research article proregenerative microenvironment...

Research ArticleProregenerative Microenvironment Triggered byDonor Mesenchymal Stem Cells Preserves Renal Function andStructure in Mice with Severe Diabetes Mellitus

Fernando Ezquer1 Maximiliano Giraud-Billoud12 Daniel Carpio3 Fabiaacuten Cabezas14

Paulette Conget1 and Marcelo Ezquer1

1Centro de Medicina Regenerativa Facultad de Medicina Clınica Alemana Universidad del Desarrollo Avenida Las Condes 12438Santiago Chile2Laboratorio de Fisiologıa (IHEM-CONICET) and Instituto de Fisiologıa Facultad de Ciencias MedicasUniversidad Nacional de Cuyo Casilla de Correo 33 Mendoza Argentina3Instituto de Anatomıa Histologıa y Patologıa Facultad de Medicina Universidad Austral de Chile Isla Teja sn Valdivia Chile4Facultad de Ciencias Biologicas Universidad Andres Bello Avenida Los Leones 745 Santiago Chile

Correspondence should be addressed to Marcelo Ezquer mezqueruddcl

Received 2 March 2015 Revised 30 April 2015 Accepted 14 May 2015

Academic Editor Florian Toegel

Copyright copy 2015 Fernando Ezquer et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of our work was to evaluate in an animal model of severe diabetes mellitus the effect of mesenchymal stem cells (MSCs)administration on diabetic nephropathy (DN) progression After diabetes induction one group of mice received the vehicle(DM) and other group received a single dose of MSCs (DM + MSCs) DM + MSCs mice showed a significant improvement infunctional parameters of the kidney compared with untreated mice While DMmice presented marked histopathological changescharacteristics of advanced stages of DN (fibrosis glomerulosclerosis glomerular basement membrane thickening capillaryocclusion decreased podocyte density and effacement of foot processes) DM +MSCs mice showed only slight tubular dilatationThe renoprotection was not associated with an improvement in diabetic condition and very low number of donor cells was foundin the kidney of DM + MSCs mice suggesting that renoprotection could be mediated by paracrine effects Indeed DM + MSCmice presented increased renal proliferation index decreased renal apoptotic index and the restoration of proregenerative factorsand anti-inflammatory cytokines levels Moreover macrophage infiltration and oxidative stress damage were also reduced in DM+MSCs mice Our data demonstrate that MSC administration triggers a proregenerative microenvironment in DN kidney whichallows the preservation of the renal function even if diabetes was uncorrected

1 Introduction

Diabetic nephropathy (DN) is the major microvascular com-plication in diabetic patients and it remains the leading causeof chronic kidney disease responsible for approximately 50of all cases of end-stage renal disease worldwide [1] Thedevelopment of DN is stimulated by sustained hypergly-caemia [2] and its key pathologic features include gradualthickening of the glomerular basement membrane (GBM)and glomerular hypertrophy accompanied by mesangialmatrix expansion with accumulation of several matrix pro-teins such as collagen type I laminin 1205731 and fibronectin [2]

These molecules contribute to the reduction in the glomeru-lar filtration surface area and to an increase in tubularinterstitial fibrosis and glomerulosclerosis Clinically thereis a decline in glomerular filtration rate with progressiveincrease in urinary albumin excretion that ultimately leadsto end-stage renal failure [3]

Hyperglycaemia induces cellular changes in various celltypes present in the kidney In the normal glomerulus thepodocytes (glomerular epithelial cells) have an arborizedphenotype and its terminal branches or foot processescover the outer wall of the glomerular capillaries Duringproteinuric stages the podocyte typical morphology is lost

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 164703 23 pageshttpdxdoiorg1011552015164703

2 BioMed Research International

and is converted into a flatter epithelial cell a process namedas ldquofoot process effacementrdquo In this process the cytoskeletonthat normally supports the delicate architecture of the footprocesses is condensed at the basal side of the flattenedpodocytes altering its functionality [4] Therefore podocyteinjury is associated with the development of albuminuriaand the appearance of more severe glomerular structuralabnormalities [4]

Throughout diabetes onset high levels of blood glucoseinduce both oxidative stress and inflammation which areintimately linked with the development of DN [5ndash7] Oxida-tive stress is caused by an imbalance between the productionof reactive oxygen species (ROS) and the biological systemrsquosability to readily detoxify the reactive intermediates or repairthe resulting damageWhen this occurs oxidation of essentialmacromolecules including proteins lipids carbohydratesand DNA is produced leading to the apoptosis of damagedcells [6]

Higher ROS levels can also induce the production ofinflammatory cytokines which stimulate the production offree radicals exacerbating DN progression [5] Thereforeintensive glycemic control is the main intervention to reduceoxidative stress

Transforming growth factor beta (TGF-120573) is a multifunc-tional cytokine that plays a critical role in the pathogenesisof DN by stimulation of the synthesis of extracellular matrixmolecules including collagen type I fibronectin and laminin1205731 leading to renal fibrosis [8]

Stem cell therapy has been envisioned as a novel strategyfor various diseases since it has the potential to be moreeffective than single-agent drug therapies [9] Multipotentmesenchymal stromal cells also referred as mesenchymalstem cells (MSCs) are a population of self-renewable andundifferentiated cells present in the bone marrow and othermesenchymal tissues of adult individuals [10] MSCs areattractive candidates for renal repair since nephrons are ofmesenchymal origin and because stromal cells are of crucialimportance for signalling leading to differentiation of bothnephrons and collecting ducts [11]

Several studies have shown that the administration ofMSCs leads to the amelioration of acute and chronic kidneyinjury [12] In those reports immune modulation and anti-apoptotic effects have been associated with the therapeuticeffect suggesting that MSCs can act through paracrinemechanisms [13 14] AdditionallyMSC-based therapies havebeen considered to be promising strategies to treat patientswith diabetes mellitus and its main complications [15] Weand others have demonstrated that the systemic adminis-tration of MSCs into immunologically induced type 1 dia-betic mice reduced microalbuminuria and preserved normalrenal histology [16ndash20] However animal models used onlydeveloped the earlier harbingers of DN furthermore in thesestudies it is difficult to distinguish whether restoration ofkidney function is based on direct effect of transplantedcells on renal tissue or indirectly through improvement ofpancreatic endocrine function To answer this question andtaking into account that hyperglycaemia is the driving forcefor the development of DN previously we used a severediabetic animal model induced by the administration of

a single high dose (200mgkg) of streptozotocin (STZ) inC57BL6 mice [21] This protocol generates an irreversibleandmassive destruction of insulin-producing cells and severehyperglycaemia leading to a rapid progression of renal failureand the appearance of most of the characteristics DN pathog-nomonic signs [21ndash23] In this model multiple systemicadministrations of MSCs did not reverse hyperglycaemia butprotect kidneys from progression to macroalbuminuria andprevent the development of marked histological alterations[21]

The aim of the present work is to further characterizethe therapeutic effect of MSC administration in an animalmodel of severe diabetesmellitus particularly regarding renalfailure

For this purpose eight weeks after the massive cytotoxicdestruction of insulin-producing cells severe diabetic micewere separated into two groups one received the vehicle (DMmice) and the other 05 times 106 MSCs (DM + MSCs mice)Two and eightweeks later the function histopathology ultra-structure cell proliferation protein profile gene expressionoxidative stress and inflammation of kidneys were examinedDonor MSCs biodistribution was also assessed

2 Material and Methods

21 Animals C57BL6 and C57BL6-Tg (ACTB-EGFP)1Osb J mice (Jackson Laboratory) were housed at constanttemperature and humidity with a 12 12 h light-dark cycleand unrestricted access to a standard diet and waterWhen required animals were slightly anaesthetized withsevoflurane (Abbott) or ketamine (Drag Pharma) plusxylazine (Centrovet) All procedures were in accordancewith institutional and international standards for the humancare and use of laboratory animals [Ethic Committee ofFacultad de Medicina Clinica Alemana Universidad delDesarrollo and Animal Welfare Assurance PublicationsA54427-01 Office for Protection from Research RisksDivision of Animal Welfare NIH (National Institute ofHealth) Bethesda MD USA]

22 Severe Diabetes Induction Eight-week-old male C57BL6 mice were slightly anesthetized and received intraperi-toneally 200mgkg STZ (Calbiochem) immediately after dis-solving in 01M citrate buffer at pH 45 (DM mice) or citratebuffer only (Normal mice) This protocol of STZ admin-istration causes massive cytotoxic destruction of insulin-producing cells generating a severe hyperglycemic conditionwhich accelerates the appearance of the complications asso-ciated to diabetes particularly DN [21ndash23]

23MSC Isolation andExVivo Expansion Eight- to 10-week-old male C57BL6 or C57BL6-Tg (ACTB-EGFP) 1OsbJmice were sacrificed by cervical dislocation Bone marrowcells were obtained by flushing femurs and tibias with sterilephosphate-buffered saline (PBS) After centrifugation cellswere resuspended in 120572-minimum essential medium (120572-MEM) (Gibco) supplemented with 10 selected fetal bovineserum (Gibco) and 016mgmL gentamicin (Sanderson Lab-oratory) and plated at a density of 1 times 106 nucleated cellscm2

BioMed Research International 3

Nonadherent cells were removed after 72 h by media changeWhen foci reached confluence adherent cells were detachedwith 025 trypsin 265mM EDTA centrifuged and sub-cultured at 7000 cellscm2 After 2 subcultures adherentcells were characterized according to their adipogenic andosteogenic differentiation potential and immunophenotypi-fied as previously described [24 25]

