The Extract of Litsea japonica Reduced the Development of Diabetic ...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 769416 9 pageshttpdxdoiorg1011552013769416

Research ArticleThe Extract of Litsea japonica Reduced the Development ofDiabetic Nephropathy via the Inhibition of Advanced GlycationEnd Products Accumulation in dbdb Mice

Eunjin Sohn1 Junghyun Kim1 Chan-Sik Kim1 Yun Mi Lee1 Kyuhyung Jo1 So Dam Shin1

Joo Hwan Kim2 and Jin Sook Kim1

1 Korean Medicine Based Herbal Drug Research Group Herbal Medicine Research Division Korea Institute of Oriental Medicine(KIOM) Daejeon 305-811 Republic of Korea

2Department of Life Science Gachon University Seongnam Gyonggi-do 461ndash701 Republic of Korea

Correspondence should be addressed to Jin Sook Kim jskimkiomrekr

Received 21 January 2013 Accepted 12 April 2013

Academic Editor Yoshiyuki Kimura

Copyright copy 2013 Eunjin Sohn et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing evidence indicates that advanced glycation end products (AGEs) contribute to the pathogenesis of diabetic nephropathyThe aim of this study was to investigate the protective effect of L japonica extract (LJE) against renal damage in the dbdb mouseLJE (100 or 250mgkg per day) was given to diabetic mice for 12 weeks Body weight blood glucose levels glycated hemoglobin(HbA1c) levels and proteinuria were examined In in vitro assay of the inhibition of AGE formation immunohistochemicalanalysis of podocyte loss and AGE accumulations were performed In 20-week-old dbdb mice severe hyperglycemia developedand proteinuria was significantly increased Diabetes induced markedly morphological alterations to the renal glomerular cellsAGE accumulations and podocyte loss were detected in renal glomeruli LJE treatment significantly reduced proteinuria and AGEaccumulations in diabetic miceMoreover the loss of nephrin an important slit diaphragm component in the kidneys was restoredby LJE treatment Our studies suggest that LJE might be beneficial for the treatment of diabetic nephropathy The ability of LJE toattenuate proteinuria and podocyte dysfunction may be mediated by the inhibition of AGE accumulation in the context of diabeticnephropathy in dbdb mice

1 Introduction

Diabetic nephropathy is one of the most serious compli-cations of diabetes mellitus [1] Experimental and clinicalresearches have revealed that the increased accumulation ofAGEs plays a significant role in the pathogenesis of diabeticnephropathy [2] The levels of AGEs are much higher inpatients with diabetes [3] The clinical hallmarks of diabeticnephropathy include progressive proteinuria followed by agradual decline in renal function and glomerular mesangialexpansion has been identified as a pathological precursor ofthese clinical changes [4] Moreover it has been reported thatAGEs induce the apoptosis of cultured murine podocytesAGEs have been hypothesized to be a potential causativeagent of podocyte damage [5]

Podocytes are one of the important components of thefiltration barrier and have special cytobiological traits and

physiological functions The injury of podocytes inevitablyleads to the occurrence of proteinuria [6] Clinically glomer-ular proteinuria is most commonly observed and this con-dition is related to structural and functional abnormalitiesin the glomerular filtration barrier [7] In recent yearsresearchers have not only gained a new understanding of theroles of glomerular endothelial cells and the glomerular base-ment membrane in the pathogenesis of proteinuria but havealso gradually discovered the close relationship between slitdiaphragm molecules such as nephrin and the pathogenesisof proteinuria [8]

Some medicinal herbs have been used for the treatmentof diabetes and diabetic complications [9 10] In the pastfew years many herbal extracts have been screened forpossible AGE inhibitory effects in vitro in our laboratoryLitsea japonica (Thunb) Jussieu is an endemic plant and

2 Evidence-Based Complementary and Alternative Medicine

grows in southern areas of Korea Sometimes this plant isalso utilized as a vegetable food and an ornamental plant [11]It was reported that this plant may have anti-inflammatoryeffects [12 13] However the effects of this plant on diabetesand diabetic complications have not yet been investigatedTherefore in this study we investigated the preventive effectof an ethanolic extract of L japonica (LJE) on renal damagein a type 2 diabetes animal model the dbdb mouse

2 Materials and Methods

21 Animals and Treatment All experiments were conductedaccording to the National Institutes of Health (NIH) Guidefor the Care and Use of Laboratory Animals and wereapproved by the Korea Institute of Oriental Medicine Insti-tutional Animal Care and Use Committee Male C57BLKsJdbdb mice (dbdb) and their age-matched lean littermates(db+ normal) were purchased from Japan SLC (ShizuokaJapan) At 7 weeks of age the dbdb mice were randomlyassigned into four groups (119899 = 10) LJE was dissolved in vehi-cle (05 wv carboxyl methylcellulose solution) at the con-centration of 5mgmL Three groups of dbdb mice receiveddaily gastric gavage with LJE (100 and 250mgkg) or met-formin (MET 350mgkg) and the other group was given thesame amount of vehicle via gavage for 12 weeks Nondiabeticlittermates received the same vehicle treatment The bloodglucose level and body weight were monitored consecutively

22 Preparation of L japonica Extract (LJE) The aerial partsofL japonicawere collected from the Jejudo an island locatedin the south of Korea and identified by botanist ProfessorJ H Kim (Department of Life Science Gachon UniversityKorea) A voucher specimen (no Diab-2008-61) of the sam-ple was deposited in the Herbarium of the Division of KoreanMedicine Research Korea Institute of Oriental Medicine(KIOM) Korea The dried and ground plant material (3 kg)was extracted with EtOH (3 times 20 L) by maceration at roomtemperature for 3 days The extracts were combined andconcentrated in vacuo at 40∘C to give an EtOHextract (390 g)The analysis of the quality and the standardization of LJEwereperformed by HPLC (data not shown) A major compoundepicatechin as well as the crude extract were analyzed usinga Shimadzu HPLC system (Shimadzu Kyoto Japan)

