Moxibustion Alleviates Injury in a Rat Focal Segmental …downloads.hindawi.com/journals/ecam/2017/7169547.pdf · 2019-07-30 · The moxa sticks or cones are ignited prior to

Research ArticleMoxibustion Alleviates Injury in a Rat Focal SegmentalGlomerulosclerosis Model

Yi Li1 Yuxia Sun1 Chunling Zhang2 KeWang3 Peicheng Shen1 Di Huang4 WenMa5

Jin Zhang6 Lin Li1 and Liqun He1

1Department of Nephrology Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghai 200021 China2Department of Cardiology Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghai 201203 China3Laboratory of Integrative Medicine Surgery Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghai 201203 China4Department of Nephrology Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineTraditional Chinese Medicine Institute of Kidney Disease Shanghai University of Traditional Chinese MedicineShanghai Key Laboratory of Traditional Chinese Clinical Medicine Shanghai 201203 China5Acupuncture Anesthesia Research Section Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese MedicineShanghai 200021 China6Shanghai University of Traditional Chinese Medicine Shanghai 201203 China

Correspondence should be addressed to Liqun He heliqun59163com

Received 19 February 2017 Accepted 9 April 2017 Published 28 May 2017

Academic Editor Thomas Lundeberg

Copyright copy 2017 Yi Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives To evaluate the therapeutic effects of moxibustion at Shenshu (BL-23) and Geshu (BL-17) acupoints in a focal segmentalglomerulosclerosis (FSGS)model in ratsMethods A FSGS rat model was established by single nephrectomy and repeated injectionof doxorubicinThe FSGS rats were randomly divided into themodel group losartan (positive control) group Shenshumoxibustiongroup and Geshu moxibustion group Molecular indicators of kidney function and renal pathological changes were monitoredResults Urinary protein serum creatinine urea nitrogen and serum uric acid were significantly reduced after 12-week interventionwith losartan Shenshu or Geshu moxibustion Renal 120572-SMA FN and TGF-120573 were also decreased while podocin and nephrinprotein and mRNA were increasedThe pathological damage in renal tissue was obviously alleviated by all three treatments whichsuggests that moxibustion may have similar efficacy to losartan in the treatment of FSGS Conclusion Moxibustion alleviatespodocyte injury and inhibits renal interstitial fibrosis in the FSGS rat model thereby minimizing the progression of glomerularsclerosis and improving renal function

1 Introduction

Focal segmental glomerulosclerosis (FSGS) is characterizedby scarring to a subset of the glomeruli of the kidney and canoccur as a result of infection drugs or systematic diseasessuch as diabetes or lupus or other glomerular diseasesThe main clinical manifestation of FSGS is proteinuria Theseverity of the proteinuria and the response to treatment areclosely associated with the kidney prognosis and patientswith nephrotic syndrome are prone to develop end stage renal

disease [1ndash3] Therefore the primary goal of FSGS treatmentis to reduce proteinuria in a short time to protect the kidney[4 5]

In recent years traditional Chinese medicines (TCMs)have been considered as effective treatments for a varietyof different physical conditions including renal diseasesAcupuncture and moxibustion are thought to contribute todisease treatment by stimulating the surface of meridian acu-points Compared to acupuncture moxibustion is motivatedby ldquowarm stimulationrdquo The main material for moxibustion

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 7169547 10 pageshttpsdoiorg10115520177169547

2 Evidence-Based Complementary and Alternative Medicine

therapy is moxa wool which is used to construct conesor sticks The moxa sticks or cones are ignited prior toapplication on the surface of meridian acupoints via scarringor nonscarring methods However rigorous randomizedcontrolled trials of moxibustion have not been describedwhich may account for the poor awareness of this therapymethod

Nevertheless animal experiments support the potentialrole of both acupuncture and moxibustion in the treatmentof renal diseases Paterno et al [6] demonstrated that elec-troacupuncture at Zusanli (ST-36) and Taixi (KI-3) acupointsand moxibustion at the Shenshu (BL-23) acupoint improvethe serum creatinine 24-h urinary protein glomeruloscle-rosis and tubulointerstitial fibrosis in a 56 nephrectomyrat model Further study by this group [7] showed that thetreatment mechanism may be related to the regulation ofrenal sympathetic nerve activity and nitric oxide levels andthe reduction of arterial blood pressure The potential roleof nitric oxide release from nerves has also been noted inthe response to local thermal stimulation on Riyue (GB-24)Weizhong (BL-40) andChengfu (BL-36) acupoints to inhibitthe physiological activities of the Oddi sphincter and theanal sphincter [8ndash10] Additionally several investigators havedemonstrated that thermal stimulation within the regionsof Qimen (LR-14) Neiguan (PC-6) and Yinmen (BL-37)acupoints increases the expression of heat shock protein 70(HSP70) and elevates reactive oxygen species which protectsliver and heartmuscles from ischemia-reperfusion injury [11ndash15] Matsumoto-Miyazaki et al [16] observed reduced renalvascular resistance after indirect moxibustion at the bilateralShenshu acupoints of 43 chronic kidney disease patients Atpresent however direct reports of moxibustion therapy forproteinuria in patients with chronic glomerular disease arerelatively rare Therefore moxibustion therapy is worthy offurther in-depth investigation

In this study we used single nephrectomy and repeatedinjections of doxorubicin [17] to establish FSGS in rats Usingthe angiotensin II receptor antagonist losartan as a positivecontrol therapeutic we evaluated the potential therapeuticeffects of mild Shenshu moxibustion (BL-23) and Geshumoxibustion (BL-17)Wemonitored the urinary protein levelkidney function expression of renal fibronectin (FN) 120572-SMA TGF-120573 podocin and nephrin and pathomorphologyof FSGS kidney tissues of different groups of rats Ourresults support the potential therapeutic value ofmoxibustionat Shenshu (BL-23) and Geshu (BL-17) acupoints in thetreatment of FSGS