24 MSC Administration Eight weeks after diabetes induc-tion 05 times 106 MSCs or 05 times 106MSCsGFP were resuspendedin 02mL of 5 mice plasma (vehicle) and administered viathe tail vein to slightly anaesthetized mice (DM + MSCsmice) Untreated animals received 02mL of vehicle (DMmice)

25 Sample Collection After four hours of fasting mice weresacrificed and blood samples were collected Kidneys werequickly removed decapsulated weighted and dissected intotwo parts one of which was immediately frozen in liquidnitrogen and stored atminus80∘C formolecular biological studieswhile the other was stored in 4 paraformaldehyde forhistopathological analysis

For 24 h urine collection animals were placed individu-ally in metabolic cages for 24 hours and urine was collectedand gravimetrically measured

26 Physical and Biochemical Assessment Body weight kid-neyweight and biochemical parameters weremeasured eightweeks after diabetes induction (before MSC administration)and two weeks and eight weeks after MSC administrationBlood glucose levels were measured with the glucometersystem Accu-Check Performa (Roche Diagnostic) Plasmainsulin levels were assayed using a mouse-specific insulinELISA kit (Ultrasensitive Mouse Insulin ELISA Mercodia)Serum triglycerides and cholesterol levels were determinedin the Architect c8000 autoanalyzer (Abbot) The renal massindex determined by the ratio of kidney weight to bodyweight was also calculated

27 Renal Function Measurements Urine samples were cen-trifuged to remove any suspended particle and the super-natant was used to detect albumin concentration using amouse-specific albumin ELISA kit (Albuwell Exocell) Urinecreatinine concentration was determined by the CreatinineCompanion kit (Exocell) Excretion of urinary albumin wasdetermined using albumin to creatinine ratio

Blood samples were centrifuged at 2000timesg for 10minplasma creatinine levels were determined using a mouse-specific enzymatic kit (Mouse Creatinine Assay CrystalChem) and blood urea nitrogen (BUN) using a quantitativecolorimetric kit (QuantiChrom Urea Assay Kit BioaAssaySystems)

28 Renal Histological and Immunofluorescence AnalysisPeriodic acid Schiff (PAS) staining was performed on 4-120583msections of paraffin-embedded slices Stained slices were usedto capture 50 photomicrographs (Leica DM2000 microscopeand Leica DFC295 digital camera) of glomeruli containing

vascular pole andor urinary pole to determine glomerularcorpuscle area glomerular tuff area andmesangial expansionin each image using Image-Pro Plus 451 software (MediaCybernetics) Massonrsquos trichrome staining was used to eval-uate the total area of collagen fibbers in the glomerular andin the tubulointerstitial area (20 photomicrographs each) andthe leukocyte infiltrate The fields were randomly chosenand analyzed in blind by the same specialist nephrologistLeukocyte infiltration was graded from 0 to 3 (0 = absentor infrequent 1 = sporadic scattered and ungrouped 2 =focalized in dense groups and 3 = dense foci + scatteredpresence) [26]

A standard point-counting method was employed toquantitate the collagen deposition [14 27 28] Under highmagnification of the 40x objective 20 consecutive nonover-lapping fields in each kidney were observed Trichrome-positive points falling on glomeruli Bowmanrsquos capsules orvessels were excluded while interstitial trichrome-positivepoints were counted for evaluation The index of interstitialcollagen volume was defined as the number of trichrome-positive points in every 2000 points evaluated

Tubular damage was graded from 0 to 4 according tothe presence or not of dilatation protein cylinders atrophyinterstitial fibrosis and leukocyte infiltration (0 = no changes1 = changes affecting lt25 of the sample 2 = changesaffecting 25 to 50 of the sample 3 = changes affecting 50to 75 of the sample and 4 = changes affecting 75 to 100of the sample) [29]

Renal markers immunoreactivity was evaluated by con-focal microscopy Sections of 4 120583m thick were deparaffinizedin Neo-Clear (Merck) rehydrated and washed in Tris-buffered saline (TBS) Antigen retrieval was performed byincubation in 10mM citrate buffer pH 6 in a boiling waterbath for 30min Sections were blocked with 2 bovineserum albumin (Sigma) in TBS and incubated with the pri-mary antibodies (WT-1 sc-192 podoplanin sc-134483 [SantaCruz Biotechnology] F480 ab74383 Ki-67 ab15589 120572-SMAab5694 [Abcam] GFP-D51XP [Cell Signaling]) in SignalStain Ab diluent (Cell Signaling) at 4∘C overnight Afterwashing sections were incubated with secondary antibody(anti-rabbit IgG Fab2 Alexa Fluor555 Molecular Probes) atroom temperature for 1 h and nuclei were counterstainedwith 410158406-diamino-2-fenilindol (DAPI) (BioChemica A1001AppliChem) Sections were examined with the FluoviewFV10i confocal microscope (Olympus)

F480 positive cells in renal tissue were quantitativelyassessed in 30 random sections per animal and expressed asF480 cellscross sections [30]

In normal kidneys 120572-SMA expression is confined to thesmooth muscle cells of efferent and afferent arterioles and itis never observed in glomeruli [31 32] Glomerulosclerosiswas defined as the presence of prominent 120572-SMA immunore-activity in the mesangial area Glomerulosclerosis indexwas quantified by determining the percentage of glomeruliexhibiting intense 120572-SMA staining in the glomerular tuft[33] One hundred consecutive glomeruli were examined foreach animal and analyzed in blind by the same specialistnephrologist


Glomerular podoplanin fluorescence area was analyzedmeasuring the relation between podoplanin staining andglomerular tuft area [34] Slides stained for WT-1 were usedfor morphometric analysis of the number of podocytes perglomerular volume WT-1-positive nuclei per glomerularcross-section were counted and the glomerular volume perpodocyte was calculated according to a previously validatedmethod [34] In all evaluations at least 50 glomeruli peranimal were randomly selected and analyzed and negativecontrols without the primary antibody were used to observetissue background Measurements of surface glomeruli areaand the immunoreactivity analysis were made using theImageJ 134 software (NIH httpimagejnihgovij)

29 Renal Ultrastructural Analysis Small fragments of kid-ney tissue were fixed in 2 glutaraldehyde (Sigma) thenpost-fixed with 1 osmium tetroxide (Ted Pella Inc) embed-ded in EMBed-812 resin (EMS Hatfield) cut stained andanalyzed under transmission electron microscopy (PhilipsTecnai 12 Bio TWIN) at 80 kV with standard magnificationsbetween times2500 and times16500

The electron micrographs of every animal were analyzedby setting the percentage of capillary loops with foot processeffacement usually along withmicrovillous transformation ofthe podocyte and irregularities of the outer surface of theglomerular basement membrane Then a comparison wasmade between micrographs of the same magnification in thedifferent groups of animals

Images were analyzed in blind by a specialist pathologist

210 Renal Gene Expression Analysis Expression levelsof type I collagen TGF-beta1 laminin-beta1 fibronectinnephrin bFGF EGF HGF IL-4 IL-10 and GAPDH fromkidney samples were assessed by quantitative qRT-PCR Forthis total RNA was purified using TRIzol (Invitrogen) andquantified spectrophotometrically (260 nm) One 120583g of totalRNA was used for reverse transcription Real time PCR wasperformed in a final volume of 20120583L containing 50 ng ofcDNA PCR LightCycler-DNA Master SYBRGreen reactionmix (Roche) 3mM MgCl

2 and 05 120583M of each specific

primer (see Supplementary Table 1 in Supplementary Mate-rial available online at httpdxdoiorg1011552015164703)using a Light-Cycler thermocycler (Roche) To ensure thatamplicons were frommRNA and do not from genomic DNAamplification controls without reverse transcription wereincluded Amplicon validation was performed based on itssize andmelting point Relative quantificationwas performedby the ΔΔCT method The mRNA level of each target genewas normalized against the mRNA level of GAPDH andexpressed as fold of change versus Normal mice

211 Quantification of Renal Mitotic and Apoptotic IndexesMitotic index was determined by immunohistofluorescenceusing an anti-Ki-67 antibody Positive nucleus indicates cellcycle activity (G1 S G2 or M phase) Apoptotic cells inkidney tissue slices were visualized using the DeadEnd Fluo-rometric TUNEL System (Promega) following the manufac-turerrsquos protocol The nuclei were counterstained with DAPIand fluorescence was evaluated by confocal microscopy

For the quantification of Ki-67 and TUNEL positive cellsat least 30 glomeruli and 30 tubules sections were examinedfor eachmouse and the number of positive stained nuclei pertotal cell nuclei was determined Data were expressed as levelof change versus Normal mice

212 Glomerular Fluorescein Angiography Mice wereinjected by tail vein with 200 120583L of PBS containing 10mgof 2 times 106 molecular weight fluorescein-dextran (Sigma)Five minutes later mice were euthanized and kidneys werefixed in 4 paraformaldehyde Four 120583m kidney slices wereobserved and photographed under confocal microscopyand at least 50 glomeruli per animal were analyzed Thefluorescence area representative of glomerular capillarylumen area of each image was quantified and normalized tothe glomerular tuft area [35] using the ImageJ 134 softwareNIH

213 Assessment of Renal Oxidative Stressand Antioxidant Capacity

2131 Total Reactive Oxygen Species Kidney slides wereincubated with 10120583M 27-dichloro-dihydro-fluorescein diac-etate (H