23 Metabolic and Morphological Analysis When the ratsreached 20 weeks of age the blood glucose levels was mea-sured using an automated analyzer (Wako Japan) Glycatedhemoglobin was determined by a commercial kit (UnimateHbA1c Roche Diagnostics Mannheim Germany) Bloodsamples were collected from the tail vein after a 16 h fastIndividual mice were placed inmetabolic cages to obtain 24 hurine collections and the urinary protein excretion levelswere measured Renal cortexes were fixed in 10 formalde-hyde and embedded in paraffin and 4 120583m thick sections werepreparedThe sections were stained with periodic acid-Schiff(PAS) reagent and hematoxylin as a counterstain

24 In Vitro Assay of the Inhibition of AGE Formation andthe Cross-Linking of AGEs with Proteins According to a well-established method [14] the reaction mixture (bovine serumalbumin [10mgmL Sigma St Louis MO USA 700mL]in 50mM phosphate buffer [pH 74] with 002 sodiumazide) was added to 02M fructose and glucose (100mL)In screw-cap tubes (15mL) the reaction mixture was thenmixed with 200mL of serial diluted LJE or aminoguanidine(AG) (Sigma) After the samples had been incubated at37∘C for 14 days the fluorescent reaction products (200mL)were transferred to 96-well plates and assayed using aspectrofluorimetric detector (BIO-TEK Synergy HT USAEx 350 Em 450 nm) The AGE assay was performed intriplicate The concentration of each test sample resulting in50 inhibition of the activity (IC

50) was estimated from the

least-squares regression line of the logarithmic concentrationplotted against the remaining activity For the AGE cross-linking inhibition assay [15] AGE-BSA (TransGenic IncKobe Japan) was incubated in either the presence or absenceof LJE or AG collagen-coated 96-well plates Collagen-AGE-BSA cross-linking was detected using a rabbit anti-BSA antibody a horseradish peroxidase-linked goat anti-rabbit IgG antibody and an H

2O2substrate containing

the ABTS chromogen The inhibition of collagen-AGE-BSAcross-linking was expressed as the percent decrease in opticaldensity (OD = 410 nm)We calculated the IC

50concentration

(120583gmL) as the concentration at which collagen-AGE-BSAcross-linking was inhibited by 50

25 Immunohistochemistry Renal sections were deparaf-finized and hydrated by sequential immersion in xylene andgraded alcohol solutions and then treated with normal serumfrom the same species that contained the secondary antibodyto block nonspecific staining The slides were incubatedovernight at 4∘C with mouse anti-AGE (1 200 TransGenicInc Kobe Japan) rabbit anti-synaptopodin (Santa Cruz CAUSA) and rabbit anti-nephrin (Abcam Inc MA USA) Theslides were viewed by light microscopy (BX51 OlympusTokyo Japan) For the detection of AGEs and nephrin thesections were incubated with the appropriate regents fromthe Envision kit (DAKO CA USA) and were visualizedusing 331015840-diaminobenzidine tetrahydrochloride (DAB sys-tem Dako CA USA) and 3-amino-9-ethylcarbazole (Imm-PACT AEC VECTOR CA USA) For morphometric analy-sis the number of positive cells or the area of positive signalper glomerulus in a total of 40 glomeruli was determinedusing Image J software (NIH Bethesda MD USA)

26 SDS-PAGE and Western Blot Analysis The isolated kid-ney cortexes were immersed in cell lysis buffer containing 1protease inhibitor cocktail and homogenized using homog-enization autolysis (Precellys Bertin Technology France)Renal tissues were separated by SDSmdashpolyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes(Bio-Rad CA USA) The membranes were incubated witha rabbit anti-AGE antibody (1 200 TransGenic Inc KobeJapan) and then the immune complexes were visualizedusing an enhanced chemiluminescence detection system

Evidence-Based Complementary and Alternative Medicine 3

(Amersham Bioscience NJ USA) and a Bio-Rad elec-trophoresis image analyzer (Bio-Rad CA USA)

27 RNA Extraction and Reverse Transcriptase PCR TotalRNA was extracted from renal cortexes using the Trizolreagent (Invitrogen Life Technologies CA USA) accordingto the manufacturerrsquos instructionsThe concentration of totalRNA was calculated using the A260 Aliquots of total RNA(2120583g each) from each sample were reverse transcribed intocDNA according to the instructions for the first strandcDNA synthesis kit (Bio-Rad CA USA) RNA (2 120583g) wasused for reverse transcription polymerase chain reaction(PCR) using a thermal cycler (TAKARA Otsu Japan) ThePCR products were run on a 15 agarose gel containing05 120583gmL ethidium bromide (EtBr) The quantification ofthe PCR products was performed using densitometry andthe relative mRNA levels were calculated and compared afternormalization to beta-actin The sequences were as followsnephrin mRNA sense 51015840-CAACAGAAGACCACCAACCG-31015840 and antisense 51015840-CCTCCACTGGGACTGAAGGT-31015840 andbeta-actinmRNA sense 51015840-GGAAAGACAACGGACAAATC-31015840 and 51015840-GCTGCTGGTTTCCAAGTTCA-31015840

