bas i c re sea r ch www.k idney - i n t e rna t i ona l . o rg

Rhein reverses Klotho repression via promoterdemethylation and protects against kidney andbone injuries in mice with chronic kidney disease

Qin Zhang1,2,3, Lin Liu1, Wenjun Lin1, Shasha Yin2, Aiping Duan1, Zhihong Liu1,3 and Wangsen Cao1,2

1National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; 2The KeyLab of Jiangsu Molecular Medicine, Nanjing University School of Medicine, Nanjing, China; and 3Division of Nephrology, Jinling Hospital,Southern Medical University, Nanjing, China

Rhein is an anthraquinone compound isolated from themedicinal plant rhubarb and mainly used in the clinicaltreatment of diabetic nephropathy. Rhein exhibits variousrenoprotective functions, but the underlying mechanismsare not fully determined. However, its renoprotectiveproperties recapitulate the role of Klotho, a renal-specificantiaging protein critical for maintaining kidneyhomeostasis. Here we explored the connections betweenrhein renoprotection and Klotho in a mouse model ofadenine-induced chronic kidney disease. In addition tobeing an impressive Klotho upregulator, rhein remarkablyreversed renal Klotho deficiency in adenine-treated mice.This effect was associated with significant improvement indisturbed serum biochemistry, profibrogenic proteinexpression, and kidney and bone damage. Furtherinvestigation of the molecular basis of Klotho loss revealedthat these kidneys displayed marked inductions of DNAmethyltransferase DNMT1/DNMT3a and Klotho promoterhypermethylation, whereas rhein treatment effectivelycorrected these alterations. The renal protective effects ofrhein were largely abolished when Klotho was knocked-down by RNA interferences, suggesting that rhein reversalof Klotho deficiency is essential for its renoprotectiveactions. Thus, our study clarifies how rhein regulation ofKlotho expression contributes to its renoprotection andbrings new insights into Klotho-targeted strategy for thetreatment of kidney diseases of various etiologies.Kidney International (2017) 91, 144–156; http://dx.doi.org/10.1016/j.kint.2016.07.040

KEYWORDS: CKD; DNA methylation; epigenetics; Klotho; rhein

Copyright ª 2016, International Society of Nephrology. Published by

Elsevier Inc. All rights reserved.

Correspondence: Zhihong Liu or Wangsen Cao, Nanjing University Schoolof Medicine, The Key Lab of Jiangsu Molecular Medicine, 22 HankouRoad, Nanjing, Jiangsu 210093, China. E-mail: liuzhihong@nju.edu.cn orwangsencao@nju.edu.cn

Received 29 February 2016; revised 19 July 2016; accepted 28 July2016; published online 28 September 2016

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R hein (1,8-dihydroxy-3-carboxyl anthraquinone) is amonomer of anthraquinone derivatives isolated fromthe rhubarb plant and widely used in Chinese tradi-

tional medicine for the treatment of various clinical disordersincluding hepatic disease, osteoarthritis, diabetes, atheroscle-rosis, various cancers, and renal diseases.1 Rhein exhibitsstrong renal protective properties, as exemplified by the treat-ment of diabetic nephropathy, IgA nephropathy, and glomer-ulosclerosis.2 Further clinical and animal studies suggest thatrhein beneficially affects many cellular processes such asglucose and lipid metabolism, oxidative stress, inflammation,tissue fibrogenesis, and oncogenesis3; however, the rheincellular targets are not identified, and the underlying molec-ular mechanisms are still not completely understood. It isintriguing to see whether rhein exerts multiple renal protec-tive functions through each individual effector molecules oracts on an upstream “master” regulator that affects multiplecellular signaling pathways. Rhein’s bioactivities sum upthe protective roles of Klotho, a newly identified kidneybiomarker that also displays various renal protective proper-ties. Because the functional connection between rhein andKlotho has not been explored, it is not clear whether the rheinprotective functions relate to Klotho.

Klotho was identified originally as an antiaging gene pref-erentially expressed in kidney convoluted tubules and thechoroid plexus in the brain.4,5 Klotho exists in 2 forms: a type Itransmembrane protein and a secreted form that is generatedthrough ectodomain shedding from the membrane Klotho orthrough differential mRNA splicing. The secreted Klothopossesses glycosidase activity and is released into blood, urine,and cerebrospinal fluid, potentially acting as an enzyme orhormone to regulate the functions of extrarenal organs.4 Klo-tho protects the kidney from various acute and chronic injuriesthrough regulation of several critical intracellular signalingpathways, including insulin/insulin-like growth factor-1,fibroblast growth factor-23 (FGF-23), cyclic adenosinemonophosphate, protein kinase C, p53/p21, Wnt signaling,and transforming growth factor-b (TGF-b).5–8 Klotho candirectly bind to the type II TGF-b receptor and interfere withTGF-b binding to the receptors, thereby inhibiting TGF-bsignaling.9 Klotho deficiency in animals leads to the develop-ment of phenotypes resembling human aging or patients withchronic kidney disease (CKD).10,11 Clinical studies reveal that

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Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

patients with kidney diseases have early decreased Klotho inboth serum and urine, and the Klotho levels inversely correlatewith disease severity and progression.12,13 More relevantly,exogenous Klotho supplementation or transgenic Klothooverexpression effectively protects against kidney and othersystematic manifestations from various animal diseasemodels,14–16 suggesting that Klotho is a renal protector and thestrategies targeting Klotho may have therapeutic potential forpatients with kidney diseases.

Klotho promoter contains typical CpG islands and distinctKlotho expression in kidney cells and silencing in other tissueare believed to be mainly controlled by epigenetic modifica-tions,9 including DNA (cytosine) methylation, histone mod-ifications, and microRNA interactions.17 DNA methylationis catalyzed by DNA methyltransferases (DNMTs). Threebioactive DNMT enzymes have been identified in mammalsincluding DNMT1, DNMT3a, and DNMT3b. DNMT1 is formaintenance methylation for the semimethylated DNAstrands after DNA replication, whereas DNMT3a andDNMT3b are for de novo methylation of unmodified cytosine

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Figure 1 | Adenine mouse kidney displays severe renal dysfunctionsadenine (Ade) mice (n ¼ 6 in each group) were monitored once a weekhematoxylin and eosin (H&E) and Masson’s trichrome–stained kidney anweeks (n ¼ 6 in each group). Bars ¼ kidney, 50 mm; bone, 200 mm. (c)creatinine (Cre), alkaline phosphatase (ALP), uric acid, phosphorus (P), fi(iPTH) from control and adenine mice at 4, 6, and 8 weeks. (d) Representlower panels are the enlarged views of framed images above. (e) KidneWestern blot (2 randomly selected samples from each time point). *P <dehydrogenase.