2DCFDA) (Invitrogen) in PBS containing 5mM

Hepes for 60min at 37∘C H2DCFDA is a nonfluorescent

molecule that in contact with ROS (hydrogen peroxideperoxynitrite and hydroxyl radicals) is rapidly converted intoa highly fluorescent derivate Kidney slidesweremechanicallylysed in RIPA buffer and fluorescence was measured in afluorometer (Turner) with excitation of 450 nm and emissionof 530 nm Data were presented as fluorescent units per mg ofrenal protein

2132 Lipid Oxidative Level Lipid peroxidation was deter-mined in urine samples measuring isoprostane levels Iso-prostanes are a type of eicosanoids produced nonenzymat-ically through the oxygen radical peroxidation of tissuephospholipids and lipoproteins Isoprostanes appear in nor-mal urine samples but are elevated by oxidative stress inrenal tissue [36] Urine samples were acidified to pH 30 byadding 110 vv of 1 N HCl and further diluted in samplediluent Level of 8-iso-prostaglandin F2120572 was determinedusing theOxiSelect 8-isoPGF2120572 ELISA kit (Cell Biolabs Inc)according to the manufacturerrsquos instructions

2133 Protein Oxidative Damage 3-Nitrotyrosine modifi-cation of proteins is a well-established marker of proteindamage by oxidative stress [37] Kidney slides were homoge-nized with an extraction buffer supplemented with proteaseinhibitor cocktail (Thermo) and protein oxidative damagewas measured with the 3-Nitrotyrosine ELISA kit (Abcam)according to the manufacturerrsquos instructions

2134 DNA Oxidative Damage The formation of 8-hydroxydeoxyguanosine (8-OHdG) is a ubiquitous marker of oxida-tive stress and its urinary excretion is associated with renalDNA oxidative damage [38] Urinary level of 8-OHdG wasevaluated by the OxiSelect Oxidative DNA Damage ELISA


Table 1 MSC administration did not improve diabetic condition

Before MSC administration 8 weeks after MSC administrationNormal DM Normal DM DM +MSCs

Body weight (g) 242 plusmn 04 202 plusmn 03lowast 269 plusmn 05 214 plusmn 05lowast 222 plusmn 05lowast

Plasma insulin (120583gL) 061 plusmn 012 003 plusmn 001lowast 051 plusmn 010 007 plusmn 003lowast 007 plusmn 003lowast

Blood glucose (mgdL) 171 plusmn 2 715 plusmn 49lowast 183 plusmn 11 766 plusmn 68lowast 740 plusmn 40lowast

Serum cholesterol (mgdL) 97 plusmn 10 173 plusmn 20lowast 146 plusmn 46 291 plusmn 32lowast 253 plusmn 52lowast

Serum triglycerides (mgdL) 72 plusmn 14 198 plusmn 35lowast 97 plusmn 39 337 plusmn 55lowast 217 plusmn 39lowast

Kidney weight (mg) 158 plusmn 4 217 plusmn 9lowast 177 plusmn 5 311 plusmn 15lowast 278 plusmn 14lowast

Kidneybody weight (mgg) 57 plusmn 02 89 plusmn 03lowast 66 plusmn 02 15 plusmn 07lowast 126 plusmn 09lowast

Urine volume (mL24 h) 13 plusmn 01 100 plusmn 16lowast 24 plusmn 09 168 plusmn 23lowast 163 plusmn 22lowast

Physical and biochemical parameters of experimental animals Before MSC administration (eight weeks after diabetes induction) mice received 200120583L of 5mice plasma (DM) or 05 times 106 MSCs resuspended in 200120583L of 5 mice plasma (DM +MSCs) via the tail vein All biochemical parameters were evaluated inurine and plasma samples obtained after four hours of fasting Data are presented as mean plusmn SEM 119899 = 12 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM forthe same time point

kit (Cell Biolabs Inc) in urine samples according to themanufacturerrsquos instructions

2135 Total Antioxidant Capacity Kidney slides werehomogenized with an extraction buffer supplemented withprotease inhibitor cocktail (Thermo) Total antioxidantcapacity was measured in tissue homogenate using theAntioxidant Assay kit (Cayman Chemicals) [39] accordingto the manufacturerrsquos instructions

214 Quantification of Systemic Cytokines and Growth FactorsCytokines (IL-1120573 IL-4 IL-6 IL-10 IL-13 and TNF-120572) andgrowth factors (EGF ET1 b-FGF HGF and VEGF) wereassessed in 25120583L of plasma using the MAGPMAG 24 kand MCYTOMAG 70 k assay kits (Luminex Milliplex MAPMillipore) respectively according to the manufacturerrsquosinstructions Plates were read on a Luminex 200 (LuminexCorp) and analyzed with the Milliplex Analyst software(VigeneTech Inc)

215 Statistical Analysis Data are presented as mean plusmnSEM To determine the statistical significance of intergroupdifferences Krustal-Wallis test was used to compare meanvalues among all groups andMann-Whitney119880 test was usedto compare mean values between two groups 119901 lt 005 wasconsidered as statistically significant

3 Results

Severe diabetes mellitus was induced in C57BL6 mice by theinjection of a single dose of 200mgkg STZ This protocolcauses rapid and massive destruction of insulin-producingcell A discrete acute nephrotoxic effect has been observedafter diabetes induction with any evidence of glomerularor tubular histopathological alterations [22 23] Diabeticmice were maintained without insulin supplementation toallow for the progression of severe diabetes mellitus and theappearance of its complications

Eight weeks after diabetes induction mice were hyper-glycemic polyuric and hypoinsulinemic (Table 1) Com-pared to normal mice they presented reduced body weightand increased serum cholesterol serum triglycerides andkidney weight (Table 1) In this condition animals wererandomly assigned into two groups one group that receivedthe vehicle (DM mice) and another group that received asingle dose of 05 times 106 bone marrow-derived MSCs (DM +MSCsmice) During the study period (16 weeks) 45 (1125)of DM mice and only 8 (225) of DM + MSCs mice diedor had to be sacrificed because of severe weight loss andcachexia These animals were excluded from data analysisDM and DM + MSCs mice remain hyperglycemic polyurichypoinsulinemic and dyslipidemic compared with normalmice (Table 1)

31 MSC Administration Prevents Renal Failure We askedwhether MSCs were able to prevent renal failure In order toaddress this question renal function was assessed in all exper-imental groups eight weeks after diabetes induction (beforeMSC administration) and two and eight weeks after MSCadministration As shown in Table 2 DM mice presentedincreased albumin excretion rate and plasmatic BUN levelsat every time point After 16 weeks from diabetes inductionthey reached values of albuminuria 10 times higher and valuesof plasmatic BUN two times higher than that observed innormal mice By contrast DM +MSCs mice maintained lowlevels of albuminuria and plasmatic BUN (similar to the pre-MSC administration stage) until the end of the study period

Plasmatic creatinine levels can be used to show signsof renal function in the end-stage of renal disease [14]During early-stage post-diabetic induction the plasmaticlevel of creatinine did not exhibited significant difference inany experimental group while this level was significantlyhigher in DM mice compared with normal mice at theend of the study period (16 weeks after diabetes induction)MSC administration to DM mice maintained the levels ofplasmatic creatinine similar to normal mice (Table 2)


Table2MSC

administratio

npreventsrenalfailure

Before

MSC

administratio

n2weeks

after

MSC

administratio

n8weeks

after

MSC

administratio

nNormal

T1DM

Normal

T1DM

T1DM

+MSC

Normal

T1DM

T1DM

+MSC

Urin

aryalbu

min

excretion(U

-Alb120583gU-C

ream

g)64plusmn14

350plusmn31lowast

67plusmn11

402plusmn43lowast

274plusmn4lowast

72plusmn08

622plusmn95lowast

309plusmn25lowast

Bloo

durea

nitro

gen(m

gdL

)301plusmn2

402plusmn33lowast

32plusmn18

421plusmn22lowast

455plusmn47lowast

319plusmn25

673plusmn38lowast

465plusmn34lowast

Plasmac

ystatin

C(m

gL)

007plusmn001

011plusmn001lowast

006plusmn001

011plusmn002lowast

008plusmn001

006plusmn002

013plusmn002lowast

008plusmn001

Plasmac

reatinine(mgL)

226plusmn006

229plusmn008

230plusmn007

227plusmn006

230plusmn013

228plusmn007

260plusmn005lowast

229plusmn005

Markersof

renalfun

ctionweree

valuated

before

MSC

administratio

n(eight

weeks

after

diabetes

indu

ction)

andaft

ertwoandeightw

eeks

after

MSC

administratio

nAllanim

alsw

ereh

ousedin

metaboliccagesto

collecturinefor

albu

minuriadeterm

inationsblood

wascollected

andused

tomeasureplasmaticcreatin

inea

ndurea

nitro

genlevels

Dataa

representedasmeanplusmnSE

M119899=12lowast119901lt005versus

Normal119901lt005

versus

DM

forthe

sametim

epoint


32 MSC Administration Prevents Renal HistopathologicalChanges To further characterize the therapeutic effect ofMSC administration we analyzed renal morphology Renalmass index determined by the kidney weight to body weightratio was significantly higher in DM mice compared withnormalmice A significant decrease of this ratio was observedin DM + MSCs mice eight weeks after MSC administration(Table 1)