28 In Situ Hybridization Analysis An 18-base-pair DNAfragment from the nephrin mRNA sequence (GenBankAF1722561) was used as a probe The sense and antisenseprimers were 51015840-CAACAGAAGACCACCAACCG-31015840 and 51015840-CCTCCACTGGGACTGAAGGT-31015840 respectively The senseand antisense oligo probes were labeled with digoxigeninwith a commercial kit (Boehringer Mannheim IN USA)according to the manufacturerrsquos instructions Renal tissuesfrom each mouse were collected in 10 neutral bufferedformalin and after 1 or 2 days of fixation they were dehy-drated using graded alcohols and a xylene step and embeddedin paraffin wax Sections were then cut at 4 120583m placedon SuperfrostPlus slides (Fisher Scientific PA USA) andstored at room temperature Just before use the sections weredewaxed in xylene and rehydrated in phosphate-bufferedsaline (PBS pH 74 001M) for 5min Deproteinization wascarried out in 02NHCl for 20min at room temperatureThetissues were then digested at 37∘C for 20min in 300 120583gmLproteinase K (Gibco BRL NY USA) in PBS and fixed in 4paraformaldehyde in PBS for 10min After being rinsed twicewith PBS the slides were acetylated in 300mL of 01mMtriethanolamine-HCl buffer (pH 80) to which 075mL ofacetic anhydride (025) had been added After 5min afurther 075mL of acetic anhydride was added and 5minlater the slides were rinsed in 2x saline sodium citrate(SSC) (1x SSC contains 50mM NaCl and 15mM sodiumcitrate pH 70) Hybridization was carried out overnight at40∘C The digoxigenin-labeled probe (50 ng) was diluted in50120583L of the standard hybridization buffer which consistedof 2x SSC containing 50 deionized formamide 10mg ofsalmon sperm DNA (Oncor MD USA) 002 sodiumdodecyl sulfate (SDS) and 50 dextran sulfate solution (50concentration) The slides were then heated for 10min ina 95∘C heating block and quenched on ice Approximately75 ng of digoxigenin-labeled probe was added to standard

Table 1 Inhibitory effect of LJE on AGE formation and cross-linking in vitro

AGE formation AGE cross-links with proteinLJE (IC50) 2121 plusmn 043 120583gmL 1734 120583gmLAminoguanidine 6240120583gmL 292 plusmn 039mgmLInhibitory activity was expressed as the mean plusmn SD of triplicate experimentsThe IC50 values were calculated from regression lines using six differentconcentrations in triplicate experiments

hybridization buffer (70 120583L) which was then layered overthe section The fluid was held in place by a coverslip (theedges of which were sealed with rubber cement) heated for10min in a 95∘C heating block and then quenched on iceAfter hybridization overnight the sections were thoroughlywashed twice in 4x SSC for 5min at room temperature twicein 2x SSC for 5min at 40∘C twice in 2x SSC for 5min at roomtemperature twice in 02x SSC for 5min at 40∘C twice in 02xSSC for 5min once inmaleic acid buffer (100mMmaleic acidand 150mMNaCl pH 75) for 5min and once in 1x blockingreagent (Boehringer Mannheim IN USA) for 40min atroom temperature For the detection of hybridization sec-tions were incubated with anti-digoxigenin conjugated withhorse-radish peroxidase (Boehringer Mannheim IN USA)After three washes in buffer substrate consisting by 331015840-diaminobenzidine tetrahydrochloride (DAB system DakoCA USA) was layered over the sections The sections werecounterstained with hematoxylin

29 Statistical Analysis Data are expressed as the mean plusmnSEM and were analyzed by one-way analysis of variance(ANOVA) followed by Tukeyrsquosmultiple comparison test or byunpaired Studentrsquos t-test using GraphPad Prism 50 software(Graph pad San Diego CA USA) Differences with a valueof 119875 lt 005 were considered statistically significant

3 Results

31 Inhibition of AGE Formation and Cross-Linking withProteins The results of the assays investigating the inhibitoryeffects on AGE formation and the cross-linking of AGEs withprotein and the IC

50values of LJE are presented in Table 1

LJE exhibited 29-fold higher inhibitory activity against AGEformation than did aminoguanidine (IC

50= 6240120583gmL) a

well-known glycation inhibitor after incubation at 37∘C for 14days (Table 1) In addition the inhibitory effect of LJE on thecross-linking of AGE-BSAwith collagen (IC

50= 1738 120583gmL)

was much stronger by 168-fold than the effect of AG (IC50=

292mgmL) (Table 1)

32 Body Weight and Blood Glucose In dbdb mice at 20weeks of age the body weight was greater than that of normalmice and the body weight was lower in the mice in the LJE-250 group The blood glucose was significantly increased indbdb mice (119875 lt 001 versus normal) LJE induced only aminor decrease of levels of blood glucose However LJE failedto reduce HbA1c levels in dbdb mice MET resulted in asignificant reduction in the blood glucose level (Table 2)


Table 2 Metabolic and physical parameters

NOR DM LJE-100 LJE-250 METBody weight (g) 2689 plusmn 115 3419 plusmn 651

lowast

3139 plusmn 323 3086 plusmn 408

3265 plusmn 055

Blood glucose (mgdL) 1202 plusmn 3574 7740 plusmn 1453lowast

6949 plusmn 1764 5861 plusmn 2168 6740 plusmn 523

HbA1c () 356 plusmn 007 731 plusmn 081lowast


NOR normal mice DM diabetic dbdb mice LJE-100 DM treated with LJE (100mgkg) LJE-250 DM treated with LJE (250mgkg) MET DM treated withmetformin (350mgkg) All data are expressed as the mean plusmn SEM lowast119875 lt 001 versus the NOR group 119875 lt 001 versus the DM group

NOR DM LJE-100 LJE-250 MET

(a)

lowast

12

10

08

06

04

02

00

Prot

einu

ria (g

dL)


(b)