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residues.18 Population-based genomewide quantitative eval-uations of DNA methylation demonstrate that DNA hyper-methylation and DNA hypomethylation occur at different lociin CKD.18 Aberrant Klotho promoter methylation has beenreported in aging-related disorders19,20 and various can-cers.21,22 Recent studies further suggest that Klotho promoterhypermethylation is associated with the pathogenesis of acutekidney disease and CKD.23,24 Because epigenetic changes arereversible, these studies provide a molecular basis for theepigenetic intervention of Klotho in the treatment of kidneydiseases.

In this study, we sought to assess the potential connectionbetween rhein renoprotection and its regulation of Klotho ina mouse model of adenine-induced CKD. We found thatrhein effectively recovered Klotho promoter hyper-methylation via reversing aberrant DNMT expression,consequently restoring the Klotho protein loss and protectingagainst kidney and bone damage in adenine mice. Therefore,our studies revealed a novel renoprotective property of rheinand demonstrated the therapeutic potential of endogenous

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d bone (femur) sections from control and adenine mice at 4, 6, and 8Average concentrations of serum Klotho, blood urea nitrogen (BUN),broblast growth factor-23 (FGF-23), and intact parathyroid hormoneative femur radiographs of control and adenine mice at 6 weeks. They levels of Klotho, b-catenin, and a-smooth muscle actin (a-SMA) by0.05, **P < 0.01 versus control. GAPDH, glyceraldehyde-3-phosphate

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Table 1 | Body weight and serum and urine biochemistry of adenine-fed mice

4 weeks 6 weeks 8 weeks

Control (n [ 6) Adenine (n [ 6) Control (n [ 6) Adenine (n [ 6) Control (n [ 6) Adenine (n [ 6)

BW (g) 28.33 � 2.10 21.58 � 2.27a 30.07 � 1.08 21.22 � 1.72b 32.5 � 1.50 21.08 � 1.67b

SerumCre (mg/dl) 0.15 � 0.06 0.49 � 0.06a 0.16 � 0.04 0.71 � 0.15b 0.17 � 0.03 0.86 � 0.14b

BUN (mg/dl) 28.15 � 4.88 101.3 � 23.33a 26.1 � 7.35 114.5 � 23.39b 21.65 � 1.77 128.67 � 15.52b

Ca (mM) 2.35 � 0.06 2.365 � 0.19 2.33 � 0.06 2.43 � 0.07 2.445 � 0.06 2.47 � 0.07P (mM) 3.95 � 0.12 4.58 � 0.49 3.83 � 0.38 4.94 � 0.25a 4.02 � 0.35 5.33 � 0.29a

ALP (U/l) 57.5 � 6.36 84.5 � 9.19 54 � 14.40 143 � 33.29a 53 � 14.14 164 � 28.16a

Klotho (ng/ml) 5.44 � 1.12 4.05 � 0.52 ND 2.02 � 0.51b ND 1.65 � 0.34b

FGF-23 (ng/ml) 0.37 � 0.14 1.59 � 1.09 ND 17.48 � 4.25a ND 26.03 � 2.39b

iPTH (ng/ml) 0.50 � 0.07 0.53 � 0.04 ND 1.23 � 0.33a ND 1.25 � 0.38a

UrineCre (mg/dl) 54.85 � 6.07 8.52 � 1.70b 44.96 � 0.71 9.48 � 0.25b 42.09 � 15.79 9.78 � 0.22b

BUN (mg/dl) 3608.5 � 422.71 695.13 � 113.63b 2769.2 � 759.01 621.4 � 56.45b 2552 � 841.17 692.6 � 56.14b

Ca (mM) 2.16 � 0.61 0.95 � 0.12b 1.41 � 0.03 0.78 � 0.13b 1.45 � 0.55 1.42 � 0.13P (mM) 16.32 � 0.15 16.39 � 1.74 16.46 � 0.18 16.96 � 0.79 16.66 � 0.42 16.95 � 0.76

Adenine, 0.2% adenine diet; ALP, alkaline phosphatase; BUN, blood urea nitrogen; BW, body weight; Ca, calcium; Control, normal diet; Cre, creatinine; FGF-23, fibroblast growthfactor-23; iPTH, intact parathyroid hormone; ND, not done; P, phosphorus.aP < 0.05 or bP < 0.01 compared with control of matching time, respectively.

bas i c re sea r ch Q Zhang et al.: Rhein restores Klotho and prevents kidney injury

Klotho intervention in relieving kidney injury and relatedbone complications.

RESULTSAdenine mouse kidney displays severe renal dysfunctionsand Klotho repressionTo gain insights into the possible link between rhein reno-protection and Klotho in CKD, we first established an adeninemouse model of CKD according to previous studies.25,26 Wefed the control mice a regular diet and adenine mice a regulardiet containing adenine (02%) for 4, 6, and 8 weeks(Figure 1a). We found that adenine mice did not gain weightduring the period (Figure 1a). The kidney histologic exami-nations revealed increasing pathologic changes such astubular atrophy and interstitial fibrosis as evidenced bycollagen deposition (Figure 1b) in adenine mouse kidney. Inaddition, the serum levels of blood urea nitrogen (BUN) andcreatinine increased early at 4 weeks, whereas phosphorus,alkaline phosphatase, FGF-23, and intact parathyroid hor-mone (PTH) increased significantly at 6 weeks after adeninefeeding, except that calcium level stayed within normal rangethroughout (Figure 1c, Table 1). Although we detected neithercardiovascular calcification nor pathologic changes in theheart after intensive examination, the mouse femurs displayedobvious osteoporosis-like changes: thinner and derangedtrabeculae with increased lacunae (Figure 1c, lower panel)and bone density loss (Figure 1d) at 6 weeks, likely due todisturbed phosphorus and intact PTH metabolism. Impres-sively, serum Klotho decreased dramatically and reachedapproximately one third of control by 8 weeks (Figure 1c).Even more impressively, renal Klotho had almost completelydisappeared at 4 weeks, whereas the markers of epithelial-mesenchymal transition such as b-catenin and a-smoothmuscle actin (a-SMA) were remarkably induced (Figure 1e).Collectively, these results suggest that adenine mice developKlotho deficiency early with serious renal damage, disturbed

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mineral metabolism, and bone complications after 6 weeks ofadenine feeding; thus, we chose 6 weeks as the experimentpoint in the later experiments.