Histological alterations in kidney tissue were evaluated byconventional PAS andMassonrsquos trichrome staining and by thepresence of the renal damage marker 120572-SMA by immuno-histofluorescence PAS stained sections were analyzed twoand eight weeks after MSC administration kidneys fromDM mice showed increased extracellular matrix depositionand glomerular hypertrophy (an increase in renal corpusclearea and glomerular tuft area) compared with kidneys fromnormal mice (Figures 1(a) 1(b) and 1(c)) Kidneys from DMmice also showed cytoplasmic vacuolation in cortical tubularcells (data not shown) By contrast kidneys from DM +MSCs mice showed decreased mesangial matrix depositionaccompanied with a reduction of the renal corpuscle area andglomerular tuft areamaintaining normal values (Figures 1(a)1(b) and 1(c)) However tubular cytoplasmic vacuolationswere still observed in the kidneys of DM + MSCs mice (datanot shown)

The renal damage marker 120572-SMA has been used as anearlymarker of glomerulosclerosis [33 40ndash42] In accordancewith previous reports immunohistofluorescence signal for120572-SMA was not detected in normal glomeruli except forsmoothmuscle cells of efferent and afferent arterioles [14 33]Compared with normal mice the percentage of glomeruliwith prominent signal for 120572-SMA in the mesangial area wasgradually increased in the kidney of DM mice leading toan increase in the glomerulosclerosis index (Figures 2(a)and 2(b)) However the percentage of glomeruli with 120572-SMA immunoreactivity in the kidneys of DM + MSCs micewas significantly reduced at every time point leading to areduction in the glomerulosclerosis index (Figures 2(a) and2(b)) Moreover the mRNA level of TGF-120573 a proscleroticcytokine was significantly reduced two weeks after MSCadministration compared with DMmice (Figure 2(c))

Eight weeks after diabetes induction Massonrsquos trichromicstained sections from DM mice showed morphologicalalterations which are signal of chronic damage such asabundant collagen fiber deposition loss of brush borderflattening of the epithelia presence of tubular detritus anda large quantity of dilated tubules compared with normalmice (Figure 2(d)) On the other hand kidney sections fromDM + MSCs mice showed more conserved morphologiesand a significant reduction in the fibrotic area comparedwith DM mice (Figures 2(d) and 2(e)) This reductionwas also reflected in the mRNA levels of some profibroticmolecules qRT-PCR studies showed that the expressionlevels of collagen type I and fibronectin in renal tissue fromDMmice were remarkably increased compared with normalmice (Figure 2(f)) Laminin 1205731 expression was also increasedin the kidneys from DM mice however this differencedid not reach a statistical significance (Figure 2(f)) Allthese profibrotic markers showed a tendency to decrease in

the kidney of DM + MSCs mice compared with DM mice(Figure 2(f)) suggesting that renal fibrosis was reduced afterMSC administration

33 MSC Administration Prevents Renal UltrastructuralChanges At the ultrastructural level normal kidneys showedscanty amount of extracellular matrix around mesangialcells and glomerular basement membrane (GBM) with athickness between 150 and 180 nm (Figures 3(a) and 3(b))Furthermore the kidneys from DM mice showed markedwidening of the GBM (range between 230 and 625 nm) withsegmental ldquohump-likerdquo localized protrusions of the capillaryloops along with up to 35 foot process effacement of thepodocytes and a significantly increased in mesangial matrixdeposition (Figures 3(c) and 3(d)) By contrast kidneysfrom DM + MSCs mice showed less extracellular mesangialexpansion than DM mice and normal thickness of the GBMand persistent protrusions of the subepithelial side of theGBM (Figures 3(e) and 3(f)) Thus MSC administrationpreserves normal renal ultrastructure

34 MSC Administration Maintains the Integrity of theGlomerular Filtration Barrier The damage caused to the fil-tration barrier triggers a slow and inexorable decline in renalfunction that can ultimately result in renal failure To identifythemolecular basis of the renoprotection observed afterMSCadministration we evaluated the glomerular capillary lumenwe performed immunostaining for WT-1 and podoplaninand we measured the expression of nephrin which are allmarker indicatives of the status of the glomerular filtrationbarrier

The ratio of glomerular capillary lumen areaglomerulartuft area was calculated at the end of the study period Innormal mice the glomeruli capillary network was stronglylabeled whereas capillary lumen area was greatly reduced inthe glomeruli of DMmice By contrast the glomeruli of DM+ MSCs mice maintained a strong capillary network similarto normal mice (Figures 4(a) and 4(d))

Podoplanin is localized on cell membranes of podocytesand is involved in the maintenance of the highly specializedstructure of the podocytes and its foot processes whichis essential to the normal functioning of the glomerularfiltration barrier [34] Since quantitative western blottingof glomerular protein extract would not show subtle seg-mental changes in the expression of podoplanin we per-formed immunohistofluorescence to detect the glomerularexpression of this marker At the end of the follow upthere was a focal and segmental loss of podoplanin proteinin the glomeruli of DM mice while MSC administrationsignificantly attenuated this loss (Figures 4(b) and 4(e))Additionally the number of podocytes per glomeruli (eval-uated by WT-1 staining) was reduced in DM mice howeverthis difference did not reach statistical significance (datanot shown) however more important than the podocytenumber per se is the podocyte number in relation to theterritory that each podocyte has to cover Accordingly theglomerular volume per podocyte was significantly greater inthe glomeruli of DM mice compared with normal animals


Normal DM2

wee

ks

after

MSC

adm

inist

ratio

n8

wee

ks

after

MSC

adm

inist

ratio

n

VP

VP VP

VP

VPVP

DM + MSCs

(a)

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

2 weeks after MSCadministration


NormalDM

DM + MSCs

lowast

lowast

Rena

l cor

pusc

le ar

ea (120583

m2)

(b)

2000

2500

3000

3500

4000

4500

5000



NormalDM

DM + MSCs

lowast

lowast

Glo

mer

ular

tuft

area

(120583m

2)

(c)

Figure 1 MSC administration reduces glomerular hypertrophic alterations Representative photomicrographs of kidney sections stainedwith PAS emphasizing in the glomeruli area two and eight weeks after MSC administration (a) Quantitative analysis of renal corpuscle area(b) and glomerular tuft area (c) two and eight weeks after MSC administration Data are presented as mean plusmn SEM 119899 = 8 lowast119901 lt 005 versusNormal for the same time point Bar = 25 120583m

while in the glomeruli of DM + MSCs mice the glomerularvolume per podocyte was significantly reduced comparedwith DMmice (Figures 4(c) and 4(f))

Finally we evaluated the expression of nephrin whichis the principal protein involved in the formation of slitdiaphragm in the filtration barrier [43 44] In line with theabove results qRT-PCR analysis showed that MSC adminis-tration significantly attenuated the diabetic-induced decreaseof nephrin expression (Figure 4(g))

35 Administered MSCs Do Not Massively Integrate intothe Kidneys In order to evaluate if the therapeutic effectobserved after MSC administration could be related to theintegration of MSCs into the damaged kidney structuresthe renal homing ofMSCsGFP was assessed inDNand normalmice two weeks and eight weeks after MSC administrationImmunohistofluorescence for GFP demonstrated that inDM mice at both time points only few MSCsGFP werepresent in the kidneymainly around renal tubulointerstitium


Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

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ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

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NormalDM

DM + MSCs

lowastlowast

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(b)

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24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

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NormalDM

DM + MSCs

lowast

(e)

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llage

n Im

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ectin

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NormalDM

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DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)

Figure 2 MSC administration prevents renal fibrosis Representative photomicrographs of kidney sections showing 120572-SMA immunoreac-tivity in glomeruli two and eight weeks after MSC administration (a) Quantitative analysis of glomerulosclerosis index evaluated by thepercentage of glomeruli exhibiting prominent signal for 120572-SMA in the mesangial area (b) TGF-120573 mRNA levels in renal tissue determinedby qRT-PCR (c) Representative photomicrographs of kidney sections stained with Massonrsquos trichrome showing deposition of collagen eightweeks after MSC administration (d) Quantitative analysis of collagen I deposition evaluated by point-counting method (e) mRNA level ofcollagen type I fibronectin and laminin 1205731 quantified by qRT-PCR eight weeks after MSC administration (f) Data are presented as mean plusmnSEM 119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for the same time point Bar = 150120583m

(Figures 5(a) and 5(b)) No MSCsGFP were found in thekidneys of normal mice (data not shown)

36 MSC Administration Triggers a Proregenerative Microen-vironment To understand if the therapeutic effects observedafter MSC administration could be related to the generationof a proregenerative microenvironment the rate of cellproliferation in the kidneys was determined by the quantifi-cation of the nuclear expression of Ki-67 and glomerular and

tubular sections were analyzed independently The glomeruliof DM + MSCs mice presented higher proliferation ratetwo weeks after MSC administration compared with normaland DM mice (Figure 6(a) and Supplementary Figure 1) Attubular level DM mice showed a significant increase in theproliferation rate comparedwith normalmice probably asso-ciated with a compensatory response to chronic injury Nev-ertheless the administration of MSCs significantly increasedthis response (Figure 6(b) and Supplementary Figure 1)


Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs

Figure 3MSC administration prevents renal ultrastructural alterations Electron photomicrographs of representative glomeruli fromnormalmice (a and b) DMmice (c and d) and DM+MSCsmice (e and f) showing marked (c) and moderate (f) increase of extracellular mesangialmatrix overt irregularities and thickening of the GBM (d) and persistent ldquohump likerdquo protrusions (f) Picture magnification (a) (c) and (e)times6000 (b) (d) and (f) times16500 Bars magnifications (a) (c) and (e) 2000 nm (b) (d) and (f) 1000 nm