2 Methods

21 Animals Fifty-one clean-grade male Sprague Daw-ley rats (bodyweight of 140ndash160 g) were purchased fromShanghai Sippr-BK Lab Animal Co Ltd (Shanghai Chinaanimal qualification certificate number SCXK [Shanghai]2013ndash0016) All experimental rats were fed common chow(containing 21 crude protein 45 crude fat and 5crude fibers purchased from Shanghai Shilin BiotechnologyCo Ltd Shanghai China) and housed in the AnimalExperimental Center of Shanghai University of TCM with

a room temperature of 22ndash25∘C and humidity of 50Animal experiments and euthanization procedures were inaccordance with Guidelines on the Treatment of ExperimentalAnimals issued by Ministry of Science and Technology in2006 and relevant animal ethics standards listed in EthicalIssues in Animal Experimentation in 2009

22 Establishment of the FSGS Model All rats were accli-mated for a week and then were weighed and numbered bytheir order in weight (from light to heavy) Correspondingto numbers generated by SPSS statistical software seven ratswere randomly selected for sham operation by laparotomyto expose the kidneys and remove the renal capsule withoutdissecting the kidney tissue followed by suturing layer bylayer These animals were then tail-vein injected with 05mlnormal saline on day 4 and day 18 after sham operationThe other 44 rats received two intravenous injections ofdoxorubicin two weeks apart and unilateral nephrectomyto model glomerulosclerosis All animals were fasted withfree access to water 12 h before operation and anesthetizedby intraperitoneal injection of 50mgkg 15 pentobarbitalsodium (Merck Drugs and Biotechnology Germany batchnumber 20130112) After the corneal reflex had subsidedthe animals were fastened on the operating table followedby hair shaving and routine disinfection A 1ndash15 cm dorsalincision was made to expose the left kidney remove the fattissues and adrenal gland around the kidney ligate the leftrenal artery and resect the left kidney The incision was thensutured The animals were tail-vein injected with 3mgkg(postoperative day 4) and 2mgkg (postoperative day 18) ofdoxorubicin which was dissolved in sterile water at a dilu-tion of 2mgml (10mg doxorubicinsyringe Shenzhen MainLuck Pharmaceuticals Inc Guangdong Province Chinabatch number national approved medicine H44024359)The urinary protein of the viable rats was quantified sevendays after the second doxorubicin injection Animals withgt100mg24 h urinary protein indicated successful modelingand were included in subsequent experiments with monitor-ing for 12 consecutive weeks

23 Grouping and Intervention Forty-four successfullymod-eled FSGS rats were numbered from 1 to 44 according to thequantity (from small to large quantity) of urinary proteinCorresponding to numbers generated by SPSS statisticalsoftware the 44 rats were randomly divided into the modellosartan Shenshu moxibustion (BL-23-MO) and Geshumoxibustion (BL-17 MO) groups (119899 = 11 rats per group)Intervention was initiated in each group on the same dayAnimals in the sham-operation model and moxibustiongroups were given 5mlkg distilled water by gavage once aday Rats in the losartan group were given losartan potas-sium tablets which were dissolved in distilled water at adilution of 5mgml immediately before the administrationby gavage once a day (100mgtablet produced by HangzhouMSD Pharmaceutical Co Ltd Hangzhou China batchnumber national approved medicine H20030654) Rats inthe Shenshu and the Geshu moxibustion groups receivedmild moxibustion at Shenshu (bilateral) acupoints or Geshu(bilateral) acupoints every other day (30min per acupoint)

Evidence-Based Complementary and Alternative Medicine 3

BL-17 BL-23

(a) (b)

Figure 1 Experimental application of moxibustion in rats (a) The body surface position of acupoints in the rat (b) Schematic presentationof the self-made device for administration of moxibustion to rats

Table 1 Primer sequences and RT-PCR condition

Gene Primer sequence Size (bp) Product GC ()

Podocin Primer F 51015840 ACCCAAGTTCTCCCAAACC 31015840 226 50Primer R 51015840 CTGCCATTTCACTGCGTTC 31015840

Nephrin Primer F 51015840 AGAGACTGGGAGAAGAAGAG 31015840 185 57Primer R 51015840 AGCAAATCGGACGACAAG 31015840

GAPDH Primer F 51015840 GTCGGTGTGAACGGATTTG 31015840 181 51Primer R 51015840 TCCCATTCTCAGCCTTGAC 31015840

Figure 1(a) shows the locations of the two acupoints To local-ize the bilateral Shenshu (BL-23) acupoints rats were fixedin a prone position with the two hind limbs straightenedThe highest points on both sides of the iliac and lumbarspinous process were located and crossed with the horizontalconnection of the spine which was 15 inches away from the2nd upper spinous process position based on the middle toe-identical unit of the rat body in the hind limb To localize thebilateral Geshu acupoints the Dazhui acupoint (GV14 at thecenter of the back between the 7th cervical vertebra and firstthoracic spine) was first located and the position 15 inchesaway from the 7th thoracic spinous process based on themiddle toe-identical unit of the rat body in the hind limbswasidentified For mild moxibustion hairs within 1 cm times 2 cm ofeach acupoint were shaved to expose and disinfect the surfaceof the skin tissue using an alcohol-soaked cotton ball A self-made moxibustion device (Figure 1(b)) was affixed to the ratand 1mm diameter of moxa stick (Nanyang Hanyi Moxa CoLtd Henan China 120mm in length containingmoxawoolwith combustion temperature of 40ndash60∘C [18]) was applied2 cm away from the skin after the rats had calmed down Ratsof the sham-operation group and model group were fastenedon the operation bench as for the moxibustion groups everyother day for a total of 12 weeks but moxibustion was notapplied to these groups

24 Specimen Processing and Detection At the end of the12-week intervention all rats were sacrificed and bloodwas collected from the abdominal aorta and centrifuged toprepare supernatant for the detection of serum creatinineurea nitrogen and uric acid The right kidney of eachanimal was dissected out to remove the renal capsule and

cut into two slices from the renal hilum with one slicefixed in 10 neutral formalin solution for histopathologyand immunohistochemistry and the other slice preserved insolution for subsequent RNA extraction and RT-PCR Oneday before sacrifice all animals were fasted inmetabolic cages(Suzhou Fengshi Laboratory Animal Equipment Co LtdJiangsu China) with free access of water and urine sampleswere collected for 24 h followed by centrifugation tomeasurethe urinary protein levels