Figure 1 Renal histopathology and function (a) Periodic acid-Schiff staining of glomerulitimes400magnification (b) proteinuria in each groupNOR normal mice DM diabetic dbdb mice LJE-100 DM treated with LJE (100mgkg) LJE-250 DM treated with LJE (250mgkg) METDM treated with metformin (350mgkg) All data are expressed as the mean plusmn SEM (119899 = 8) lowast119875 lt 001 versus the NOR group

119875 lt 001

versus the DM group

33 Morphology and Renal Function Mesangial matrixexpansion is hallmark of diabetic nephropathy At 20 weeksof age dbdbmice showed evidence of focalmesangialmatrixexpansion and proteinuria was significantly increased indbdb mice The mice in the LJE-250 and MET groupsexhibited markedly ameliorated mesangial expansion andproteinuria compared with untreated dbdb mice (Figures1(a) and 1(b))

34 Accumulation and Expression of AGEs In our in vitrostudy LJE exhibited an inhibitory effect on AGE formationand the cross-linking of AGEs with proteins To investigatethe effect of LJE on AGE accumulation we performedimmunoassays and western blot analysis Immunohisto-chemical staining of AGEs in glomeruli demonstrated a sig-nificant increase in dbdb mice compared with normal miceThis increase was attenuated by LJE and MET (Figure 2(a))In the western blot analysis the AGE levels in the renal cortexwere also markedly increased in dbdb mice LJE and MET

reduced these diabetes-induced increases in AGE expressionin a dose-dependent manner (Figures 2(b) and 2(c))

35 Podocyte Loss in dbdb Mice To investigate the effect ofLJE on renal podocyte loss an immunoassay was performedThe average number of podocytes per glomerulus was deter-mined by counting cells and measuring the areas that werepositively labeled by a podocyte marker synaptopodin [16]In dbdbmice the number of synaptopodin-positive cells wassignificantly lower than that in age-matched normal miceTreatmentwith LJE-250 orMETvisibly increased the numberof positive cells and the positively stained areas in the renalglomeruli (Figures 3(a) and 3(b))

36 Nephrin Expression in dbdb Mice Nephrin is a novelpodocyte-specific protein that localizes to the slit diaphragm[17] We investigated the expression of the protein and RNAlevels of nephrin in the renal cortex by performing an



(a)

AGEs

Actin

NOR DM 100 250 MET

LJE

(b)

lowast


30

20

10

0

AGEs

pro

tein

den

sity

(au

)

(c)

Figure 2 Effect of LJE on AGE accumulation in the renal glomeruli (a) Immunohistochemical staining of AGEs times400 magnification (b)AGE protein expression in each group (c) Quantitative analysis of the AGE signals in histological sections All data are expressed as the meanplusmn SEM (119899 = 8) lowast119875 lt 001 versus the NOR group

119875 lt 001 versus the DM group


(a)

lowast

70

60

50

40

30

20

10

0NOR DM LJE-100 LJE-250 MET

Posit

ive a

rea o

f syn

apto

podi

n (

)

(b)

Figure 3 Effect of LJE on podocyte loss in the renal glomeruli (a) Renal cortexes from mice were stained using a specific antibody forsynaptopodin which is a specific marker of podocytes times400 magnification (b) Quantitative analysis of the podocyte signals in histologicalsections All data are expressed as the mean plusmn SEM (119899 = 8) lowast119875 lt 001 versus the NOR group




(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)

Figure 4 Effect of LJE on nephrin expression in the renal glomeruli (a) Immunohistochemical signal for nephrin (b) In situ hybridizationof nephrin mRNA times400 magnification (c) Quantitative analysis of the nephrin protein signals in histological sections (d) RT-PCR using anephrin-specific primer All data are expressed as the mean plusmn SEM (119899 = 8) lowast119875 lt 005 versus the NOR group lowastlowast119875 lt 001 versus the NORgroup

119875 lt 005 versus the DM group 119875 lt 001 versus the DM group

immunoassay in situ hybridization and RT-PCR Nephrinin normal mice was strongly expressed in renal glomeruliIn untreated dbdb mice the expression of nephrin wasweaker than that in the normal mice However the stainingof nephrin in the renal glomeruli of LJE- and MET-treateddbdb mice showed much higher nephrin levels than thoseof the untreated dbdb mice (Figure 4(a)) To confirm theresults obtained from immunohistochemistry we performedan in situ hybridization analysis and RT-PCR Untreateddbdb mice had significantly lower levels of nephrin than

normalmice did and the decreased nephrin expression in theglomeruli of diabeticmicewas restored by treatmentwith LJEand MET (Figures 4(b) 4(c) and 4(d)) These results suggestthat LJE prevents nephrin loss in the context of diabeticnephropathy by stabilizing slit diaphragm proteins

4 Discussion

Theresults of this study indicate that the use of LJE an indige-nous plant in Korea may reduce the severity of diabetic renal


complication Our study showed that LJE exhibits strongerinhibitory activity against AGE formation and the cross-linking of AGEs with proteins than AG in vitro Our resultsalso showed that LJE an herbal AGE inhibitor reducedthe development of diabetic nephropathy in type 2 diabeticdbdbmice Our current study demonstrated that LJE-treateddiabeticmice showed significant improvements in renal func-tion markers such as proteinuria In addition LJE preventedAGE accumulation in diabetic kidney and reduced podocyteloss and the decreased expression of nephrin which is a slitdiagram protein that plays an important role in podocytefunction LJE induced only aminor decrease of levels of bloodglucose but this difference was not statistically significantIn addition LJE failed to reduce HbA1c levels in dbdbmice LJE has significant effects on any parameters of renalstructure and function without the strong reduction of bloodglucoseThese findings suggest that even in hyperglycemia itis possible to attenuate diabetic nephropathy by LJE

