study was supported by Research Grants Council of Hong Kong(GRF 14106518, 14117418, 14117815, 14121816, 14163317, C7018-16G, TRST12-402/13N), Health and Medical Research Fund (03140486, 14152321),Direct Grant for Research CUHK (2017.002), Lui Che Woo Institute ofInnovative Medicine (CARE), and the Major State Basic Research Devel-opment Program of China (2012CB517705).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Y.-y.Z., P.M.-K.T., P.C.-T.T., J.X., X.-R.H., C.Y.,R.C.W.M., and H.-Y.L. approved the final version of the manuscript. Y.-y.Z. draftedthe original manuscript. Y.-y.Z. and P.M.-K.T. analyzed the data and created thefigures. Y.-y.Z., P.M.-K.T., P.C.-T.T., J.X., and X.-R.H. performed experiments.P.M.-K.T. and H.-Y.L. reviewed and edited the writing. X.-R.H., C.Y., and R.C.W.M.supported resources. R.C.W.M. and H.-Y.L. covered the funding. H.-Y.L. designedthe study. H.-Y.L. is the guarantor of this work and, as such, had full access to allthe data in the study and takes responsibility for the integrity of the data and theaccuracy of the data analysis.

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