The urinary protein level serum creatinine urea nitro-gen and serumuric acidwere detected by the BeckmanCoul-terAU580 automatic biochemical analyzer in the biochemicallaboratory of the Shanghai University of TCM The assaykits were purchased from Beckman Coulter ExperimentalSystem Co (Suzhou China) and World Connaught ClinicalDiagnostic Product Co Ltd (Japan) Expression of renalFN 120572-SMA and TGF-120573 was detected by immunohisto-chemistry and analyzed using Image-Pro Plus 60 imageprocessing software Antibodies for immunohistochemistrywere purchased from Abcam (Cambridge UK) Expressionof renal podocin and nephrin was detected by Western blotanalysis Anti-podocin antibodywas purchased fromAbcamand anti-nephrin antibody was purchased from Santa CruzBiotechnology Anti-GAPDH antibody was purchased fromCell Signaling Technology (Danvers MA) Goat anti-rabbitdonkey anti-goat and goat anti-mouse secondary antibodieswere purchased from Beyotime Institute of Biotechnology(Shanghai China) Renal podocin and nephrin mRNAs weremeasured by RT-PCR The primers for PCR were synthe-sized by Shanghai Generay Biotech Co Ltd (ShanghaiChina Table 1) The pathomorphology of the glomeruli andtubulointerstitium of the kidney tissues was observed under


Table 2 Comparison of 24-h urinary protein in each group of rats (mean plusmn standard deviation mg24 h)

Group 119899 0th week 4th week 8th week 12th weekSham 7 1119 plusmn 334 3560 plusmn 1070 2883 plusmn 705 1170 plusmn 390

Model 6 25832 plusmn 4241 39717 plusmn 7997 43173 plusmn 4499 40415 plusmn 2277

Losartan 6 26228 plusmn 4775 32681 plusmn 7713 28423 plusmn 7740 24809 plusmn 9367

BL-23 MO 6 26164 plusmn 3678 37316 plusmn 4287 35650 plusmn 6562 30436 plusmn 8714

BL-17 MO 6 26299 plusmn 3804 39353 plusmn 3008 37516 plusmn 7651∘ 27227 plusmn 6339

119875 lt 005 and

119875 lt 001 versus model group ∘119875 lt 005 versus losartan group 119875 lt 005 and 119875 lt 001 versus week 0 within the group

light microscopy (Olympus Tokyo Japan) after conventionaldehydration clearing paraffin-embedding sectioning into3 120583m and Masson staining

25 Statistical Analysis SPSS 170 software (SPSS IncChicago IL) was used for statistical analysis in this studyThe normally distributed data were presented as means plusmnstandard deviation (119909 plusmn 119904) One-way ANOVA was used forcomparison among groups The least-significant difference(LSD) test was used to make comparisons between any twogroups The urinary protein was quantitatively analyzed byrepeated measurement of variance When 120572 = 005 119875 lt 005was considered as an indication of statistical significance ofthe test data 119875 lt 001 was considered extremely statisticallysignificant

3 Results

31 Establishment of a Rat FSGS Model To establish a modelfor rat FSGS we subjected rats to single nephrectomy fol-lowed by repeated injection of doxorubicinThe urinary pro-tein levels for all FSGS rats were more than 100mg24 h indi-cating FSGS model was successfully replicated No rats diedduring the surgery or doxorubicin treatment but 13 rats diedafter the FSGS modeling (2955 mortality) mostly occur-ring in the 6thweek after the second injection of doxorubicinAnatomical analysis suggested that the deaths may be causedby doxorubicin-induced cardiotoxicity adverse reactions indigestive system and glomerular sclerosis These resultsconfirm the successful establishment of the FSGS model

32 Effects of Moxibustion on Urinary Protein in the FSGSRat Model To assess the effects of moxibustion on FSGS werandomly divided the FSGS rats into four groups the modelgroup (no therapeutic treatment) losartan group (positivecontrol therapy) Shenshu moxibustion group (BL-23 MO)and Geshu moxibustion group (BL-17 MO) The urinaryprotein levels in the four groups of FSGS rats were comparedto each other and that of sham-operated rats (Figure 2 andTable 2) As expected the urinary protein levels in the fourFSGS groups were uniformly greater than in the sham groupOver time the urinary protein levels increased in the modelgroup reaching a peak at 8 weeks and remaining high at12 weeks However for the losartan Shenshu moxibustionand Geshu moxibustion groups the urinary protein levelsdeclined after week 4 Repeated measures ANOVA indicated

600

400

200

0

Urin

ary

prot

ein

(mg

24h)

0 4 8 12

Time (weeks)

ShamModelLosartan

BL-23 MOBL-17 MO

998779998779 998779998779998779998779998779998779

998779998779998779

∘

Figure 2 Moxibustion ameliorates proteinuria in FSGS rats Dataare expressed as the mean plusmn SD Comparison between groups at thesame time point

119875 lt 005 and 119875 lt 001 versus model group

∘119875 lt 005 versus losartan group 119875 lt 005 and 119875 lt 001 versusweek 0 within the group MO moxibustion

that time and group factors significantly affected the urinaryprotein levels Comparison over time demonstrated thatthe urinary protein levels within the FSGS model groupwere significantly higher at the end of the 4th 8th and12th weeks than at week 0 (119875 lt 005) however for thelosartan BL-23MO and BL-17 MO groups the urinaryprotein content declined by week 12 to levels that were notsignificantly different than the levels at week 0 within eachgroup Comparison among groups indicated that at the endof the 12-week intervention period urinary protein levels ofthe losartan Shenshu moxibustion and Geshu moxibustiongroups were significantly lower than those of the FSGSmodelgroup (119875 lt 005 119875 lt 001) No significant difference ofthe urinary protein level was observed between the losartanShenshu moxibustion and Geshu moxibustion groups at theend of the 12-week intervention period (119875 gt 005) Becauseurinary protein is a primary indication of kidney functionthese results indicate that Shenshu moxibustion and Geshumoxibustion protected the rats from the damaging effects ofFSGS on the kidneys


400

300

200

100

0

Seru

m cr

eatin

ine (

120583m

olL

)

Sham Model Losartan BL-23 MO BL-17 MO

lowastlowast

lowast

lowast

lowast

(a)

lowastlowast

50

40

30

20

10

0

Seru

m u

rea n

itrog

en (m

mol

L)


(b)

150

100

50

0

Seru

m u

ric ac

id (120583

mol

L)


lowastlowast

(c)