We used an animal model of type 2 diabetes to investigatediabetic nephropathyThe dbdb mouse develops glomerularhypertrophy significant proteinuria glomerulosclerosis andinsulin resistancewith abnormalities that resemble the courseof type 2 diabetic nephropathy in humans [18 19] In dbdbmice AGEs induce mesangial expansion and proteinuria[20] Although various initiators of diabetic nephropathyhave been proposed including glycation the polyol pathwayand oxidative stress one of the major consequences of hyper-glycemia is the formation of AGEs The formation of AGEsin renal tissue is closely correlated with the developmentof diabetic nephropathy [21 22] The irreversible formationof AGEs affects proteins and lipids such as hemoglobincollagen and lipoprotein and causes damage to the kidneyseyes and blood vessels [23] Moreover AGEs induce theapoptosis of cultured murine podocytes and have beenproposed to be a potential pathogenic factor of podocyteloss [5] Podocytes appear to be sensitive targets of theaction of AGEs because they express the receptor for AGEs(RAGE) [24] Podocyte damage has been demonstrated tocorrelate with worsening proteinuria and albuminuria [25]Consistent with this interpretation our results showed thatthe expression of synaptopodin a podocyte marker [26] waslower in the AGE-accumulated glomeruli in dbdb mice withsevere proteinuria However treatment with LJE amelioratedthe enhanced diabetic nephropathy in dbdb mice

There is considerable interest in compounds that inhibitAGE formation because of the therapeutic potential of suchcompounds [27] Several natural and synthetic compoundshave been proposed as inhibitors of AGE formation AGEinhibitors such as AG and LR-90 attenuate mesangialexpansion and proteinuria in diabetic animal models [28ndash30] Our previous studies showed that KIOM-79 a naturalAGE inhibitor prevented the decrease in renal function dueto podocyte damage in type 2 diabetes animal models [31]Epicatechin a marker compound isolated from L japonicais a chemical standard for LJE and has a preventive effect onthe formation of AGEs [32 33]Therefore the ability of LJE toprotect against renal damage may be due to the effect of thiscompound

In this study we also found that the treatment ofdbdb mice with LJE ameliorated the podocyte loss and thedecreased nephrin mRNA and protein levels in AGE-accu-mulated glomeruli in the kidney The slit diaphragms (SDs)formed by podocytic processes are important components ofthe glomerular filtration barrier Slit diaphragms are zipper-like membranous electrosense structures that are formedby the linkage of podocytic processes in a zigzag mannerNephrin is a single-spanning transmembrane Ig superfamilyprotein and an integral component of the podocyte slitdiaphragm (SD) a structure that is critical to the glomerularselectivity filter [34]The SD is thewidest part of the podocytestructure and the deletion of these structures may relax thepodocytes leading to their disappearance thus disrupting thefiltration barrier and inducing proteinuria [8 35] Recently ithas been reported that nephrin deficiency is due to the loss ofpodocyte function [8 36]

In this study we also compared the effect of LJE withthat of metforminMetformin has been widely used for treat-ing type 2 diabetes without stimulating insulin production[37] Metformin significantly decreases the urine albuminexcretion rate in patients with type 2 diabetes [38] Theprevious studies also have shown that metformin is a potentinhibitor of AGE formation [39] Our study showed that LJEtreatment afforded a level of inhibitory effects similar to thatof metformin These observations demonstrate that LJE haspotential clinical uses to prevent diabetic nephropathy

Although amajor chemical standard of LJE is epicatechinthe most active compound of LJE needs to be identifiedNevertheless our study still sheds light on a potential treat-ment strategy for diabetic nephropathy In this study LJEsuppressed the generation of AGEs which are an indicatorof diabetic complications and enhanced the stabilization ofpodocytes in the kidneys of dbdb mice In addition theexpression of nephrin a slit diagram protein that plays animportant role in podocyte function was restored Althoughthe pathogenesis of diabetic nephropathy is multifactorialmicroinflammation and subsequent extracellular matrixexpansion are common pathways for the progression ofdiabetic nephropathy [40] In previous study LJE also hadan anti-inflammatory effect [12 13] Therefore LJE is likelyexerting some of its effects via improvement renal inflam-matory stress It could be reasonable to assume that therenoprotective effect of LJEmight be at least partly attributedto its influence on AGEs formation and inflammatory statusLJE could be beneficial agent by protecting against renalcomplications of diabetes

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This research was supported by a grant [K12040] from theKorea Institute of Oriental Medicine (KIOM)


References

[1] M D Vestra A Saller E Bortoloso M Mauer and P FiorettoldquoStructural involvement in type 1 and type 2 diabetic nephropa-thyrdquo Diabetes and Metabolism vol 26 supplement 4 pp 8ndash142000

[2] K Kalia S Sharma and K Mistry ldquoNon-enzymatic glycosy-lation of immunoglobulins in diabetic nephropathyrdquo ClinicaChimica Acta vol 347 no 1-2 pp 169ndash176 2004

[3] M Brownlee ldquoAdvanced protein glycosylation in diabetes andagingrdquo Annual Review of Medicine vol 46 pp 223ndash234 1995

[4] S M Mauer M W Steffes and E N Ellis ldquoStructural-functional relationships in diabetic nephropathyrdquo The Journalof Clinical Investigation vol 74 no 4 pp 1143ndash1155 1984

[5] P Y ChuangQ YuW Fang J Uribarri and J CHe ldquoAdvancedglycation endproducts induce podocyte apoptosis by activationof the FOXO4 transcription factorrdquo Kidney International vol72 no 8 pp 965ndash976 2007

[6] Y Zhang B Chen X H Hou et al ldquoEffects of mycophenolatemofetil valsartan and their combined therapy on preventingpodocyte loss in early stage of diabetic nephropathy in ratsrdquoChinese Medical Journal vol 120 no 11 pp 988ndash995 2007