Rhein upregulates Klotho and reverses Klotho loss in adeninemouse kidneyTo investigate whether rhein regulates Klotho expression, wefirst examined Klotho protein in mouse kidneys that weretreated with rhein for 3 days. Impressively, rhein treatmentnotably increased Klotho protein in healthy mouse kidneys(Figure 2a). We next tested rhein regulation of Klotho inkidney tubular HK2 cells. TGF-b is a known pathologic factormediating renal injury during CKD and inhibiting Klotho.9,27

We found that rhein dose-dependently inhibited TGF-b–induced Klotho repression (Figure 2b and c). We also con-structed a mouse Klotho promoter luciferase reporter con-taining part CpG island (up to þ88 related to thetranscription starting site) and found that rhein dose-dependently increased Klotho promoter basal activity andinhibited the TGF-b–incurred promoter repression(Figure 2d). In adenine mice, rhein treatment reversed serumand urine Klotho loss, as evidenced by immunofluorescentstaining (Figure 2e), enzyme-linked immunosorbent assay(Figure 2f) and Western blot assay (Figure 2g). In addition,rhein also significantly reversed the abnormal expression ofE-cadherin, a-SMA, b-catenin, and phosphorylated Smad3(Figure 2g and h). Taken together, these results demonstratethat rhein is able to effectively inhibit Klotho loss andimprove the expression of key kidney-injury markers underpathologic conditions.

Rhein protects renal function and prevents kidney and boneinjuries in adenine miceAdenine mice were reported to exhibit extrarenal complica-tions such as bone injuries and cardiovascular calcificationsdue to extensive disturbance of mineral and growth factor

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Figure 2 | Rhein reverses Klotho repression in adenine mice. (a) Western blot examination of Klotho levels in mouse kidneys treated withrhein (Rh) for 3 days (3 mice in each group). (b) Rhein reverses transforming growth factor-b (TGF-b)–induced Klotho suppression in HK2 cellstreated with TGF-b in presence or absence of increasing doses of rhein for 48 hours. Klotho protein was assayed by Western blot. (c)Quantification for Figure 2b. (d) Luciferase (luc) assay of mouse Klotho promoter reporter. HEK293 cells were transfected with the reporterplasmid and a renilla luciferase reporter, then treated with TGF-b (5 ng/ml) and/or increasing doses of rhein (1, 5, 10 mg/ml) for 48 hours. Theluciferase activities of Klotho promoter reporter were normalized with renilla luciferase activities. (e) Representative photomicrographs ofimmunochemistry staining of kidney sections from control, rhein, adenine, and rhein-treated adenine mice (6 weeks). (f) Serum Klotho levelsfrom control, rhein, and adenine mice with or without treatment for 6 and 12 weeks determined by enzyme-linked immunosorbent assay. (g)Kidney expression of E-cadherin, a-smooth muscle actin (a-SMA), b-catenin, phosphorylated Smad3, and Klotho from control, adenine-, andrhein-treated adenine mice, assayed by Western blot (3 samples in each group). (h) Quantification analyses of Figure 2g. *P < 0.05, **P < 0.01versus Control; #P < 0.05, ##P < 0.01 versus TGF-b/adenine treatment.

Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

metabolism.28 To determine whether rhein treatment protectsagainst renal malfunctions as well as the extrarenal compli-cations, we randomly divided mice into 4 groups: control,rhein, adenine, and rhein-treated adenine mice and admin-istered rhein for 6 and 12 weeks, respectively. The bodyweight of rhein mice did not notably increase during theexperiment, probably due to its energy expenditure–promoting and lipid-lowering effects29; however, rheinsignificantly blocked body weight loss of adenine mice(Figure 3a, which compares rhein/adenine mice with adeninemice). Adenine caused obvious kidney shrinkage with smallopaque nodules on the kidney surface, whereas rhein treat-ment eliminated the abnormal morphologies (Figure 3b). Thekidney histologic examinations by hematoxylin and eosin andMasson trichrome staining showed that adenine mouse kid-ney displayed tubular atrophy, increased cell density(Figure 3c, upper panel) due to the infiltration of myofibro-blast,30 and fibrosis (Figure 3c, middle panel); however, rhein

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treatment effectively reduced these pathologic changes after 6weeks of treatment (Figure 3c). Rhein also significantly cor-rected the disturbance of BUN, creatinine, alkaline phos-phates, uremic acid, phosphorus, FGF-23, and intact PTH(Figure 3d and Table 2) at both 6 and 12 weeks (Figure 3d andTable 2). In addition, rhein reversed the osteoporosis-likechanges in femurs: the thinner and deranged trabeculaewith increased lacuna (Figure 3c, lower panel) and bonedensity loss (Figure 3e and f). Taken together, these resultsindicate that rhein can effectively protect renal functions andprevent adenine-induced kidney and bone injuries.

Rhein post-administration mitigates CKD progressionTo gain further insights into the rhein treatment potential forprevious renal damage, we fed mice adenine for 2 or 4 weeksfirst and then treated mice with rhein for an additional 4weeks. The results showed that rhein treatment after 2 weeksof adenine feeding still effectively attenuated kidney tubule

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Figure 3 | Rhein protects against kidney and bone injuries in adenine mice. (a) Mouse body weights of control (Ctl), rhein (Rh), adenine(Ade), and rhein-treated adenine (Rh/Ade) mice (n¼ 6 in each group) were monitored once a week for a total of 6 weeks. (b) Representative grossappearance of kidneys from indicated groups. (c) Representative photomicrographs of hematoxylin and eosin (H&E) and Masson’s trichrome–stained kidney or femur (lower panel) sections of control, rhein, adenine, and rhein-treated adenine mice (6 weeks). The fibrotic lesions wereindicated by blue color areas in the middle panel. (d) Average concentrations of serum blood urea nitrogen (BUN), creatinine (Cre), alkalinephosphatase (ALP), uric acid (UA), phosphorus (P), and fibroblast growth factor-23 (FGF-23), and intact parathyroid hormone (iPTH) from control,rhein, adenine, and rhein-treated adenine mice of 6 and 12 weeks, respectively. (e) Femur radiographs from control, rhein, adenine, and rhein-treated adenine mice of 6 weeks. The lower panels are the enlarged views of images above. (f) Quantification of bone density of femurs (tibialside). *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus adenine mice.

bas i c re sea r ch Q Zhang et al.: Rhein restores Klotho and prevents kidney injury

atrophy and fibrosis on histologic examination (Figure 4a);significantly improved the serum parameters of BUN, creat-inine, and phosphorus (Figure 4b); and mitigated abnormalexpression of Klotho, b-catenin, E-cadherin, a-SMA, andphosphorylated Smad3 (Figure 4c and d), indicating thatrhein has the treatment potential to protect the kidney fromprevious damage and functional abnormalities. However,these beneficial effects were only marginal when rhein treat-ment started after 4 weeks of adenine feeding (Figure 4c, rightpanel), probably due to the fact that some irreversible path-ologic changes had occurred.