The apoptotic index was evaluated in glomerular andtubular sections As expected DM mice showed a markedincrease in the number of apoptotic nuclei in both areas(Figures 6(c) and 6(d) and Supplementary Figure 2) Never-theless this increase was significantly reduced in the kidneyof DM + MSCs mice after two weeks and eight weeks afterMSC administration

MSCs are known to produce both in vitro and in vivoa broad range of trophic factors that have been associatedwith tissue regeneration by induction of proliferation andreduction of apoptosis [45] Systemic bFGF and EGF levelswere markedly reduced in DM mice compared with normalmice while the administration of MSCs restored the normalplasmatic level of both regenerative factors (bFGF two weeksafter MSC administration and EGF two and eight weeks afterMSC administration) (Figures 7(a) and 7(b)) Plasmatic HGFlevels were increased both in DM mice and DM + MSCsmice comparedwith normalmice (Figure 7(c)) and plasmaticlevels of VEGF and ET1 were unchanged in any experimentalcondition (data not shown)

The renal tissue has high regeneration capacity associatedat least in part to the secretion of trophic factors [46] Toevaluate if MSC administration could induce a proregenera-tivemicroenvironment the local expression of trophic factorswas evaluated by qRT-PCR In accordance with plasmaticlevels the renal expression of EGF decreased significantly indiabetic animals and MSC administration restored normalEGF mRNA levels (Figure 7(e)) Additionally diabetic micepresented highmRNA levels of bFGF andHGF in the kidney

however MSC administration significantly enhanced thisresponse (Figures 7(d) and 7(f))

37 MSC Administration Reduces Oxidative Stress DamageOxidative stress plays an important role at both early and latestages of DN [6] and it has been postulated that MSCs couldefficiently scavenge reactive species [47] Total oxidativespecies were evaluated in renal tissue two and eight weeksafter MSC administration The diabetic condition induced asignificant increase in the amount of total oxidative speciesin the kidney that was significantly reduced eight weeksafter MSC administration (Figure 8(a)) The reduction inROS level was correlated with a significant reduction inlipid peroxidation level (Figure 8(b)) and protein oxidativedamage (Figure 8(c)) By contrast theDNAoxidative damageremained significantly elevated irrespective of MSC adminis-tration (Figure 8(d))

Total antioxidant capacity was reduced in DMmice witha trend to be higher in the treatedmice eight weeks afterMSCadministration (Figure 8(e))

38 MSC Administration Prevents Renal Inflammation Theinflammatory influx is present in the diabetic kidney and isone of the driving forces to induce tissue damage [48 49]Multifocal inflammatory cellular foci were observed in thekidney of DM mice (Figure 9(a)) By contrast no leukocyteinfiltration foci were observed in the kidney of DM + MSCsmice (Figure 9(a)) Since macrophages account for most ofthe infiltrating leukocytes (gt90) [30] and they correlate


Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)

Figure 4 MSC administration maintains the integrity of the glomerular filtration barrier Representative photomicrographs of kidneysections eight weeks after MSC administration showing glomerular capillary lumen area (green) labeled by the endovenous administrationof 2 times 106MW FITC-dextran (a) glomerular immunoreactivity of podoplanin (red) to evaluate podocyte foot processes integrity (b) andglomerular immunoreactivity of WT-1 (nuclei in red) to evaluate glomerular podocytes (white arrows) (c) In all cases total nuclei werecounterstainedwithDAPI (blue) Quantitative analysis of capillary lumen area normalized by glomerular tuft area (d) glomerular podoplaninarea normalized by glomerular tuft area (e) and glomerular volume per podocyte (f) mRNA level of nephrin quantified by qRT-PCR (g)Data are presented as mean plusmn SEM 119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for the same time point Bar = 25120583m

2 weeks after MSC administration

(a)


(b)

Figure 5 Administered MSCs do not massively integrate into the kidney structure Two and eight weeks after MSC administration MSCGFP

were localized by confocal microscopy Representative photomicrographs of kidney sections form DM mice showing the localization ofMSCsGFP white arrows indicate donor cells immunolabeled with anti-GFP-Alexa555 (red) cell nuclei were counterstain with DAPI (blue)Bars = 15120583m

with increased levels of albuminuria and plasma creatinine[50 51] F480 cells in renal tissue were evaluated In accor-dance with the above result diabetic animals presented anincreased number of macrophages per sections and this was

significantly reduced by MSC administration (Figures 9(b)and 9(c))

MSCs have been recognized as immunomodulatory cellswith potent immunosuppressive properties [13] Based on


00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

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12

16

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32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

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1

2

3

4

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lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)

Figure 6 MSC administration induces kidney tissue regeneration Quantitative analysis of the Ki-67 proliferation index in glomeruli (a) andtubules (b) two and eight weeks after MSC administration Quantitative analysis of the apoptotic index in glomeruli (c) and tubules (d) twoand eight weeks after MSC administration Data are presented as mean plusmn SEM 119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for thesame time point

these properties we measured the serum levels and the renalexpression of cytokines related to proinflammatory and anti-inflammatory responses

The administration ofMSCs did not change the plasmaticlevel of the proinflammatory cytokines TNF-120572 and IL-1120573(data not shown) However MSC-treated mice showed aplasmatic cytokine profile compatible with a Th2 polarizedimmune response since plasmatic levels of IL6 IL-4 and IL-10 were restored comparedwithDMmice (Figures 9(d) 9(e)and 9(f))

The same pattern was observed in renal tissues sinceMSC-treated animals showed a renal cytokine expression

profile compatible with a Th2 polarized immune responsewith a high expression of IL-4 and IL-10 (Figures 9(g) and9(h)) Therefore MSC administration induces a protectivemicroenvironment

4 Discussion

The major findings of this study were that MSC adminis-tration preserved renal function and ameliorate histopatho-logical alterations in an animal model of severe diabetesmellitus The possible mechanisms underlying these effectsinvolved the temporal inhibition of the TGF-120573 expression


lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

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lowast

2 weeks post-MSCadministration


NormalDM

DM + MSCs

(f)

Figure 7MSC administration increases local and systemic levels of proregenerative factors Quantitative analysis of plasmatic levels of bFGF(a) EGF (b) and HGF (c) two and eight weeks after MSC administration determined by Luminex Multiplex system Renal mRNA levels ofbFGF (d) EGF (e) and HGF (f) quantified by qRT-PCR two and eight weeks after MSC administration Data are presented as mean plusmn SEM119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for the same time point

and the triggered of a proregenerative microenvironmentwhich include the production of protective trophic factorsand the reduction of both the oxidative stress damage and theproinflammatory response in the kidney

Using an animal model of nonimmunological severediabetes mellitus induced by the administration of a singlehigh dose of STZ (200mgkg) we previously showed thatthe administration of multiple doses of MSCs was able toprevent albuminuria development despite the persistence ofhyperglycaemia [21] suggesting a direct effect of MSCs inthe prevention of renal damage In the present study usingthe same animal model we further characterize the effects ofMSC administration on renal function and structure along

with the possiblemechanisms associatedwith the therapeuticeffects

MSC administration effectively reduced urinary albuminexcretion and plasmatic BUN and creatinine levels Thepreservation of renal function lasted at least up to the endof the follow-up study (eight weeks after MSC adminis-tration) This improvement in renal function was clearlyassociated with the preservation of renal structure sinceuntreated diabetic mice presented glomerular hypertrophymesangial expansion tubular dilatation glomerulosclerosistubulointerstitial fibrosis and GBM thickening while theadministration ofMSCs prevented all these histopathologicalalterations


0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast

lowast lowast lowast



Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)

Figure 8 MSC administration reduces oxidative stress Quantitative analysis of total reactive oxygen species expressed as total ROS per mgof kidney proteins (a) lipid peroxidation level expressed as pg of urinary 8-iso-PFG2120572 per mg of urinary creatinine (b) oxidative proteindamage level expressed as ng of 3-nitrotyrosine per 120583g of kidney proteins (c) oxidative DNAdamage level expressed as ng of urinary 8-OHdGper mg of urinary creatinine (d) and total antioxidant capacity expressed as mM of Trolox equivalents per mg of kidney proteins (e) two andeight weeks after MSC administration Data are presented as mean plusmn SEM 119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for thesame time point


Normal

DM

scoreLeucocyte infiltration

DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)

Figure 9 MSC administration prevents renal inflammation Representative photomicrographs of kidney sections showing inflammatoryinfiltrates (white arrow) eightweeks afterMSCadministration Bar = 100120583m(a) Representative photomicrographs of kidney sections showingmacrophages (white arrows) (F480-red) eight weeks after MSC administration Barr = 40 120583m (b) Nuclei were counterstained with DAPI(blue) Quantitative analysis of macrophages per tissue section eight weeks after MSC administration (c) Quantitative analysis of plasmaticlevels of the anti-inflammatory cytokines IL-6 (d) IL-10 (e) and IL-4 (f) determined by the Luminex Multiplex system two and eight weeksafterMSCadministration RenalmRNA levels of IL-10 (g) and IL-4 (h) quantified by qRT-PCR two and eightweeks afterMSCadministrationData are presented as mean plusmn SEM 119899 = 8 lowast119901 lt 005 versus Normal 119901 lt 005 versus DM for the same time point