Figure 3 Moxibustion ameliorates renal dysfunction in FSGS rats The levels of creatinine (a) urea nitrogen (b) and uric acid (c) in serumfrom different groups of rats Data represent at least 6 rats per group and are expressed as the mean plusmn SD lowast119875 lt 005 and lowastlowast119875 lt 001 versussham group 119875 lt 005 and

119875 lt 001 versus model group

33 Effects of Moxibustion on Kidney Function in the FSGSRat Model To further evaluate the effects of moxibustionon kidney damage by FSGS we measured serum markersin the different groups of rats at 12 weeks (Figure 3) Serumcreatinine urea nitrogen and uric acid were significantlyincreased in the FSGS model group compared to the sham-operation group (119875 lt 001) Additionally serum creatininelevels were significantly increased in the losartan Shenshumoxibustion and Geshumoxibustion groups compared withthe sham-operation group (119875 lt 005) however the increasesrelative to the sham group were less pronounced for thesethree treatment groups than for the FSGS model group (119875 lt005) Additionally the serum urea nitrogen and uric acid

contents were significantly reduced in the losartan BL-23MO and BL-17 MO groups compared to the model groupand were not significantly different from that of the shamgroup These results are consistent with the results of theurinary protein analysis and provide additional evidence thatmoxibustion ameliorates kidney damage

34 Effects of Moxibustion on FN 120572-SMA and TGF-120573 Expres-sion in Kidney Tissues of FSGS Rats To further examinethe effects of moxibustion on kidney toxicity we performedimmunohistochemistry for biomolecules that are associatedwith renal disease (Figure 4) Compared with the sham-operation group expression of renal FN 120572-SMA and TGF-120573



FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin


lowastlowastlowastlowast lowast

lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0


lowastlowastlowastlowast

lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)

Figure 4 Moxibustion attenuates renal fibrotic lesions in vivo Immunohistochemical staining of fibronectin (a) 120572-SMA (b) and TGF-120573 (c)in the kidneys Data represent at least 6 rats per group and are expressed as the mean plusmn SD lowast119875 lt 005 and lowastlowast119875 lt 001 versus sham group119875 lt 005 and


was significantly elevated in the FSGS model group and theShenshumoxibustion group (119875 lt 005119875 lt 001) Expressionof renal FN and 120572-SMA was also significantly elevated in thelosartan group (119875 lt 001) and renal TGF-120573 expression wasalso elevated in the Geshu moxibustion group (119875 lt 005 119875 lt001) Compared with the FSGS model group the expressionof renal FN 120572-SMA and TGF-120573 of the losartan group theShenshu moxibustion and Geshu moxibustion groups wassignificantly reduced (119875 lt 005 119875 lt 001) Furthermorecompared with the losartan group expression of renal FNand120572-SMAof theGeshumoxibustion groupwas significantlyreduced (119875 lt 005 119875 lt 001) These results are consistent

with the ability of moxibustion to attenuate the effects ofFSGS in rats

35 Effects of Moxibustion on Renal Podocin and NephrinmRNAandProtein Expression in FSGSRats To further assessthe effects ofmoxibustion on FSGS progression we examinedthe mRNA and protein expression of podocin and nephrin(Figure 5) which are components of a complex that mediateskidney filtration [19] Comparedwith expression in the sham-operation group expression of renal podocin and nephrinmRNA was significantly reduced in the FSGS model groupthe losartan group the Shenshu moxibustion and Geshu


4

3

2

1

0

Sham Model Losartan BL-23 BL-17

PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)

Figure 5 Moxibustion attenuates podocyte injury in vivo (a) Real-time PCR analyses of podocin and nephrin in rat kidney tissues (b)Western blot analyses of podocin and nephrin in the kidneys Data represent groups of 3 rats and are expressed as the mean plusmn SD lowast119875 lt 005and lowastlowast119875 lt 001 versus sham group 119875 lt 005 and

119875 lt 001 versus model group ∘119875 lt 005 and ∘∘119875 lt 001 versus losartan group ◻119875 lt 005versus BL-23 group

(a) (b) (c) (d) (e)

Figure 6 Moxibustion attenuates glomerular lesions in vivo Representative micrographs demonstrate kidney injury at 12 weeks aftertreatment in different groups Kidney sections were subjected to Massonrsquos trichrome staining

moxibustion groups (119875 lt 005 119875 lt 001) however thereduction was less pronounced in the losartan Shenshumoxibustion and Geshu moxibustion groups compared tothe model group (119875 lt 005 119875 lt 001) Similar results wereobtained for podocin and nephrin proteinThese results sup-port the protective role of moxibustion on kidney filtration

36 Effects of Moxibustion on the Renal Pathomorphologyof FSGS Rats To directly examine the pathological effectsof FSGS to the kidneys and the ability of moxibustion to

attenuate these effects we examined the histomorphologyof kidney sections (Figure 6) In the sham-operation group(Figure 6(a)) the glomerular capillary loops were openedand glomerular sclerosis was not observed the renal tubularepithelial cells were arranged neatly few fibroblasts werefound in the interstitium and inflammatory cells wereonly occasionally seen In the rats of FSGS model group(Figure 6(b)) glomerular capillary loops were collapsedsome capillaries were narrow or even occluded and seg-mental glomerulosclerosis was observed In addition tubular


lesions were relatively severe with swelling of tubular epithe-lial cells granular degeneration necrosis loss of obvioustubular expansion a large number of tubes and sometubular atrophy interstitial fibrosis and inflammatory cellinfiltration were also obvious Comparatively the areas ofthe glomerular capillaries were shrunk in the rats of losartangroup (Figure 6(c)) which had some narrowed capillariesand improved glomerular segmental sclerosis Renal tubularepithelial cells in this group of rats were slightly swollenshowing interstitial fibrosis and small amount of inflamma-tory cell infiltration Similarly glomerular capillaries in therats of the Shenshu moxibustion (Figure 6(d)) and Geshumoxibustion (Figure 6(e)) were partially narrowed withmildglomerular segmental sclerosis obvious interstitial fibrosisand small amount of inflammatory cell infiltration Theseresults provide morphological evidence for the protectiveeffects of moxibustion