Rhein reverses Klotho promoter hypermethylation in adeninemouse kidneyHuman and mouse Klotho promoters contain typical CpGislands (Figure 5a). To gain additional insight into thepossible involvement of DNA methylation alteration in Klo-tho reduction in adenine kidney, we first examined the KlothomRNA levels. The results showed that adenine mouse kidneyexhibited reduced Klotho mRNA, and rhein treatment almostcompletely reversed the reduction (Figure 5b and c). We then

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examined the Klotho promoter methylation by methylation-specific polymerase chain reaction (PCR) on a CpG-richarea (þ497/þ685) as selected by MethPrimer software. Theresults showed that adenine mouse kidney displayed increasedmethylation (28%–60%, P < 0.05), whereas rhein treatmentbrought the methylation level back to 35% (P < 0.05)(Figure 5d and e). To confirm this result, we performed anadditional methylation analysis from a different locus (�242/þ14) by bisulfate-specific PCR (3 mice randomly selectedfrom each group and 5 clones from each mouse). The resultsshowed that adenine mice had significant increase inmethylation at this site, and rhein treatment reversed theincrement to the control level (Figure 5f and g), indicatingthat rhein reversal of Klotho loss is likely due to its deme-thylating effects.

Rhein mitigates aberrant DNMT expression in adenine mousekidneyTo explore the molecular basis of Klotho promoter hyper-methylation in adenine mice, we examined the DNMTexpression by Western blot. We found that adenine mouse

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Table 2 | Body weight and serum and urine biochemistry

Normal (n [ 8) Rhein (n [ 6)

6 Weeks 12 Weeks

Adenine (n [ 8) Rh/Ade (n [ 8) Adenine (n [ 8) Rh/Ade (n [ 6)

BW (g) 32.13 � 2.81 25.43 � 1.46 23.76 � 1.08c 26.21 � 1.18d 19.23 � 1.15c 26.73 � 1.03d

SerumCre (mg/dl) 0.11 � 0.02 0.09 � 0.04 0.77 � 0.07c 0.12 � 0.02d 1.06 � 0.14c 0.17 � 0.04d

BUN (mg/dl) 28.03 � 5.04 24.62 � 4.71 125.72 � 14.49c 34.8 � 5.72d 146.63 � 11.68c 28.08 � 4.85d

Ca (mM) 2.18 � 0.24 1.97 � 0.24 2.45 � 0.25 2.23 � 0.18 2.17 � 0.24 1.92 � 0.11P (mM) 3.63 � 0.47 3.20 � 0.24 5.16 � 0.28c 3.02 � 0.48d 4.80 � 0.54c 2.93 � 0.28d

ALP (U/l) 72.33 � 13.01 60.67 � 8.03 170.67 � 15.82c 90.33 � 9.62b 162.67 � 32.55c 78.75 � 13.30b

UA (mM) 110.67 � 32.50 81.33 � 15.31 267.67 � 19.45c 118.67 � 24.70b 199.67 � 14.57c 117 � 16.33d

FGF-23 (ng/ml) 0.37 � 0.14 0.33 � 0.08 17.40 � 2.08c 0.37 � 0.10d 28.04 � 3.00c 0.36 � 0.09d

iPTH (ng/ml) 0.50 � 0.07 0.48 � 0.08 1.23 � 0.33a 0.51 � 0.07b 1.84 � 0.50a 0.45 � 0.05b

Klotho (ng/ml) 5.44 � 1.32 5.55 � 0.62 1.69 � 0.32c 4.89 � 0.92b 1.85 � 0.38c 5.43 � 1.72b

UrineCre (mg/dl) 36.76 � 14.21 18.32 � 2.24 9.54 � 0.18 16.24 � 2.56d 8.09 � 0.43c 16.72 � 2.41d

BUN (mg/dl) 2363.3 � 884.48 843.3667 � 93.6 605.57 � 83.28c 1286.97 � 233.78d 525.23 � 11.05c 870.27 � 79.31b

Ca (mM) 1.17 � 0.42 0.78 � 0.05 0.82 � 0.19 1.11 � 0.61 0.92 � 0.29 1.22 � 0.39P (mM) 15.72 � 1.30 11.23 � 2.38 18.24 � 1.46 16.80 � 0.08 17.61� 0.30 15.20 � 4.15UA (mM) 377 � 62.75 187.67 � 5.51 69 �3.61c 223.33 � 20.79d 14 � 6.56c 172.33 � 60.47d

ALP, alkaline phosphatase; BUN, blood urea nitrogen; BW, body weight; Ca, calcium; Cre, creatinine; FGF-23, fibroblast growth factor-23; iPTH, intact parathyroid hormone;P, phosphorus; Rh/Ade, rhein/adenine; UA, uric acid.aP < 0.05 or cP < 0.01 compared with control for each time point.bP < 0.05 or dP < 0.01 compared with adenine treatment alone for each time point.

Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

kidney displayed dramatic increases of DNMT1 andDNMT3a and a slight decrease of DNMT3b. Rhein treatmentreturned DNMT levels to control levels (Figure 6a and b).Because DNA hypermethylation correlates with a gain of

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Figure 4 | Rhein protects kidney from previous renal injuries. (a) Repsections of control and mice treated with on adenine for 2 weeks (A2Wtreatment. (b) Average levels of serum blood urea nitrogen (BUN), creatin(c) Kidney expression of Klotho, E-cadherin (E-cad), a-smooth muscle actiand mice treated with adenine for 2 or 4 weeks followed by 4 weeks of rhALP, alkaline phosphatase; UA, uric acid. *P < 0.05, **P < 0.01 versus co

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function of DNMT, we speculated that DNMT1 andDNMT3a are responsible for the Klotho promoter hyper-methylation. To test this idea, we examined the effect ofDNMT inhibition on rhein restoration of Klotho reduction

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resentative photomicrographs of Masson’s trichrome–stained kidney) or 4 weeks (A4W) followed by 4 weeks with or without rhein (Rh)ine (Cre), phosphorus (P), and calcium (Ca) from mice as in (a) (n ¼ 3).n (a-SMA), b-catenin (b-cat), phosphorylated Smad3 from control miceein treatment, assayed by Western blot. (d) Quantification of Figure 4c.ntrol; #P < 0.05, ##P < 0.01 versus adenine mice.

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Figure 5 | Rhein attenuates Klotho promoter hypermethylation. (a) Schematic diagram of mouse Klotho promoter. The CpG island is in gray.The relative locations of methylation-specific polymerase chain reaction (MSP) and bisulfite-sequencing polymerase chain reaction (BSP)primers are indicated. (b) Klotho mRNA levels from control (Ctl), rhein (Rh), adenine (Ade), and rhein-treated adenine (Rh/Ade) mouse kidneys(6 weeks) were examined by reverse transcriptase polymerase chain reaction (PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) wasthe internal control. (c) Quantifications of Figure 4b. (d) MSP analysis of kidney Klotho promoter methylation (Methy) of control, adenine, andrhein-treated adenine mice (6 weeks, 2 randomly selected samples in each group). (e) Quantifications of Figure 5d. (f) BSP analysis of control,adenine, and rhein-treated adenine mouse kidneys (6 weeks). Each box represents the indicated mouse kidney (M); each row of circles in theboxes represents the Klotho CpG island; each dot represents a single CpG site. Open circles indicate unmethylated (Unmethy) CpGs; filled circlesindicate methylated CpGs. Each row represents a single sequenced clone (5 clones for each mouse were presented). (g) Quantitative analysis of(f). The results are presented as percentages of methylation. The statistical analysis was based on at least 6 mice in each group, and therepresentative results are shown. *P < 0.05 versus control; #P < 0.05 versus adenine mice. GC, guanine-cytosine content; M1, mouse 1; M2,mouse 2; M3, mouse 3.

bas i c re sea r ch Q Zhang et al.: Rhein restores Klotho and prevents kidney injury

incurred by TGF-b, the crucial profibrogenic growth factorthat promotes epithelial-mesenchymal transition during CKDprogression.31 We verified that adenine mouse kidneyexpressed increased TGF-b by reverse transcriptase PCR(Figure 6c and d). We then treated HK2 cells with TGF-b inpresence or absence of rhein and/or a DNMT inhibitor 5-aza2-deoxicytidine (decitabine, 5-Aza 2-deoxicytidine). The re-sults showed that rhein and 5-Aza 2-deoxicytidine similarlyblunted TGF-b inhibition of Klotho and the induction ofphosphorylated Smad3, a TGF-b–signaling activation markerthat was reported to be inhibited by Klotho (Figure 6e and f).These results suggest that enhanced Klotho promotermethylation in adenine mice is due to increased DNMT1 andDNMT3a, and rhein reversal of DNMT1 and DNMT3 con-tributes to the reversal of Klotho loss.

Rhein derepression of Klotho protects against renal damagein an adenine mouse modelTo assess whether rhein reversal of Klotho loss contributes toits renal protection, we first tested the impact of Klothoknockdown on rhein improvement of epithelial-mesenchymal transition marker expressions. We surmisedthat if rhein provided renal protection through Klothorestoration, then a lack of Klotho would reduce or eliminatethe rhein improvements of aberrant epithelial-mesenchymaltransition marker expressions. We constructed a shorthairpin RNA (shRNA) plasmid that can effectively reduceKlotho protein level when transfected into HK2 cells

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(Figure 7c) and then performed the experiment with a controlplasmid containing a scrambled oligo and an shRNA plasmidspecific for Klotho in HK2 cells. The results showed thatshRNA-Klotho plasmid-transfected cells exhibited increasedbasal levels of a-SMA, b-catenin, phosphorylated Smad3, andfibronectin, as well as reduced E-cadherin, compared to thatin control plasmid-transfected cells (Figure 7a, comparinglane 5 with lane 1), indicating that Klotho controls their basalexpression. Although rhein significantly alleviated TGF-b–induced abnormal expression of the epithelial-mesenchymaltransition markers in control cells, the effects were largelyabolished when Klotho was knocked down by short hairpinRNA (Figure 7a and b).

We next wanted to verify whether rhein restoration ofKlotho protects against renal damage in adenine mice. Forefficient Klotho knockdown in kidney, we used a small,interfering RNA (siRNA)–mediated RNA interference tech-nique. Mice received either a scrambled control RNA orsiRNA specific for Klotho once a week before and duringrhein treatment and/or adenine feeding for a total of 6 weeks.The results showed that Klotho knockdown (Figure 8c, toppanel) induced pathologic changes in renal and bone mor-phologies (Figure 8a), BUN levels, creatinine and alkalinephosphatase (Figure 8b), changes in the expression ofE-cadherin, a-SMA, b-catenin, and phosphorylated Smad3(Figure 8c and d, comparing the controls between siRNA-control and siRNA-Klotho-treated mice) and exacerbatedadenine-induced pathologic alterations (Figure 8, comparing

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Figure 6 | Rhein ameliorates aberrant DNA methyltransferase (DNMT) expression. (a) Kidney expression of DNMT1, DNAM3a, and DNMT3bwere assayed from control (Ctl), adenine, and rhein (Rh)-treated adenine (Ade) mouse kidneys by Western blot (3 randomly selected samples ineach group). (b) Quantifications of a. (c) Reverse transcriptase polymerase chain reaction examination of transforming growth factor-b (TGF-b)from control, rhein, and adenine mouse kidneys (2 randomly selected samples from each group). (d) Quantifications of c. (e) Expression ofKlotho and phosphorylated Smad3 in HK2 cells treated with TGF-b (5 ng/ml) in presence or absence of rhein or 5-Aza 2-deoxicytidine (5Aza2d)(–) for 48 hours, assayed by Western blot. (f) Quantifications of e. The statistical analysis was based on at least 6 mice or 3 independent cellexperiments, and representative results are shown. *P < 0.05, **P < 0.01 versus control; #P < 0.05, ##P < 0.01 versus adenine mice. GAPDH,glyceraldehyde-3-phosphate dehydrogenase.

Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

adenine mice between siRNA-control and siRNA-Klothotreatment). Although rhein treatment effectively reducedrenal and bone damage (Figure 8a) and improved the serumlevels of BUN, creatinine, phosphorus, and alkaline phos-phatase (Figure 8b) and the protein expression (Figure 8c andd), the protective effects were largely abolished in Klothoknockdown kidneys (Figure 8a–d). Altogether, these resultsstrongly demonstrate that rhein restoration of Klotho is a

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Figure 7 | Rhein Klotho-dependently improves profibrogenic proteinspecific short hairpin RNA (shRNA) plasmid, and then transforming growfollowed by Western blot assay for the expression of a-smooth muscleSmad3, and fibronectin (FN). (b) Quantifications for Figure 6a. (c) Klothofected in duplicates with control (containing scrambled [Scrm.] sequencesblot 24 hours later. All statistics from cell experiments were based on acontrol in shRNA-control cells; #P < 0.05, ##P < 0.01 versus TGF-b treatmeTGF-b treatment in shRNA-Klotho cells.

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critical mechanism that confers rhein’s renal protection inadenine-fed CKD mice.

DISCUSSIONOur study tests the hypothesis that rhein protection againstrenal damage is related to Klotho. Our major novel findingsinclude the following: (i) rhein is an outstanding Klothoupregulator and effectively reverses Klotho loss in adenine

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expressions. (a) HK2 cells were transfected with control or Klotho-th factor-b (TGF-b) (5 ng/ml) and/or rhein (Rh) (5 mg/ml) for 48 hours,actin (a-SMA), E-cadherin (E-cad), b-catenin (b-cat), phosphorylatedshRNA reduces Klotho protein in kidney cells. HK2 cells were trans-) or shRNA plasmid specific for Klotho. Klotho was assayed by Westernt least 3 independent experiments. *P < 0.05, **P < 0.01 versus thent; OP < 0.05, OOP < 0.01 versus shRNA control, :P < 0.05 versus

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Figure 8 | Klotho is essential for rhein renal protection. (a) Representative hematoxylin and eosin– (H&E) and Masson’s trichrome–stainedkidney and bone sections from control or small, interfering RNA (siRNA)-Klotho mice that were subjected to adenine or adenine treated withrhein (n ¼ 6, 6-week treatments). (b) Serum levels of blood urea nitrogen (BUN), creatinine (Cre), phosphorus (P), and alkaline phosphatase (ALP)from the experimental mice in a. (c) Western blot examination of renal expression of Klotho, a-smooth muscle actin (a-SMA), E-cadherin (E-cad),phosphorylated and total Smad3, and b-catenin (b-cat) from the experimental mice in a (2 randomly selected samples from each group). (d)Quantifications of c. *P < 0.05, **P < 0.01 versus control, #P < 0.05 versus adenine in small, interfering RNA (siRNA) control (Con) kidneys; OP <0.05, OOP < 0.01 versus control in siRNA-Klotho kidneys.

bas i c re sea r ch Q Zhang et al.: Rhein restores Klotho and prevents kidney injury

mouse kidney and reduces renal and bone pathogenesis, (ii)rhein reverses Klotho loss through inhibiting aberrant DNMTexpression and Klotho promoter hypermethylation, and (iii)Klotho is an essential molecule that mediates, at least in part,the rhein renoprotective functions. Therefore, our studyrevealed an epigenetic mechanism of Klotho repression inan animal model of CKD and demonstrated a novel DNAdemethylating property of rhein in renoprotection.

An adenine animal model of obstructive nephropathy wasoriginally established in the rat for the study of chronic renalfailure.32 Adenine in the diet is readily absorbed from theintestine and converted to 2,8-dihydroxyadenine, becominginsoluble crystals that block renal tubules and consequentlycausing extensive kidney injuries, disturbance of serumbiochemistry, bone damage, and cardiovascular calcification,28

which resembles the pathologic alterations in patients withCKD-mineral bone disorder, a clinical syndrome that en-compasses patients with CKD with biochemical alterations in

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calcium, phosphorus, and PTH, as well as bone abnormalitiesand vascular calcification. Therefore, the adenine model dis-plays more severe renal damage associated with external organcomplications. Adenine mice in our experiments seemed tohave normal calcium and did not display apparent cardio-vascular manifestations such as vascular calcification andcardiac hypertrophy even after 12 weeks of adenine feeding(data not shown), probably due to the fact that the consistentcardiovascular calcifications are only present with high phos-phorus or with a reduced protein diet.25 Further, the bodyweight of adenine mice did not increase during our study andthe others,33–35 similar to that observed in Klotho knockoutmice.4 The influence of growth retardation on renal patho-genesis is currently unclear. Other than that, the miceexhibited characteristics of CKD-mineral bone disorder withapparent bone complications after 6 weeks of adenine feeding.

The kidney is the major organ involved in the regulation ofcalcium and phosphate homeostasis, which is essential for

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Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

bone mineralization and development. Many substancessynthesized by the kidney including Klotho are involved indifferent stages of bone formation, remodeling, and repair.Some cytokines that can be affected by the kidney, such asFGF-23 and PTH, also play important roles in bone meta-bolism.36,37 Klotho-deficient mice show low bone formationand resorption activities, which result in osteopenia, indi-cating that Klotho can directly affect bone formation andremodeling.4 Impressively, rhein treatment effectively allevi-ated disturbed serum and uric biochemistry, serum FGF-23,and intact PTH and attenuated renal and bone injuries in aKlotho-dependent manner, consistent with previous obser-vations that Klotho-targeted strategies with chemical agentssuch as rapamycin, resveratrol, and testosterone remarkablyprotected against kidney and kidney damage–associatedvascular calcifications via upregulating Klotho,38–41 indicatingthat rhein reversal of Klotho loss contributes significantly toits renal and bone protective functions.