TGF-120573 upregulation is considered one of the mainfactors involved in the development and progression ofDN [8] It stimulates renal cell hypertrophy and extra-cellular matrix deposition which causes GBM thickeningglomerular capillary occlusion and may promote podocyteapoptosis or detachment [35] In the present severe diabetesmellitus model we observed an early reduction in the renal

expression of TGF-1205731 after MSC administration suggestingthat glomerulosclerosis and renal interstitial fibrosis wereefficiently prevented The same observation was made indiabetic spontaneous hypertensive rats where the inhibitionof TGF-120573 expression by Tranilast an antiallergic drug thatimpedes the release of cytokines from immune cells led to areduction in glomerular hyperfiltration and glomerular tuff


area expansion despite persistent hyperglycaemia [52] In thesame line in vitro and in vivo studies showed thatMSCs couldinhibit the upregulation of TGF-120573 expression stimulated byhigh glucose in mesangial cells by the secretion of trophicfactors [53] Thus we can assume that the early modulationof TGF-120573 expression after MSC administration is related atleast in part to a reduction in kidney fibrosis

Under diabetic condition high glucose levels compro-mise the integrity of the glomerular filtration barrier viaeffacement of the podocyte foot processes and loss ofpodocytes from the glomerular basement membrane Thedamage caused to the filtration barrier triggers a slow andinexorable decline in renal function that can ultimatelyresult in renal failure [54ndash56] Podoplanin is a glycopro-tein expressed on the base of podocyte foot processes andis involved in the maintenance of the highly specializedstructure of the podocytes and its foot processes duringhypertrophy [34] In great contrast to the general pattern ofmost of the podocyte-associated proteins podoplanin hasshowed changes in expressionwith clear temporal associationto the development of proteinuria [57] In the same waynephrin is the principal protein involved in the formationof slit diaphragm of the filtration barrier [43 44 58 59] Toidentify the molecular basis of the renoprotection observedafter MSC administration we performed immunostainingfor WT-1 and podoplanin and evaluated the renal expressionof nephrin which are indicatives of the status of the glomeru-lar filtration barrier At the end of the study period untreatedmice showed a significant increase in the glomerular volumeper podocyte that was accompanied by focal and segmentalloss of glomerular podoplanin protein expression whileMSC administration restored the normal level of podoplaninnephrin and the normal glomerular volume per podocyte

It has been hypothesized that the expansion of theglomerular tuft requires the adaptation of the glomerularepithelial cells to cover a wide area of glomerular capillarywall a process that requires the reorganization of the actincytoskeleton and a higher podocyte workload The increasedworkload may lead to podocyte damage and eventuallypodocyte loss [60] Several studies have reinforced thepoint that the primary issue of concern is probably notthe podocyte number per se but the podocyte number inrelation to the territory that each podocyte has to coverdesignated as glomerular volume per podocyte [61ndash64] Theglomerular hypertrophy observed in DM mice added tothe diabetic microenvironment could result in an increasedpodocyte workload followed by the loss of the filtrationbarrier integrity and the development of albuminuria Bycontrast the preservation of the integrity of the glomerularfiltration barrier in the kidney of MSC-treated mice could beassociated to the reduction of glomerular hypertrophy andthe production of trophic factors According to this in vitrostudies showed that human adipose-derived MSCs couldprevent podocyte injury induced by high glucose mainlythrough secreting EGF [65] Additionally MSCs contributeto podocyte regeneration by trophic factor secretion whenadministered in an animal model of Alport syndrome [66]

In this study we found that after MSC administrationa small amount of donor cells were present in the kidney

mainly in the tubulointerstitial space The selectivity of thiscell recruitment was supported by the fact that no donor cellswere detected whenMSCsGFP were administered into normalmice Thus in agreement with already published data MSCrecruitment to kidney depends on the presence of injury [67]Therapeutics effects with reduced MSCs homing have beenreported in other studies of tissue damage [24 68ndash71] Soit is likely reasonable to speculate that MSCs protected thediabetic kidney primarily via paracrine mechanisms

In our study the improvement of functional parameterswas associatedwith an acceleration of the regeneration phaseas demonstrated by higher amounts of positive Ki-67 nucleiand increased expression of trophic factors in the kidney ofMSC-treated mice Additionally this recovery was correlatedwith an important inhibition of the apoptotic process

It is well recognized that MSCs can secrete a broad rangeof trophic factors such as HGF bFGF IGF-1 and EGF allknown to improve the renal function in animal models ofkidney injury due to their antiapoptotic mitogenic andmorphogenic activities on intrarenal cells [45 66] Basedon these observations we speculate that the mechanismsthat mediate protective effects after MSC administrationmight be primarily related to the paracrine secretion of thesefactors creating a proregenerative microenvironment thatcould induce the endogenous regeneration of the tissue

In our severe diabetic animalmodelwe showed a systemicincrease in the plasmatic levels of bFGF (two weeks afterMSC administration) and EGF and HGF (2 and 8 weeksafter MSC administration) and also a local increase in theexpression levels of these proregenerative factors Both invitro and in vivo experiments EGF can reduce apoptosis andinduce the repair of the damage renal tubular epithelial cells[72] According to this it has been found in an in vitro studythat human adipose-derived MSCs could prevent podocyticapoptosis induced by high glucose mainly through secretingEGF [65] and thatMSCs contribute to podocyte regenerationby trophic factor secretion when administered in an animalmodel of Alport syndrome [66] Moreover bFGF can alsoinhibit the apoptotic process and accelerate renal tissueregeneration when is exogenously administrated [73] Theeffect of bFGF on renal injury is particularly important sinceit has been suggested that bFGF can induce the expressionof several tubulogenicepitheliogenic and angiogenic proteinslike Pax-2 bone morphogen protein-7 Noggin Smad 1-5-8p-Smad and hypoxia-inducible factor-1 which in turn areresponsible for kidney regeneration [73] On the other handrenal damage is exacerbated when bFGF is inhibited [74]

Glucose is the primary fuel source for ROS generation[6] and it is known that oxidative stress condition increasethe vulnerability of renal cells [6] We showed that afterMSC transplantation renal cell death and lipid peroxidationwere significantly reduced despite the sustained high bloodglucose levels

There is growing evidence indicating that MSCs can effi-ciently control oxidative stress Previously we have demon-strated that the low susceptibility of MSCs to the harmfuleffect of ROS correlates with the ability of these cells to effec-tively scavenge peroxide and peroxynitrite due to constitutive


expression of the enzymes superoxide dismutases 1 and 2catalase and glutathione peroxidase [47] Moreover MSCspossess the main enzymatic mechanisms to detoxify reactivespecies guaranteeing an efficient regulation of oxidativestress [75]

Previous reports of clinical and preclinical studies haveindicated that inflammatory cytokines play an impor-tant role in the onset and progression of DN in whichmacrophages infiltrate the kidney and produce a proin-flammatory microenvironment [5] In this sense it hasbeen demonstrated that anti-inflammatory treatments andimmunotherapy could attenuate podocyte injury renal fibro-sis and alleviate albuminuria [14 76] Here we showed thatthe number of infiltrating macrophages in diabetic mice wasmarkedly reduced after MSC administration [13] Addition-ally at molecular level a significant reduction in the plasmaticlevels of IL-4 IL-10 and IL-6 was observed in untreated dia-betic mice compared with normal animals These levels werenormalized inMSC-treatedmice IL-4 and IL-10 are themainanti-inflammatory and immunosuppressive cytokines whichare produced by several types of immune cells such as regu-latory T cells and Th2 lymphocytes [5] These cytokines playa key role in the regulation of immune responses acting aspotent deactivator of the synthesis of monocytemacrophageproinflammatory cytokine and inhibiting leukocyte infil-tration inflammation and tissue damage [14] In differentanimalmodels it has been reported thatMSC administrationreduces macrophage infiltration into the target tissues [1477] and switches macrophages from M1 to M2 phenotypecontributing to tissue homeostasis [78] MSCs can secrete IL-4 and IL-10 generating a protective microenvironment thatmight avoid the immunological destruction of renal cells [9]Strikingly plasmatic IL-6 reached normal levels two weeksafter MSC administration This fast recovery may be due inpart to the release of IL-6 by the administered MSCs IL-6 isa pleiotropic cytokine that regulates immune responses andinflammatory reactions Djouad et al showed that murineMSCs secrete high level of IL-6 and this level is directlycorrelated to the inhibition of the proliferation of T-cells [79]

In the kidney of MSC-treated mice we also observeda skewing toward a Th2 profile as demonstrated by theincreased expression of IL-4 and IL-10 The local attenuationof inflammation has been associated to a reduction in fibrosisand the generation of a protective microenvironment thatsupports the tissue regeneration [13 14 77 80]

5 Conclusions

In summary our study provides further evidence that MSCscan not only exert antialbuminuric effects but also preventglomerular capillary occlusion and maintain podocyte den-sity and foot process in the glomerular filtration barrier Inthis animal model of severe diabetes mellitus the improve-ment of the glomerular function and the morphologicalrecoveries observed after MSC administration could bemediated at least in part by the promotion of endogenousregeneration of the kidney tissue due to changes in thelocal microenvironment and the inhibition of oxidative stressdamage and inflammatory reactions skewing aTh2 profile

We think that the successful treatment of DN withMSCs demonstrated here holds a substantial promise forthe development of novel MSC-based interventions that canprevent the progression of DN These findings should beconfirmed in clinical trial since MSC transplantations inhuman patients is feasible and safe