4 Discussion

In this study we assessed the ability of moxibustionmonotherapy to intervene with disease progression in theFSGS rat model and monitored its impacts on urinaryprotein levels renal function and relevant biomolecules Ourresults demonstrate that the 24-h urinary protein serumcreatinine urea nitrogen and serum uric acid of FSGS ratswere improved to certain degrees after administration oftwo different moxibustion protocols (MO-BL-23 and MO-BL-17) In addition the renal expression of FN 120572-SMATGF-120573 podocin and nephrin and the pathomorphologyof the kidney tissues also showed positive changes aftermoxibustion therapy

To evaluate the effects of moxibustion we first examinedits effects on urinary protein levels which is a common indi-cator of kidney failureWe selected losartan as a positive con-trol therapy in these experiments because it is used in patientswith primary FSGS to delay progression of chronic kidneydisease to end stage kidney disease when corticosteroids andimmunosuppressive therapy fail to induce remission [20] Byblocking the renin-angiotensin-aldosterone system losartanprovides regulation of tubulointerstitial injury and fibrosisto improve the glomerular filtration rate reduce tubuloint-erstitial injury and fibrosis and enable renal protection Theurinary protein levels of the FSGS rats were significantlyhigher than those of the sham-operation group at all timepoints regardless of therapeutic application Furthermorethe protein showed a time-dependent pattern with continuedelevation on weeks 0 4 and 8 and a slight drop on week12 in the model group In contrast the 24-h urinary proteinlevels of the losartan group the Shenshu moxibustion andthe Geshu moxibustion groups showed a pattern of reduc-tion over time While the therapeutic effects in this assaywere most obvious for losartan these results suggest thatmoxibustion may also have therapeutic potential Given thatmoxibustion is applied externally and functions by enhancinga natural system within the body it could provide a less toxicand less invasive alternative therapy with similar effect to oralor injectable pharmaceuticals

The production of urinary proteins is inseparable frompodocyte injury [21] and elevation of urinary protein furtherenhances the severity of podocyte injury in turn resultingin the progression of glomerular sclerosis This study showedthat expression of renal podocin and nephrinmRNA in FSGSrats of different treatment groups was significantly lower thanthat of the sham group These results confirm that the renaltoxicity of doxorubicin caused glomerular podocyte injuryand reduced podocyte marker protein expression whichled to podocyte loss or apoptosis The expression of renalpodocin and nephrin protein and mRNA of the losartanShenshu moxibustion and Geshu moxibustion groups wassignificantly higher than that of the FSGS model group sug-gesting that moxibustion alleviates the shedding and missingof podocyte marker proteins in kidney tissues after FSGSThus the relevant mechanism of moxibustion in reducingthe elevation in urinary protein content after FSGS inductionmay involve an increase in renal podocin and nephrin proteinexpression thereby maintaining the structural integrity ofpodocyte septa and subsequently alleviating podocyte injury

We also demonstrated that the serum creatinine ureanitrogen and uric acid of the losartan ShenshumoxibustionandGeshumoxibustion groupswere significantly higher thanthose of the sham-operation group but lower than those of theFSGS model group suggesting that FSGS rats had differentdegrees of renal dysfunction after therapeutic application Inaddition these three indicators had no significant differencein different treatment groups as assessed by pairwise com-parison suggesting that Shenshu moxibustion and Geshumoxibustion significantly improve the renal function of FSGSrats with similar effects to losartan treatment

Thedegree of renal interstitial fibrosis is closely associatedwith the renal function Doxorubicin nephrotoxicity andstimulation of excessive proteinuria induce the release of avariety of cytokines and inflammatory mediators producedby glomerular intrinsic cells and other inflammatory cells tostimulate the proliferation of glomerular mesangial cells andincrease of themesangial matrix alleviating the developmentand formation of glomerulosclerosis [22] In addition byinducing interstitial inflammatory cell infiltration doxoru-bicin nephrotoxicity and proteinuria renal tubular injury canlead to interstitial fibrosis ultimately causing renal fibrosiswhich could progress to end stage renal failure In this studywe demonstrated that expression of renal 120572-SMA TGF-120573and FN in rats of the FSGS model group losartan groupShenshu moxibustion group and Geshu moxibustion groupwas higher than in the sham-operation group suggesting thatrenal interstitial fibrosis occurs in rats after FSGS modelingIn the Shenshu moxibustion and Geshu moxibustion groupsthe above indexes were relatively lower than in the FSGSmodel group while expression of renal 120572-SMA and FN of theGeshumoxibustion group was lower than the losartan groupsuggesting that Shenshu moxibustion Geshu moxibustionand losartan treatment improve the renal interstitial fibrosisof FSGS rats The regulatory effect of moxibustion on renaltubular epithelial-mesenchymal transition and extracellu-lar matrix (ECM) integrity in FSGS rats was similar forthe losartan treatment Therefore according to this mea-sure the inhibitory effect of 120572-SMA and FN expression


by Geshu moxibustion may be more significant than bylosartan

Podocytes are themajor cells participating in the develop-ment of FSGS Denaturation occurs after podocyte injury andthe separation of glomerular basementmembrane (shedding)causes expansion of the capillary loop exposure of thetubular basementmembrane andBowman capsule adhesionThrough tearing stretching and other changes gradualprogression of the disease occurs to the entire glomerulusand its connected tubules In addition ECM production ofthe focal lesion continuously increases and is characterized bycompression of the capillary loop occlusion which eventuallyleads to the formation of glomerular sclerosis In this studydoxorubicin-induced FSGS modeling in different groupsof rats caused different degrees of glomerular structuraldisorder collapse and atrophy and some of the rats showedglomerular sclerosis Pathological injury of rat kidney tissuesin the losartan Shenshu moxibustion and Geshu moxibus-tion groups was relatively less severe compared to the FSGSmodel group which is consistent with the pattern of expres-sion of renal fibrosis- and podocyte injury-related markersThese findings confirm that podocyte injury and ECMaccumulation (renal interstitial fibrosis) are closely associatedwith the incidence of glomerular sclerosis in our model

Moxibustion has been extensively applied but a lack ofsufficient data is available to demonstrate its effectivenessin the scientific research field Most previous studies havefocused on randomized controlled trials of acupuncture com-binedwithmoxibustion howevermoxibustionmonotherapyis rarely reported Our study evaluated the effects of mox-ibustion without acupuncture and compared the acupointcompatibilities of moxibustion monotherapy for the BL-23and BL-17 acupoints The BL-23 acupoint has been morefrequently used in studies of chronic kidney diseases [6ndash8]while BL-17 acupoint has been reported in the treatment ofa variety of diseases [23ndash25] Randomized controlled trialsthat have evaluated these acupoints involved a large numberand different combinations of acupoints which has made itdifficult to accurately determine the role of single-acupointintervention for specific acupoint