The molecular events that lead to Klotho deficiency afterkidney injuries are not fully understood. Several pathologicprocesses and cellular factors have been reported or impliedto be associated with Klotho repression, including oxidativestress,42 proinflammatory cytokine tumor necrosis factor-a,43–45 interleukin-6,46 TGF-b,47 and epigenetic in-fluences.9,23,24 Klotho promoter contains a functional nuclearfactor-kB binding site that is believed to be responsible forproinflammatory cytokine–mediated earlier Klotho tran-scriptional repression.45 Epigenetic modification, especiallythe DNA methylation, is emerging as a critical contributor toboth acute and CKD progression.18,48 Klotho promoter ischaracterized by typical CpG islands, and its differentialmethylation is associated with aging-related disorders20 andvarious cancer conditions.21,47 Elevated Klotho promotermethylation has been linked to the severity of CKD in patientsand in uremic toxin-induced CKD in experimental ani-mals.23,24 Our results not only confirmed that Klotho pro-moter demonstrated increased methylation but alsodemonstrated increased DNMT1 and DNMT3a, whichaccounted for the increased Klotho promoter hyper-methylation and probably epigenetic alterations associatedwith other profibrogenic genes reported previously.47 Moreimportantly, we demonstrated that rhein can almostcompletely reverse both aberrant DNMT expression andKlotho promoter hypermethylation, and Klotho knockdowncompromised rhein protective activity toward profibrogenicprotein expression. Therefore, our results reveal a novelfeature of rhein in renoprotection and suggest that many ofrenoprotective activities reported previously are probably due,at least in part, to rhein restoration of Klotho because Klothoacts upstream of many renal pathologic signaling pathways.

It is interesting that, unlike increased DNMT1 andDNMT3a, DNMT3b protein is decreased in adenine mousekidney, whereas rhein seems to be able to simultaneouslyreverse both up- or downregulated DNMTs (Figure 6A), sug-gesting a more complicated regulatory mechanism. Itis currently unclear how rhein affects both up- and

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downregulated DNMTs, which definitely warrants furtherinvestigation. Nevertheless, the overall effect of rhein on Klo-tho promoter seems to be the correction of hypermethylation;therefore, we propose that rhein has a strong demethylatingfunction, and rhein can confer its protein regulation and renalprotection through an epigenetic mechanism.

The results of our study have several important clinicalimplications. First, we clearly show that DNMT aberrationis associated with CKD progression and causes epigeneticmodification of an important renal protective gene, Klotho,providing a molecular basis for Klotho-targeted interventionsin potential treatments of CKD. Second, demethylating agentssuch as azacitidine and decitabine might lack specificity andefficacy in the treatment of renal diseases1; hence, explorationof novel demethylating agents such as rhein holds great po-tential for effective retardation or treatment of kidney diseases.Third, Klotho epigenetic alteration is also involved in variousaging and cancer conditions8,22,46,47,49; thus, our study opensup new prospects for rhein’s potential applications in not onlykidney diseases, but also the antiaging and anticancer therapies.

In conclusion, we have uncovered an important feature ofrhein in the prevention and treatment of kidney disease withbone complications and identified a novel epigenetic mech-anism of rhein renoprotection—Klotho promoter demethy-lation and subsequent reversal of Klotho loss. The resultsfrom this study provide strong evidence that Klotho-targetedstrategies with agents such as rhein have powerful treatmentpotential for patients with CKD and the related externalorgan complications.

MATERIALS AND METHODSAnimal modelsAnimal maintenance and experimental procedures were in accor-dance with the experimental animal use guidelines and approved bythe Animal Care Committee of Nanjing University (Nanjing, China).C57BL/6 male mice were from the Model Animal Research Center ofNanjing University and housed in the animal facility on site. Mousebody weight was measured every week. The adenine mouse modelwas established with C57BL/6 male mice by 0.2% adenine-containing (Sigma-Aldrich, St. Louis, MO) diet feeding at varioustimes. Rhein (Sigma-Aldrich) was administered by oral gavage. Theexperiments were basically composed of 4 groups of mice: (i) control(mice receiving normal diet, n ¼ 8), (ii) Rhein (mice receiving rheinat 120 mg/kg daily by oral gavage, n ¼ 12), (iii) adenine (mice fed a0.2% adenine-containing diet, n ¼ 12), and (iv) rhein þ adenine(mice on adenine diet plus rhein treatment, n ¼ 12).

Serum and urine biochemistryMouse serum creatinine, BUN, calcium, phosphorus, alkalinephosphatase, and uric acid were measured with a Hitachi 7180automatic analyzer (Hitachi Ltd, Tokyo, Japan). Serum Klotho(SEH757Mu, USCN Life Science Inc., Wuhan, China), PTH, andFGF-23 (C-term) (Immutopics, San Clemente, CA) were measuredwith the respective enzyme-linked immunosorbent assay (ELISA)kits according to the manufacturers’ instructions.

Western blot analysisWestern blot was performed following the regular procedure withfollowing primary antibodies: antibodies to Klotho (TransGenic,

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Table 3 | Oligonucleotide list

Name Oligonucleotides

PlasmidspKlothoF CGACGCGTGTAACACAGGAGTCCTTACTCpKlothoR CCGCTCGAGGCAGATGCAGCAACAGCAAAC

shRNA-Klotho1 ctTCCTTATTTCACTGAAGATCTCGAGATCTTCAGTGAAATAAGGAAG

shRNA-Klotho2 gtTGTTGACAACTACATTCAACTCGAGTTGAATGTAGTTGTCAACAAC

MSP/BSP primersmKL-MF GGTATCGCGGGTATTTTTAATCmKL-MR CGACATAAT CCCTAAAATAATCGACmKL-unMF TTAATGGTATTGTGGGTATTTTTAATTGmKL-unMR CAACATAATCCCTAAAATAATCAACmInp-F TAGTTTTAGGAAGGTAAAGGGAGTGmInp-R AAATCCC A AAAAAAACACAACAAAhKL-MF AAAGAGAATGAATTTGAGC GTTTAChKL-MR ACTCCGCTAACAATAATTACCTACG

hKL-unMf AAGAGAAT GAATTTGAGTGTTTATGAhKL-unMr TCCACTAACAATAATTACCTACAAAhInp-F CCAACTCCAAATCCCCTCTCTAThInp-R TGATTAATTTAGATTGGGTTTAGAGAA GGABSP-F TAGTTTTAGGAAGGTAA AGGGAGTGBSP-R AACAATAATTATCCAAAACAAACRT-PCR primersKlotho-F GGCTTTCCTCCTTTACCTGAAAAKlotho-R CACATCCCACAGATAGACATTCG