[7] R O Estacio and R W Schrier ldquoDiabetic nephropathy patho-genesis diagnosis and prevention of progressionrdquo Advances inInternal Medicine vol 46 pp 359ndash408 2001

[8] A Zhang and S Huang ldquoProgress in pathogenesis of protein-uriardquo International Journal of Nephrology vol 2012 Article ID314251 14 pages 2012

[9] F J Alarcon-Aguilara R Roman-Ramos S Perez-GutierrezA Aguilar-Contreras C C Contreras-Weber and J L Flores-Saenz ldquoStudy of the anti-hyperglycemic effect of plants used asantidiabeticsrdquo Journal of Ethnopharmacology vol 61 no 2 pp101ndash110 1998

[10] W L Li H C Zheng J Bukuru and N De Kimpe ldquoNaturalmedicines used in the traditional Chinese medical system fortherapy of diabetesmellitusrdquo Journal of Ethnopharmacology vol92 no 1 pp 1ndash21 2004

[11] E KimH J Boo J HHyun et al ldquoThe effects of Litsea japonicaon the induction of apoptosis in HL-60ADRrdquo Korean Journalof Pharmacognosy vol 40 no 1 pp 65ndash69 2009

[12] H N T Tanaka T Nakamura K Ichino K Ito and T TanakaldquoButanolides from Litsea japonicardquo Phytochemistry vol 29 no3 pp 857ndash859 1990

[13] B S Min S Y Lee J H Kim et al ldquoLactones from the leavesof Litsea japonica and their anti-complement activityrdquo Journalof Natural Products vol 66 no 10 pp 1388ndash1390 2003

[14] I Miwa T Tsugawa K Koyasu and Y Terada ldquoInhibition ofadvanced protein glycation by 8-quinolinecarboxylic hydraz-iderdquo Pharmacology vol 52 no 5 pp 314ndash320 1996

[15] J M Forbes T Soulis V Thallas et al ldquoRenoprotective effectsof a novel inhibitor of advanced glycationrdquoDiabetologia vol 44no 1 pp 108ndash114 2001

[16] P Mundel and J Reiser ldquoNew aspects of podocyte cell biologyrdquoKidney and Blood Pressure Research vol 20 no 3 pp 173ndash1761997

[17] M Toyoda D Suzuki T Umezono et al ldquoExpression of humannephrin mRNA in diabetic nephropathyrdquo Nephrology DialysisTransplantation vol 19 no 2 pp 380ndash385 2004

[18] A A Like R L Lavine P L Poffenbarger and W L ChickldquoStudies in the diabetic mutant mouse VI Evolution ofglomerular lesions and associated proteinuriardquo The AmericanJournal of Pathology vol 66 no 2 pp 193ndash224 1972

[19] F N Ziyadeh B B Hoffman D C Han et al ldquoLong-termprevention of renal insufficiency excessmatrix gene expressionand glomerular mesangial matrix expansion by treatmentwith monoclonal antitransforming growth factor-120573 antibody indbdb diabetic micerdquo Proceedings of the National Academy ofSciences of the United States of America vol 97 no 14 pp 8015ndash8020 2000

[20] M J Soler M Riera and D Batlle ldquoNew experimental modelsof diabetic nephropathy in mice models of type 2 diabetesefforts to replicate human nephropathyrdquo Experimental DiabetesResearch vol 2012 Article ID 616313 19 pages 2012

[21] M T Coughlan A LMibus and J M Forbes ldquoOxidative stressand advanced glycation in diabetic nephropathyrdquo Annals of theNew York Academy of Sciences vol 1126 pp 190ndash193 2008

[22] K Fukami S I Yamagishi S Ueda and S Okuda ldquoRole ofAGEs in diabetic nephropathyrdquoCurrent Pharmaceutical Designvol 14 no 10 pp 946ndash952 2008

[23] M Brownlee ldquoThe pathobiology of diabetic complications aunifying mechanismrdquo Diabetes vol 54 no 6 pp 1615ndash16252005

[24] T M Wendt N Tanji J Guo et al ldquoRAGE drives thedevelopment of glomerulosclerosis and implicates podocyteactivation in the pathogenesis of diabetic nephropathyrdquo TheAmerican Journal of Pathology vol 162 no 4 pp 1123ndash11372003

[25] K Susztak A C RaffM Schiffer and E P Bottinger ldquoGlucose-induced reactive oxygen species cause apoptosis of podocytesand podocyte depletion at the onset of diabetic nephropathyrdquoDiabetes vol 55 no 1 pp 225ndash233 2006

[26] P Mundel H W Heid T M Mundel M Kruger J Reiserand W Kriz ldquoSynaptopodin an actin-associated protein intelencephalic dendrites and renal podocytesrdquo Journal of CellBiology vol 139 no 1 pp 193ndash204 1997

[27] P J Thornalley S Battah N Ahmed et al ldquoQuantitativescreening of advanced glycation endproducts in cellular andextracellular proteins by tandem mass spectrometryrdquo The Bio-chemical Journal vol 375 no 3 pp 581ndash592 2003

[28] N Ahmed ldquoAdvanced glycation endproductsmdashrole in pathol-ogy of diabetic complicationsrdquo Diabetes Research and ClinicalPractice vol 67 no 1 pp 3ndash21 2005

[29] S Rahbar and J L Figarola ldquoNovel inhibitors of advancedglycation endproductsrdquoArchives of Biochemistry and Biophysicsvol 419 no 1 pp 63ndash79 2003