As a limitation this study did not include nonspecificacupoint moxibustion control groups for rats without FSGSHowever the results affirm the therapeutic effect of themoxibustion in FSGS Additionally because no statisti-cally significant differences were found between the two-moxibustion groups in this study we could not validatethe specific therapeutic effect of moxibustion at the twoacupoints Studies that assess the efficacy of moxibustion inameliorating proteinuria that results from chronic kidneydiseases are rare as is acupoint-specificity-related researchTherefore our work provides a unique cross-section of a fieldthat is in need of further investigation Future studies willbe based on supporting the current findings to improve theexperimental design and conduct further in-depth study

5 Conclusions

In this study we applied single nephrectomy and repeatedinjection of doxorubicin to a FSGS model in rats and used

moxibustion at Shenshu and Geshu acupoints to confirmthat the moxibustion improved urinary protein levels andrenal function Shenshu and Geshu moxibustion also allevi-ated podocyte injury and inhibited renal interstitial fibrosisthereby improving the glomerulosclerosis

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Authorsrsquo Contributions

Yi Li designed the experiments performed experiments andwrote the manuscript Yuxia Sun performed experimentsand wrote the manuscript Chunling Zhang designed theexperiments and wrote the manuscript Ke Wang wrotethe manuscript Peicheng Shen Di Huang Wen Ma andJin Zhang designed the experiments Lin Li performedexperiments Liqun He designed the experiments wrote themanuscript and served as the lead investigator

Acknowledgments

This work was supported by the grant from the NationalNatural Science Foundation of China (no 81302923)

References

[1] M J Chun SMKorbetMM Schwartz and E J Lewis ldquoFocalsegmental glomerulosclerosis in nephrotic adults presentationprognosis and response to therapy of the histologic variantsrdquoJournal of the American Society of Nephrology vol 15 no 8 pp2169ndash2177 2004

[2] E Alexopoulos M Stangou A Papagianni A Pantzaki andM Papadimitriou ldquoFactors influencing the course and theresponse to treatment in primary focal segmental glomeru-losclerosisrdquo Nephrology Dialysis Transplantation vol 15 no 9pp 1348ndash1356 2000

[3] S Troyanov C A Wall J A Miller J W Scholey and D CCattran ldquoFocal and segmental glomerulosclerosis definitionand relevance of a partial remissionrdquo Journal of the AmericanSociety of Nephrology vol 16 no 4 pp 1061ndash1068 2005

[4] V D DrsquoAgati F J Kaskel and R J Falk ldquoFocal segmentalglomerulosclerosisrdquo The New England Journal of Medicine vol365 no 25 pp 2398ndash2411 2011

[5] S M Korbet ldquoTreatment of primary FSGS in adultsrdquo Journal ofthe American Society of Nephrology vol 23 no 11 pp 1769ndash17762012

[6] J C Paterno A O Freire M F Soares M F Franco N Schorand V P C Teixeira ldquoElectroacupuncture and moxibustionattenuate the progression of renal disease in 56 nephrec-tomized ratsrdquo Kidney and Blood Pressure Research vol 31 no6 pp 367ndash373 2009

[7] J C Paterno C T Bergamaschi R R Campos et al ldquoElec-troacupuncture and moxibustion decrease renal sympatheticnerve activity and retard progression of renal disease in ratsrdquoKidney and Blood Pressure Research vol 35 no 5 pp 355ndash3642012


[8] J-H Chiu W-Y Lui Y-L Chen and C-Y Hong ldquoLocalsomatothermal stimulation inhibits the motility of sphincterof Oddi in cats rabbits and humans through nitrergic neuralrelease of nitric oxiderdquo Life Sciences vol 63 no 6 pp 413ndash4281998

[9] J-K Jiang J-H Chiu and J-K Lin ldquoLocal thermal stimulationrelaxes hypertonic anal sphincter evidence of somatoanalreflexrdquoDiseases of the Colon and Rectum vol 42 no 9 pp 1152ndash1159 1999

[10] J-K Jiang J-H Chiu and J-K Lin ldquoLocal somatothermalstimulation inhibits motility of the internal anal sphincterthrough nitrergic neural release of nitric oxiderdquo Diseases of theColon and Rectum vol 43 no 3 pp 381ndash388 2000

[11] Y-H Lin J-H ChiuH-H TungM-T TsouW-Y Lui andC-W Wu ldquoPreconditioning somatothermal stimulation on rightseventh intercostal nerve territory increases hepatic heat shockprotein 70 and protects the liver from ischemiamdashreperfusioninjury in ratsrdquo Journal of Surgical Research vol 99 no 2 pp328ndash334 2001

[12] J H Chiu M T Tsou H H Tung et al ldquoPreconditionedsomatothermal stimulation onmedian nerve territory increasesmyocardial heat shock protein 70 and protects rat heartsagainst ischemia-reperfusion injuryrdquo Journal of Thoracic andCardiovascular Surgery vol 125 no 3 pp 678ndash685 2003

[13] M-T Tsou J-Y Ho C-H Lin and J-H Chiu ldquoProteomicanalysis finds different myocardial protective mechanisms formedian nerve stimulation by electroacupuncture and by localsomatothermal stimulationrdquo International Journal of MolecularMedicine vol 14 no 4 pp 553ndash563 2004

[14] P-J Pan R-C Chan A-H Yang C-L Chou Y-F Chengand J-H Chiu ldquoProtective effects of preconditioned localsomatothermal stimulation on neuromuscular plasticity againstischemia-reperfusion injury in ratsrdquo Journal of OrthopaedicResearch vol 26 no 12 pp 1670ndash1674 2008

[15] P J Pan C F Hsu J J Tsai and J H Chiu ldquoThe role of oxidativestress response revealed in preconditioning heat stimulation inskeletal muscle of ratsrdquo Journal of Surgical Research vol 176 no1 pp 108ndash113 2012