TGF-b-F CCACCTGCAAGACCATCGACTGF-b-R CTGGCGAGCCTTAGTTTGGACGlyceraldehyde-3-phosphatedehydrogenase-F

AGGCCCGG TGCTGAGTATGTC

Glyceraldehyde-3-phosphatedehydrogenase-R

TGCCTGCTTCACCACCTTCT

BSP, bisulfite-sequencing polymerase chain reaction; MSP, methylation-specific po-lymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction;shRNA, short hairpin RNA; TGF-b, transforming growth factor-b.

bas i c re sea r ch Q Zhang et al.: Rhein restores Klotho and prevents kidney injury

Kumamoto, Japan), DNMT1 (Cell Signaling Technology, Danvers,MA), DNMT3a (GeneTex, Irvine, CA), DNMT3b (EpiGentek,Farmingdale, NY), E-cadherin (BD Biosciences, San Jose, CA),a-SMA (Abcam, Cambridge, UK), phosphorylated Smad3 (CellSignaling Technology), b-catenin (Invitrogen, Carlsbad, CA), andfibronectin (Proteintech, Wuhan, China).

Histology stainingKidney sections (3 mm) were stained with hematoxylin and eosin orMasson’s trichrome. For immunochemistry, staining was performedon kidney sections with Klotho antibody and horseradishperoxidase–conjugated secondary antibody (Jackson Immuno-Research Labs, West Grove, PA). The images were captured with lightmicroscopy.

Bone analysisMouse right femurs were isolated by surgery and cut in 10-mm slicesand stained with hematoxylin and eosin. X-ray analysis was per-formed with a radiography system (Faxitron X-ray Corp., Lincoln-shire, IL). The bone images were analyzed by ImageJ software(National Institutes of Health, Bethesda, MD).

Klotho promoter reporter plasmid constructionA mouse Klotho promoter reporter plasmid was constructed by PCRamplification of genomic DNA from mouse RAW264.7 cells withprimer pKlothoF/pKlothoR, which contained 2321 base pairscovering �2233 to þ88 relative to the transcription starting site. Theprimer sequences are listed in Table 3. The fragment was insertedinto pGL3-basic vector, and the cloned sequences were confirmed byDNA sequencing.

RNA interferenceKlotho knockdown was performed with 2 shRNA plasmids specificfor Klotho (shRNA-KL1 and shRNA-KL2) and a control plasmid(shRNA-scrambled) constructed in a GV248 vector (GeneChem,Shanghai, China). The oligo sequences for shRNA-KL1 and shRNA-KL2 are described in the following. Human proximal renal tubularepithelial HK2 cells were transfected with the control or Klothointerference plasmids first. At 8 hours post-transfection, cells weretreated with rhein (10 mg/ml) and/or TGF-b (5 ng/ml) for 48 hoursbefore the cells were analyzed for protein expression by Western blot.

For the in vivo assay, siRNA-mediated Klotho knockdown wasused for better efficiency. The siRNA targeted 50-GCGACTACCCA-GAG AGTAT-30 in mouse Klotho gene as described previously.50 Ascrambled RNA CGUACGCGGAAUACUUCGA dTdT was used ascontrol. A single dose of siRNA (10 nm in 200 ml of phosphate-buffered saline) was injected in each mouse through a tail veinonce a week before and during adenine feeding.

Cell cultureHK2 or HEK293 (human embryo kidney cells) (American TypeCulture Collection, Manassas, VA) were maintained in Dulbecco’smodified Eagle’s medium with 10% fetal bovine serum and 1%penicillin/streptomycin (GIBCO Cell Culture, Big Cabin, OK) at37 �C in a humidified atmosphere of 5% CO2.

Methylation-specific PCR and bisulfite-sequencing PCRThe prediction of a CpG island in the Klotho promoter and theprimer designed for methylation-specific PCR were performed withMethPrimer software. Mouse genomic sequences near the Klothotranscription starting site (�300 to þ900) were analyzed. The

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relative locations of CpG islands and methylation-specific PCRprimers are plotted in Figure 4. Mouse genomic DNA was extractedfrom the kidney and modified by bisulfate treatment. Methylation-specific PCR primers were designed to amplify the þ497/þ685locus, and PCR was performed with specific methylated or unme-thylated primers mKL-MF, mKL-MR, mKL-unMF, mKL-unMR,mInp-F, and mInp-R listed in Table 3. Methylation-specific PCR forhuman Klotho amplifies �315/�99 region with primers hKL-MF,hKL-MR, hKL-unMF, hKL-unMR, hInp-F, and hInp-R (Table 3).Genomic DNA without bisulfate treatment was used as the inputcontrol. The PCR products were analyzed on a 2% agarose gel andvisualized under ultraviolet light. The densitometry analysis wasperformed using ImageJ software. The percentages of methylated orunmethylated PCR products over the total PCR products werecalculated. For bisulfite-sequencing PCR (BSP), bisulfite-treatedDNA was amplified by PCR with BSP primers BSP-F and BSP-R(Table 3). Amplified PCR products were gel-purified and cloned intopCR-TOPO vector (Invitrogen) for sequencing. At least ten colonieswere randomly chosen for sequencing.

Reverse transcriptase PCRReverse transcriptase PCR was performed with a routine procedureto analyze renal mRNA levels of Klotho, TGF-b, and GAPDH withprimers Klotho-F, Klotho-R, TGF-b-F, TGF-b-R, GAPDH-F, andGAPDH-R (Table 3).

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Q Zhang et al.: Rhein restores Klotho and prevents kidney injury ba s i c re sea r ch

Transfection and luciferase assayCells were transfected with Klotho promoter reporter plasmid orshRNA plasmid with Lipofectamine 2000 (Invitrogen) following themanufacturer’s instructions. A renilla luciferase plasmid wascotransfected as an internal control for luciferase assay. The reporterluciferase activities were normalized with Renilla luciferase activity.

Statistical analysesData are presented as the mean � SD. The statistical analyses wereperformed using the Student t test or 1-way analysis of variance; P <0.05 or P < 0.01 was considered statistically significant or verysignificant.

DISCLOSUREAll the authors declared no competing interests.

ACKNOWLEDGMENTSThis work was supported by research grants from National BasicResearch Program of China 973Program No.2012CB517606, NationalNature Science Foundation of China (81271301, 81470940, and81670672), and the Major International (Regional) Joint ResearchProject (81320108007).

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