[30] M Peppa H Brem W Cai et al ldquoPrevention and reversal ofdiabetic nephropathy in dbdb mice treated with alagebrium(ALT-711)rdquo American Journal of Nephrology vol 26 no 5 pp430ndash436 2006

[31] Y S Kim J Kim C S Kim et al ldquoKIOM-79 an inhibitor ofAGEs-protein cross-linking prevents progression of nephropa-thy in zucker diabetic fatty ratsrdquo Evidence-Based Complemen-tary and Alternative Medicine vol 2011 Article ID 761859 10pages 2011

[32] P V A Babu K E Sabitha and C S Shyamaladevi ldquoEffect ofgreen tea extract on advanced glycation and cross-linking of tailtendon collagen in streptozotocin induced diabetic ratsrdquo Foodand Chemical Toxicology vol 46 no 1 pp 280ndash285 2008

[33] H Matsuda T Wang H Managi and M Yoshikawa ldquoStruc-tural requirements of flavonoids for inhibition of protein glyca-tion and radical scavenging activitiesrdquoBioorganic andMedicinalChemistry vol 11 no 24 pp 5317ndash5323 2003

[34] K Tryggvason J Patrakka and J Wartiovaara ldquoHereditaryproteinuria syndromes and mechanisms of proteinuriardquo The


New England Journal of Medicine vol 354 no 13 pp 1387ndash14012006

[35] K Tryggvason ldquoUnraveling the mechanisms of glomerularultrafiltration nephrin a key component of the slit diaphragmrdquoJournal of the American Society of Nephrology vol 10 no 11 pp2440ndash2445 1999

[36] J Juhila M Lassila R Roozendaal et al ldquoInducible nephrintransgene expression in podocytes rescues nephrin-deficientmice from perinatal deathrdquoThe American Journal of Pathologyvol 176 no 1 pp 51ndash63 2010

[37] C J Bailey and R C Turner ldquoMetforminrdquo The New EnglandJournal of Medicine vol 334 no 9 pp 574ndash579 1996

[38] N Amador-Licona J M Guızar-Mendoza E Vargas GSanchez-Camargo and L Zamora-Mata ldquoThe short-term effectof a switch from glybenclamide tometformin on blood pressureandmicroalbuminuria in patientswith type 2 diabetesmellitusrdquoArchives of Medical Research vol 31 no 6 pp 571ndash575 2000

[39] TKihoMKato SUsui andKHirano ldquoEffect of buformin andmetformin on formation of advanced glycation end products bymethylglyoxalrdquo Clinica Chimica Acta vol 358 no 1-2 pp 139ndash145 2005

[40] A Fornoni A Ijaz T Tejada and O Lenz ldquoRole of inflamma-tion in diabetic nephropathyrdquo Current Diabetes Reviews vol 4no 1 pp 10ndash17 2008

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


grows in southern areas of Korea Sometimes this plant isalso utilized as a vegetable food and an ornamental plant [11]It was reported that this plant may have anti-inflammatoryeffects [12 13] However the effects of this plant on diabetesand diabetic complications have not yet been investigatedTherefore in this study we investigated the preventive effectof an ethanolic extract of L japonica (LJE) on renal damagein a type 2 diabetes animal model the dbdb mouse

2 Materials and Methods

21 Animals and Treatment All experiments were conductedaccording to the National Institutes of Health (NIH) Guidefor the Care and Use of Laboratory Animals and wereapproved by the Korea Institute of Oriental Medicine Insti-tutional Animal Care and Use Committee Male C57BLKsJdbdb mice (dbdb) and their age-matched lean littermates(db+ normal) were purchased from Japan SLC (ShizuokaJapan) At 7 weeks of age the dbdb mice were randomlyassigned into four groups (119899 = 10) LJE was dissolved in vehi-cle (05 wv carboxyl methylcellulose solution) at the con-centration of 5mgmL Three groups of dbdb mice receiveddaily gastric gavage with LJE (100 and 250mgkg) or met-formin (MET 350mgkg) and the other group was given thesame amount of vehicle via gavage for 12 weeks Nondiabeticlittermates received the same vehicle treatment The bloodglucose level and body weight were monitored consecutively

22 Preparation of L japonica Extract (LJE) The aerial partsofL japonicawere collected from the Jejudo an island locatedin the south of Korea and identified by botanist ProfessorJ H Kim (Department of Life Science Gachon UniversityKorea) A voucher specimen (no Diab-2008-61) of the sam-ple was deposited in the Herbarium of the Division of KoreanMedicine Research Korea Institute of Oriental Medicine(KIOM) Korea The dried and ground plant material (3 kg)was extracted with EtOH (3 times 20 L) by maceration at roomtemperature for 3 days The extracts were combined andconcentrated in vacuo at 40∘C to give an EtOHextract (390 g)The analysis of the quality and the standardization of LJEwereperformed by HPLC (data not shown) A major compoundepicatechin as well as the crude extract were analyzed usinga Shimadzu HPLC system (Shimadzu Kyoto Japan)

23 Metabolic and Morphological Analysis When the ratsreached 20 weeks of age the blood glucose levels was mea-sured using an automated analyzer (Wako Japan) Glycatedhemoglobin was determined by a commercial kit (UnimateHbA1c Roche Diagnostics Mannheim Germany) Bloodsamples were collected from the tail vein after a 16 h fastIndividual mice were placed inmetabolic cages to obtain 24 hurine collections and the urinary protein excretion levelswere measured Renal cortexes were fixed in 10 formalde-hyde and embedded in paraffin and 4 120583m thick sections werepreparedThe sections were stained with periodic acid-Schiff(PAS) reagent and hematoxylin as a counterstain