Abbreviations

Alb AlbuminCrea CreatininebFGF Basic fibroblast growth factorBUN Blood urea nitrogenDAPI 410158406-Diamino-2-fenilindolDM Severe diabetes mellitusDN Diabetic nephropathyEGF Epidermal growth factorGAPDH Glyceraldehyde-3-phosphate dehydrogenaseGBM Glomerular basement membraneGFP Green fluorescent proteinHGF Hepatocyte growth factorMSCs Mesenchymal stem cellsNphs NephrinPAS Periodic acid SchiffPBS Phosphate-buffered salineROS Reactive oxygen speciesSTZ StreptozotocinTGF-120573 Transforming growth factor 120573TUNEL Terminal uridine nucleotide end-labelingVP Vascular poleWT-1 Wilmsrsquo tumor protein 1120572-SMA Smooth muscle actin 120572

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thank Dr Amelina Albornoz for the Englishediting of the paper This work was supported by GrantFondecyt Regular [1120133] to Marcelo Ezquer

References

[1] A J Collins R N Foley C Herzog et al ldquoUS renal datasystem2012 annual data reportrdquoTheAmerican Journal of KidneyDiseases vol 61 no 1 supplement 1 p A7 2013

[2] Y-M Sun Y Su J Li and L-F Wang ldquoRecent advances inunderstanding the biochemical and molecular mechanism ofdiabetic nephropathyrdquo Biochemical and Biophysical ResearchCommunications vol 433 no 4 pp 359ndash361 2013

[3] A K H Lim ldquoDiabetic nephropathymdashcomplications and treat-mentrdquo International Journal of Nephrology and RenovascularDisease vol 7 pp 361ndash381 2014

[4] K E White ldquoResearch into the glomerular podocytemdashis itrelevant to diabetic nephropathyrdquo Diabetic Medicine vol 23no 7 pp 715ndash719 2006


[5] A A Elmarakby and J C Sullivan ldquoRelationship betweenoxidative stress and inflammatory cytokines in diabeticnephropathyrdquo Cardiovascular Therapeutics vol 30 no 1 pp49ndash59 2012

[6] J M Forbes M T Coughlan and M E Cooper ldquoOxidativestress as a major culprit in kidney disease in diabetesrdquoDiabetesvol 57 no 6 pp 1446ndash1454 2008

[7] S-I Yamagishi S Maeda T Matsui S Ueda K Fukami andS Okuda ldquoRole of advanced glycation end products (AGEs)and oxidative stress in vascular complications in diabetesrdquoBiochimica et Biophysica Acta vol 1820 no 5 pp 663ndash671 2012

[8] F N Ziyadeh ldquoMediators of diabetic renal disease the case forTGF-120573 as the major mediatorrdquo Journal of the American Societyof Nephrology vol 15 supplement 1 pp S55ndashS57 2004

[9] F Togel and C Westenfelder ldquoAdult bone marrow-derivedstem cells for organ regeneration and repairrdquo DevelopmentalDynamics vol 236 no 12 pp 3321ndash3331 2007

[10] A I Caplan ldquoThe mesengenic processrdquo Clinics in PlasticSurgery vol 21 no 3 pp 429ndash435 1994

[11] F Anglani M Forino D del Prete E Tosetto R Torregrossaand A DrsquoAngelo ldquoIn search of adult renal stem cellsrdquo Journalof Cellular and Molecular Medicine vol 8 no 4 pp 474ndash4872004

[12] S Sedrakyan S Angelow R E de Filippo and L Perin ldquoStemcells as a therapeutic approach to chronic kidney diseasesrdquoCurrent Urology Reports vol 13 no 1 pp 47ndash54 2012

[13] P Semedo C G Palasio C D Oliveira et al ldquoEarly modulationof inflammation by mesenchymal stem cell after acute kidneyinjuryrdquo International Immunopharmacology vol 9 no 6 pp677ndash682 2009

[14] P Semedo M Correa-Costa M A Cenedeze et al ldquoMes-enchymal stem cells attenuate renal fibrosis through immunemodulation and remodeling properties in a rat remnant kidneymodelrdquo Stem Cells vol 27 no 12 pp 3063ndash3073 2009

[15] G C Davey S B Patil A OrsquoLoughlin and T OrsquoBrienldquoMesenchymal stem cell-based treatment for microvascularand secondary complications of diabetes mellitusrdquo Frontiers inEndocrinology vol 5 article 86 2014

[16] F E Ezquer M E Ezquer D B Parrau D Carpio A J Yanezand P A Conget ldquoSystemic administration of multipotentmesenchymal stromal cells reverts hyperglycemia and preventsnephropathy in type 1 diabetic micerdquo Biology of Blood andMarrow Transplantation vol 14 no 6 pp 631ndash640 2008

[17] R H Lee M J Seo R L Reger et al ldquoMultipotent stromalcells from human marrow home to and promote repair ofpancreatic islets and renal glomeruli in diabetic NODscidmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 46 pp 17438ndash17443 2006

[18] S Lv J Cheng A Sun et al ldquoMesenchymal stem cells transplan-tation ameliorates glomerular injury in streptozotocin-induceddiabetic nephropathy in rats via inhibiting oxidative stressrdquoDiabetes Research and Clinical Practice vol 104 no 1 pp 143ndash154 2014

[19] J Ouyang G Hu Y Wen and X Zhang ldquoPreventive effects ofsyngeneic bone marrow transplantation on diabetic nephropa-thy in micerdquo Transplant Immunology vol 22 no 3-4 pp 184ndash190 2010

[20] H Zhou H-M Tian Y Long et al ldquoMesenchymal stemcells transplantation mildly ameliorates experimental diabeticnephropathy in ratsrdquo Chinese Medical Journal vol 122 no 21pp 2573ndash2579 2009

[21] F Ezquer M Ezquer V Simon et al ldquoEndovenous admin-istration of bone-marrow-derived multipotent mesenchymalstromal cells prevents renal failure in diabetic micerdquo Biology ofBlood andMarrowTransplantation vol 15 no 11 pp 1354ndash13652009

[22] M Katoh Y Ohmachi Y Kurosawa H Yoneda N Tanaka andH Narita ldquoEffects of imidapril and captopril on streptozotocin-induced diabetic nephropathy in micerdquo European Journal ofPharmacology vol 398 no 3 pp 381ndash387 2000

[23] Y-C Tay Y Wang L Kairaitis G K Rangan C Zhang and DC H Harris ldquoCan murine diabetic nephropathy be separatedfrom superimposed acute renal failurerdquo Kidney Internationalvol 68 no 1 pp 391ndash398 2005

[24] F Ezquer M Ezquer D Contador M Ricca V Simonand P Conget ldquoThe antidiabetic effect of mesenchymal stemcells is unrelated to their transdifferentiation potential but totheir capability to restore Th1Th2 balance and to modify thepancreatic microenvironmentrdquo Stem Cells vol 30 no 8 pp1664ndash1674 2012

[25] M Ezquer F Ezquer M Ricca C Allers and P Conget ldquoIntra-venous administration of multipotent stromal cells preventsthe onset of non-alcoholic steatohepatitis in obese mice withmetabolic syndromerdquo Journal of Hepatology vol 55 no 5 pp1112ndash1120 2011

[26] B D Kubiak S P Albert L A Gatto et al ldquoPeritoneal negativepressure therapy prevents multiple organ injury in a chronicporcine sepsis and ischemiareperfusion modelrdquo Shock vol 34no 5 pp 525ndash534 2010

[27] J Ma H Nishimura A Fogo V Kon T Inagami and IIchikawa ldquoAccelerated fibrosis and collagen deposition developin the renal interstitium of angiotensin type 2 receptor nullmutantmice during ureteral obstructionrdquoKidney Internationalvol 53 no 4 pp 937ndash944 1998

[28] D Sheehan and B HrapchakTheory and Practice of Histotech-nology Battelle Press Columbus Ohio USA 1980

[29] A Benigni C Zoja C Zatelli et al ldquoVasopeptidase inhibitorrestores the balance of vasoactive hormones in progressivenephropathyrdquoKidney International vol 66 no 5 pp 1959ndash19652004

[30] GHTesch ldquoMacrophages anddiabetic nephropathyrdquo Seminarsin Nephrology vol 30 no 3 pp 290ndash301 2010

[31] C E Alpers K L Hudkins A M Gown and R J JohnsonldquoEnhanced expression of lsquomuscle-specificrsquo actin in glomeru-lonephritisrdquo Kidney International vol 41 no 5 pp 1134ndash11421992

[32] Y Utsunomiya T Kawamura A Abe et al ldquoSignificanceof mesangial expression of 120572-smooth muscle actin in theprogression of IgA nephropathyrdquo American Journal of KidneyDiseases vol 34 no 5 pp 902ndash910 1999

[33] T J M Geleilete R S Costa M Dantas and T M CoimbraldquoAlpha-smooth muscle actin and proliferating cell nuclearantigen expression in focal segmental glomerulosclerosis func-tional and structural parameters of renal disease progressionrdquoBrazilian Journal of Medical and Biological Research vol 34 no8 pp 985ndash991 2001

[34] D H T Ijpelaar A Schulz K Koop et al ldquoGlomerular hyper-trophy precedes albuminuria and segmental loss of podoplaninin podocytes in Munich-Wistar-Fromter ratsrdquo The AmericanJournal of PhysiologymdashRenal Physiology vol 294 no 4 ppF758ndashF767 2008