[16] J Matsumoto-Miyazaki N Miyazaki I Murata et al ldquoTra-ditional thermal therapy with indirect moxibustion decreasesrenal arterial resistive index in patients with chronic kidneydiseaserdquo Journal of Alternative and Complementary Medicinevol 22 no 4 pp 306ndash314 2016

[17] X H Ma and L Q He ldquoEffect of Jianpi Qinghua decoctions oninterleukin-6 and monocyte chemotactic protein-1 in rats withfocal segmental glomurularsclerosis nephropathy role of theinflammatory signaling pathwaysrdquo Journal of Southern MedicalUniversity vol 33 no 11 pp 1577ndash1582 2013

[18] A O Freire G C M Sugai M M Blanco A Tabosa YYamamura and L E A M Mello ldquoEffect of moxibustionat acupoints Ren-12 (Zhongwan) St-25 (Tianshu) and St-36 (Zuzanli) in the prevention of gastric lesions induced byindomethacin in Wistar ratsrdquo Digestive Diseases and Sciencesvol 50 no 2 pp 366ndash374 2005

[19] T B Huber and T Benzing ldquoThe slit diaphragm a signalingplatform to regulate podocyte functionrdquo Current Opinion inNephrology and Hypertension vol 14 no 3 pp 211ndash216 2005

[20] H Trachtman S Vento D Gipson et al ldquoNovel therapies forresistant focal segmental glomerulosclerosis (FONT) phase IIclinical trial study designrdquo BMC Nephrology vol 12 no 1article 8 2011

[21] J Patrakka and K Tryggvason ldquoNew insights into the role ofpodocytes in proteinuriardquo Nature Reviews Nephrology vol 5no 8 pp 463ndash468 2009

[22] P Nistico I Capone B Palermo et al ldquoChemotherapyenhances vaccine-induced antitumor immunity in melanomapatientsrdquo International Journal of Cancer vol 124 no 1 pp 130ndash139 2009

[23] M Lu K Li and J Wang ldquoAcupuncture for distal symmetricmultiple peripheral neuropathy of diabetes mellitus a random-ized controlled trialrdquo Chinese Acupuncture amp Moxibustion vol36 no 15 pp 481ndash484 2016

[24] M Lu Y Wang D Yu D Cao Y Teng and J Li ldquoEffectsof acupuncture-moxibustion on contents of IL-12 and TNF-120572in spleen of cyclophosphamide- induced cancer-bearing micerdquoChinese Acupuncture amp Moxibustion vol 35 no 11 pp 1145ndash1148 2015

[25] B-S Ouyang J Gao G Sun et al ldquoImpact of acupoint heat-sensitive moxibustion on lung function and life quality ofpatients with chronic persistent bronchial asthma a random-ized controlled studyrdquoChinese Acupuncture ampMoxibustion vol31 no 11 pp 965ndash970 2011

Submit your manuscripts athttpswwwhindawicom

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


therapy is moxa wool which is used to construct conesor sticks The moxa sticks or cones are ignited prior toapplication on the surface of meridian acupoints via scarringor nonscarring methods However rigorous randomizedcontrolled trials of moxibustion have not been describedwhich may account for the poor awareness of this therapymethod

Nevertheless animal experiments support the potentialrole of both acupuncture and moxibustion in the treatmentof renal diseases Paterno et al [6] demonstrated that elec-troacupuncture at Zusanli (ST-36) and Taixi (KI-3) acupointsand moxibustion at the Shenshu (BL-23) acupoint improvethe serum creatinine 24-h urinary protein glomeruloscle-rosis and tubulointerstitial fibrosis in a 56 nephrectomyrat model Further study by this group [7] showed that thetreatment mechanism may be related to the regulation ofrenal sympathetic nerve activity and nitric oxide levels andthe reduction of arterial blood pressure The potential roleof nitric oxide release from nerves has also been noted inthe response to local thermal stimulation on Riyue (GB-24)Weizhong (BL-40) andChengfu (BL-36) acupoints to inhibitthe physiological activities of the Oddi sphincter and theanal sphincter [8ndash10] Additionally several investigators havedemonstrated that thermal stimulation within the regionsof Qimen (LR-14) Neiguan (PC-6) and Yinmen (BL-37)acupoints increases the expression of heat shock protein 70(HSP70) and elevates reactive oxygen species which protectsliver and heartmuscles from ischemia-reperfusion injury [11ndash15] Matsumoto-Miyazaki et al [16] observed reduced renalvascular resistance after indirect moxibustion at the bilateralShenshu acupoints of 43 chronic kidney disease patients Atpresent however direct reports of moxibustion therapy forproteinuria in patients with chronic glomerular disease arerelatively rare Therefore moxibustion therapy is worthy offurther in-depth investigation

In this study we used single nephrectomy and repeatedinjections of doxorubicin [17] to establish FSGS in rats Usingthe angiotensin II receptor antagonist losartan as a positivecontrol therapeutic we evaluated the potential therapeuticeffects of mild Shenshu moxibustion (BL-23) and Geshumoxibustion (BL-17)Wemonitored the urinary protein levelkidney function expression of renal fibronectin (FN) 120572-SMA TGF-120573 podocin and nephrin and pathomorphologyof FSGS kidney tissues of different groups of rats Ourresults support the potential therapeutic value ofmoxibustionat Shenshu (BL-23) and Geshu (BL-17) acupoints in thetreatment of FSGS

2 Methods

21 Animals Fifty-one clean-grade male Sprague Daw-ley rats (bodyweight of 140ndash160 g) were purchased fromShanghai Sippr-BK Lab Animal Co Ltd (Shanghai Chinaanimal qualification certificate number SCXK [Shanghai]2013ndash0016) All experimental rats were fed common chow(containing 21 crude protein 45 crude fat and 5crude fibers purchased from Shanghai Shilin BiotechnologyCo Ltd Shanghai China) and housed in the AnimalExperimental Center of Shanghai University of TCM with

a room temperature of 22ndash25∘C and humidity of 50Animal experiments and euthanization procedures were inaccordance with Guidelines on the Treatment of ExperimentalAnimals issued by Ministry of Science and Technology in2006 and relevant animal ethics standards listed in EthicalIssues in Animal Experimentation in 2009