24 In Vitro Assay of the Inhibition of AGE Formation andthe Cross-Linking of AGEs with Proteins According to a well-established method [14] the reaction mixture (bovine serumalbumin [10mgmL Sigma St Louis MO USA 700mL]in 50mM phosphate buffer [pH 74] with 002 sodiumazide) was added to 02M fructose and glucose (100mL)In screw-cap tubes (15mL) the reaction mixture was thenmixed with 200mL of serial diluted LJE or aminoguanidine(AG) (Sigma) After the samples had been incubated at37∘C for 14 days the fluorescent reaction products (200mL)were transferred to 96-well plates and assayed using aspectrofluorimetric detector (BIO-TEK Synergy HT USAEx 350 Em 450 nm) The AGE assay was performed intriplicate The concentration of each test sample resulting in50 inhibition of the activity (IC

50) was estimated from the

least-squares regression line of the logarithmic concentrationplotted against the remaining activity For the AGE cross-linking inhibition assay [15] AGE-BSA (TransGenic IncKobe Japan) was incubated in either the presence or absenceof LJE or AG collagen-coated 96-well plates Collagen-AGE-BSA cross-linking was detected using a rabbit anti-BSA antibody a horseradish peroxidase-linked goat anti-rabbit IgG antibody and an H

2O2substrate containing

the ABTS chromogen The inhibition of collagen-AGE-BSAcross-linking was expressed as the percent decrease in opticaldensity (OD = 410 nm)We calculated the IC

50concentration

(120583gmL) as the concentration at which collagen-AGE-BSAcross-linking was inhibited by 50

25 Immunohistochemistry Renal sections were deparaf-finized and hydrated by sequential immersion in xylene andgraded alcohol solutions and then treated with normal serumfrom the same species that contained the secondary antibodyto block nonspecific staining The slides were incubatedovernight at 4∘C with mouse anti-AGE (1 200 TransGenicInc Kobe Japan) rabbit anti-synaptopodin (Santa Cruz CAUSA) and rabbit anti-nephrin (Abcam Inc MA USA) Theslides were viewed by light microscopy (BX51 OlympusTokyo Japan) For the detection of AGEs and nephrin thesections were incubated with the appropriate regents fromthe Envision kit (DAKO CA USA) and were visualizedusing 331015840-diaminobenzidine tetrahydrochloride (DAB sys-tem Dako CA USA) and 3-amino-9-ethylcarbazole (Imm-PACT AEC VECTOR CA USA) For morphometric analy-sis the number of positive cells or the area of positive signalper glomerulus in a total of 40 glomeruli was determinedusing Image J software (NIH Bethesda MD USA)

26 SDS-PAGE and Western Blot Analysis The isolated kid-ney cortexes were immersed in cell lysis buffer containing 1protease inhibitor cocktail and homogenized using homog-enization autolysis (Precellys Bertin Technology France)Renal tissues were separated by SDSmdashpolyacrylamide gelelectrophoresis and transferred to nitrocellulose membranes(Bio-Rad CA USA) The membranes were incubated witha rabbit anti-AGE antibody (1 200 TransGenic Inc KobeJapan) and then the immune complexes were visualizedusing an enhanced chemiluminescence detection system









3 Results




50= 6240120583gmL) a


50= 1738 120583gmL)


292mgmL) (Table 1)





lowast

3139 plusmn 323 3086 plusmn 408

3265 plusmn 055







(a)

lowast

12

10

08

06

04

02

00

Prot

einu

ria (g

dL)


(b)


119875 lt 001

versus the DM group








(a)

AGEs

Actin

NOR DM 100 250 MET

LJE

(b)

lowast


30

20

10

0

AGEs

pro

tein

den

sity

(au

)

(c)




(a)

lowast

70

60

50

40

30

20

10


Posit

ive a

rea o

f syn

apto

podi

n (

)

(b)





(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)





4 Discussion











Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























3 Results




50= 6240120583gmL) a


50= 1738 120583gmL)


292mgmL) (Table 1)





lowast

3139 plusmn 323 3086 plusmn 408

3265 plusmn 055







(a)

lowast

12

10

08

06

04

02

00

Prot

einu

ria (g

dL)


(b)


119875 lt 001

versus the DM group








(a)

AGEs

Actin

NOR DM 100 250 MET

LJE

(b)

lowast


30

20

10

0

AGEs

pro

tein

den

sity

(au

)

(c)




(a)

lowast

70

60

50

40

30

20

10


Posit

ive a

rea o

f syn

apto

podi

n (

)

(b)





(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)





4 Discussion











Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















lowast

3139 plusmn 323 3086 plusmn 408

3265 plusmn 055







(a)

lowast

12

10

08

06

04

02

00

Prot

einu

ria (g

dL)


(b)


119875 lt 001

versus the DM group








(a)

AGEs

Actin

NOR DM 100 250 MET

LJE

(b)

lowast


30

20

10

0

AGEs

pro

tein

den

sity

(au

)

(c)




(a)

lowast

70

60

50

40

30

20

10


Posit

ive a

rea o

f syn

apto

podi

n (

)

(b)





(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)





4 Discussion











Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

AGEs

Actin

NOR DM 100 250 MET

LJE

(b)

lowast


30

20

10

0

AGEs

pro

tein

den

sity

(au

)

(c)




(a)

lowast

70

60

50

40

30

20

10


Posit

ive a

rea o

f syn

apto

podi

n (

)

(b)





(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)





4 Discussion











Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)


(b)

8

6

4

2

0

Posit

ive a

rea o

f nep

hrin

()

lowastlowast


(c)

NOR DM 100 250 MET

LJE

lowast


125

100

075

050

025

000

Nep

hrin

mRN

A ex

pres

sion

(au

)

Nephrin

Actin

(d)





4 Discussion











Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgment



References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















The Extract of Litsea japonica Reduced the Development of Diabetic ...

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