22 Establishment of the FSGS Model All rats were accli-mated for a week and then were weighed and numbered bytheir order in weight (from light to heavy) Correspondingto numbers generated by SPSS statistical software seven ratswere randomly selected for sham operation by laparotomyto expose the kidneys and remove the renal capsule withoutdissecting the kidney tissue followed by suturing layer bylayer These animals were then tail-vein injected with 05mlnormal saline on day 4 and day 18 after sham operationThe other 44 rats received two intravenous injections ofdoxorubicin two weeks apart and unilateral nephrectomyto model glomerulosclerosis All animals were fasted withfree access to water 12 h before operation and anesthetizedby intraperitoneal injection of 50mgkg 15 pentobarbitalsodium (Merck Drugs and Biotechnology Germany batchnumber 20130112) After the corneal reflex had subsidedthe animals were fastened on the operating table followedby hair shaving and routine disinfection A 1ndash15 cm dorsalincision was made to expose the left kidney remove the fattissues and adrenal gland around the kidney ligate the leftrenal artery and resect the left kidney The incision was thensutured The animals were tail-vein injected with 3mgkg(postoperative day 4) and 2mgkg (postoperative day 18) ofdoxorubicin which was dissolved in sterile water at a dilu-tion of 2mgml (10mg doxorubicinsyringe Shenzhen MainLuck Pharmaceuticals Inc Guangdong Province Chinabatch number national approved medicine H44024359)The urinary protein of the viable rats was quantified sevendays after the second doxorubicin injection Animals withgt100mg24 h urinary protein indicated successful modelingand were included in subsequent experiments with monitor-ing for 12 consecutive weeks

23 Grouping and Intervention Forty-four successfullymod-eled FSGS rats were numbered from 1 to 44 according to thequantity (from small to large quantity) of urinary proteinCorresponding to numbers generated by SPSS statisticalsoftware the 44 rats were randomly divided into the modellosartan Shenshu moxibustion (BL-23-MO) and Geshumoxibustion (BL-17 MO) groups (119899 = 11 rats per group)Intervention was initiated in each group on the same dayAnimals in the sham-operation model and moxibustiongroups were given 5mlkg distilled water by gavage once aday Rats in the losartan group were given losartan potas-sium tablets which were dissolved in distilled water at adilution of 5mgml immediately before the administrationby gavage once a day (100mgtablet produced by HangzhouMSD Pharmaceutical Co Ltd Hangzhou China batchnumber national approved medicine H20030654) Rats inthe Shenshu and the Geshu moxibustion groups receivedmild moxibustion at Shenshu (bilateral) acupoints or Geshu(bilateral) acupoints every other day (30min per acupoint)


BL-17 BL-23

(a) (b)


















119875 lt 005 and




3 Results



600

400

200

0

Urin

ary

prot

ein

(mg

24h)

0 4 8 12

Time (weeks)

ShamModelLosartan

BL-23 MOBL-17 MO

998779998779 998779998779998779998779998779998779

998779998779998779

∘






400

300

200

100

0

Seru

m cr

eatin

ine (

120583m

olL

)


lowastlowast

lowast

lowast

lowast

(a)

lowastlowast

50

40

30

20

10

0

Seru

m u

rea n

itrog

en (m

mol

L)


(b)

150

100

50

0

Seru

m u

ric ac

id (120583

mol

L)


lowastlowast

(c)








FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin



lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0



lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)







4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















BL-17 BL-23

(a) (b)


















119875 lt 005 and




3 Results



600

400

200

0

Urin

ary

prot

ein

(mg

24h)

0 4 8 12

Time (weeks)

ShamModelLosartan

BL-23 MOBL-17 MO

998779998779 998779998779998779998779998779998779

998779998779998779

∘






400

300

200

100

0

Seru

m cr

eatin

ine (

120583m

olL

)


lowastlowast

lowast

lowast

lowast

(a)

lowastlowast

50

40

30

20

10

0

Seru

m u

rea n

itrog

en (m

mol

L)


(b)

150

100

50

0

Seru

m u

ric ac

id (120583

mol

L)


lowastlowast

(c)








FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin



lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0



lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)







4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























119875 lt 005 and




3 Results



600

400

200

0

Urin

ary

prot

ein

(mg

24h)

0 4 8 12

Time (weeks)

ShamModelLosartan

BL-23 MOBL-17 MO

998779998779 998779998779998779998779998779998779

998779998779998779

∘






400

300

200

100

0

Seru

m cr

eatin

ine (

120583m

olL

)


lowastlowast

lowast

lowast

lowast

(a)

lowastlowast

50

40

30

20

10

0

Seru

m u

rea n

itrog

en (m

mol

L)


(b)

150

100

50

0

Seru

m u

ric ac

id (120583

mol

L)


lowastlowast

(c)








FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin



lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0



lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)







4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















400

300

200

100

0

Seru

m cr

eatin

ine (

120583m

olL

)


lowastlowast

lowast

lowast

lowast

(a)

lowastlowast

50

40

30

20

10

0

Seru

m u

rea n

itrog

en (m

mol

L)


(b)

150

100

50

0

Seru

m u

ric ac

id (120583

mol

L)


lowastlowast

(c)








FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin



lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0



lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)







4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















FN120572

-SM

ATG

F-120573

(a)

800

600

400

200

0

Kidn

ey ti

ssue

fibr

onec

tin



lowast

(b)

Kidn

ey ti

ssue

120572-S

MA

800

600

400

200

0



lowast

(c)

Kidn

ey ti

ssue

TG

F-120573

800

600

400

200

0



lowast

(d)







4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4

3

2

1

0


PodocinNephrin


lowastlowast

lowastlowast

lowast

∘

lowastlowast

∘∘

lowastlowast

∘∘

lowastlowast

Relat

ive m

RNA

expr

essio

n(lowast

10minus2)

(a)


Podocin

Nephrin

GAPDH

15

10

05

00

Relat

ive p

rote

in ex

pres

sion

(Wes

tern

blo

t gra

y va

lue)


lowastlowast


∘

lowastlowast

PodocinNephrin

(b)



(a) (b) (c) (d) (e)







4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















4 Discussion











5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















5 Conclusions







Acknowledgments


References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Moxibustion Alleviates Injury in a Rat Focal Segmental …downloads.hindawi.com/journals/ecam/2017/7169547.pdf · 2019-07-30 · The moxa sticks or cones are ignited prior to