[35] M Notoya T Shinosaki T Kobayashi T Sakai and HKurihara ldquoIntussusceptive capillary growth is required for


glomerular repair in ratThy-11 nephritisrdquoKidney Internationalvol 63 no 4 pp 1365ndash1373 2003

[36] A Salahudeen V Poovala W Parry et al ldquoCisplatin inducesN-acetyl cysteine suppressible F2 isoprostane production andinjury in renal tubular epithehal cellsrdquo Journal of the AmericanSociety of Nephrology vol 9 no 8 pp 1448ndash1455 1998

[37] DA Butterfield andCM Lauderback ldquoLipid peroxidation andprotein oxidation in Alzheimerrsquos disease brain potential causesand consequences involving amyloid 120573-peptide-associated freeradical oxidative stressrdquo Free Radical Biology and Medicine vol32 no 11 pp 1050ndash1060 2002

[38] H Zhou A Kato T Miyaji et al ldquoUrinary marker for oxidativestress in kidneys in cisplatin-induced acute renal failure in ratsrdquoNephrology Dialysis Transplantation vol 21 no 3 pp 616ndash6232006

[39] R Mahfouz R Sharma D Sharma E Sabanegh and AAgarwal ldquoDiagnostic value of the total antioxidant capacity(TAC) in human seminal plasmardquo Fertility and Sterility vol 91no 3 pp 805ndash811 2009

[40] T Shimo M Ohshita J Katayama et al ldquoSignificance ofintraglomerular expression of 120572-smooth muscle actin desminand PDGF receptor on glomerulosclerosis in the rat remnantkidney modelrdquo Journal of Toxicologic Pathology vol 11 no 4pp 229ndash234 1998

[41] J Floege M W Burns C E Alpers et al ldquoGlomerular cellproliferation and PDGF expression precede glomerulosclerosisin the remnant kidney modelrdquo Kidney International vol 41 no2 pp 297ndash309 1992

[42] Y Kaneko K Nakazawa M Higuchi K Hora and HShigematsu ldquoGlomerular expression of 120572-smooth muscle actinreflects disease activity of IgA nephropathyrdquo Pathology Interna-tional vol 51 no 11 pp 833ndash844 2001

[43] G IWelsh andM A Saleem ldquoNephrinmdashsignaturemolecule ofthe glomerular podocyterdquo Journal of Pathology vol 220 no 3pp 328ndash337 2010

[44] H Yuan E Takeuchi andD J Salant ldquoPodocyte slit-diaphragmprotein nephrin is linked to the actin cytoskeletonrdquoThe Amer-ican Journal of Physiology Renal Physiology vol 282 no 4 ppF585ndashF591 2002

[45] F Togel KWeiss Y Yang ZHu P Zhang andCWestenfelderldquoVasculotropic paracrine actions of infusedmesenchymal stemcells are important to the recovery from acute kidney injuryrdquoTheAmerican Journal of PhysiologymdashRenal Physiology vol 292no 5 pp F1626ndashF1635 2007

[46] M H Little ldquoRegrow or repair potential regenerative therapiesfor the kidneyrdquo Journal of the American Society of Nephrologyvol 17 no 9 pp 2390ndash2401 2006

[47] A Valle-Prieto and P A Conget ldquoHuman mesenchymal stemcells efficiently manage oxidative stressrdquo Stem Cells and Devel-opment vol 19 no 12 pp 1885ndash1893 2010

[48] J F Navarro-Gonzalez and C Mora-Fernandez ldquoThe role ofinflammatory cytokines in diabetic nephropathyrdquo Journal of theAmerican Society of Nephrology vol 19 no 3 pp 433ndash442 2008

[49] D Nguyen F Ping W Mu P Hill R C Atkins and S JChadban ldquoMacrophage accumulation in human progressivediabetic nephropathyrdquo Nephrology vol 11 no 3 pp 226ndash2312006

[50] F Y Chow D J Nikolic-Paterson R C Atkins andGH TeschldquoMacrophages in streptozotocin-induceddiabetic nephropathypotential role in renal fibrosisrdquo Nephrology Dialysis Transplan-tation vol 19 no 12 pp 2987ndash2996 2004

[51] F Y Chow D J Nikolic-Paterson E Ozols R C AtkinsB J Rollin and G H Tesch ldquoMonocyte chemoattractantprotein-1 promotes the development of diabetic renal injury instreptozotocin-treated micerdquo Kidney International vol 69 no1 pp 73ndash80 2006

[52] H Akahori T Ota M Torita H Ando S Kaneko and TTakamura ldquoTranilast prevents the progression of experimentaldiabetic nephropathy through suppression of enhanced extra-cellular matrix gene expressionrdquo Journal of Pharmacology andExperimental Therapeutics vol 314 no 2 pp 514ndash521 2005

[53] S Lv G Liu A Sun et al ldquoMesenchymal stem cells amelioratediabetic glomerular fibrosis in vivo and in vitro by inhibitingTGF-120573 signalling via secretion of bone morphogenetic protein7rdquoDiabetes and Vascular Disease Research vol 11 no 4 pp 251ndash261 2014

[54] M Danilewicz and M Wagrowska-Danilewicz ldquoMorphome-tric and immunohistochemical insight into focal segmentalglomerulosclerosis in obese and non-obese patientsrdquo Nefrolo-gia vol 29 no 1 pp 35ndash41 2009

[55] H Kawachi N Miyauchi K Suzuki D H Gi M Orikasaand F Shimizu ldquoRole of podocyte slit diaphragm as a filtrationbarrierrdquo Nephrology vol 11 no 4 pp 274ndash281 2006

[56] F N Ziyadeh and G Wolf ldquoPathogenesis of the podocytopathyand proteinuria in diabetic glomerulopathyrdquo Current DiabetesReviews vol 4 no 1 pp 39ndash45 2008

[57] K Koop M Eikmans M Wehland et al ldquoSelective loss ofpodoplanin protein expression accompanies proteinuria andprecedes alterations in podocyte morphology in a spontaneousproteinuric rat modelrdquoThe American Journal of Pathology vol173 no 2 pp 315ndash326 2008

[58] R G Langham D J Kelly A J Cox et al ldquoProteinuria and theexpression of the podocyte slit diaphragm protein nephrin indiabetic nephropathy effects of angiotensin converting enzymeinhibitionrdquo Diabetologia vol 45 no 11 pp 1572ndash1576 2002

[59] P Mundel and J Reiser ldquoProteinuria an enzymatic disease ofthe podocyterdquo Kidney International vol 77 no 7 pp 571ndash5802010

[60] J E Wiggins M Goyal S K Sanden et al ldquoPodocytehypertrophy lsquoadaptationrsquo and lsquodecompensationrsquo associated withglomerular enlargement and glomerulosclerosis in the agingrat prevention by calorie restrictionrdquo Journal of the AmericanSociety of Nephrology vol 16 no 10 pp 2953ndash2966 2005

[61] D B Bhathena ldquoGlomerular basement membrane length topodocyte ratio in human nephronopenia implications for focalsegmental glomerulosclerosisrdquo American Journal of KidneyDiseases vol 41 no 6 pp 1179ndash1188 2003

[62] A B Fogo ldquoGlomerular hypertension abnormal glomerulargrowth and progression of renal diseasesrdquoKidney InternationalSupplement vol 57 no 75 pp S15ndashS21 2000

[63] Y H Kim M Goyal D Kurnit et al ldquoPodocyte depletionand glomerulosclerosis have a direct relationship in the PAN-treated ratrdquo Kidney International vol 60 no 3 pp 957ndash9682001

[64] M E Pagtalunan P LMiller S Jumping-Eagle et al ldquoPodocyteloss and progressive glomerular injury in type II diabetesrdquoTheJournal of Clinical Investigation vol 99 no 2 pp 342ndash348 1997

[65] D Li NWang L Zhang et al ldquoMesenchymal stem cells protectpodocytes from apoptosis induced by high glucose via secretionof epithelial growth factorrdquo Stem Cell Research andTherapy vol4 no 5 article 103 2013


[66] E I Prodromidi R Poulsom R Jeffery et al ldquoBone marrow-derived cells contribute to podocyte regeneration and amelio-ration of renal disease in a mouse model of Alport syndromerdquoStem Cells vol 24 no 11 pp 2448ndash2455 2006

[67] Y Zhang C Ye G Wang et al ldquoKidney-targeted trans-plantation of mesenchymal stem cells by ultrasound-targetedmicrobubble destruction promotes kidney repair in diabeticnephropathy ratsrdquo BioMed Research International vol 2013Article ID 526367 13 pages 2013

[68] E BOliveira-Sales EMaquigussa P Semedo et al ldquoMesenchy-mal stem cells (MSC) prevented the progression of renovascularhypertension improved renal function and architecturerdquo PLoSONE vol 8 no 11 Article ID e78464 2013

[69] A I Caplan and J E Dennis ldquoMesenchymal stem cells astrophic mediatorsrdquo Journal of Cellular Biochemistry vol 98 no5 pp 1076ndash1084 2006

[70] D J ProckopD J Kota N Bazhanov andR L Reger ldquoEvolvingparadigms for repair of tissues by adult stemprogenitor cells(MSCs)rdquo Journal of Cellular andMolecularMedicine vol 14 no9 pp 2190ndash2199 2010

[71] Y Wang H E Johnsen S Mortensen et al ldquoChanges incirculating mesenchymal stem cells stem cell homing factorand vascular growth factors in patients with acute ST elevationmyocardial infarction treated with primary percutaneous coro-nary interventionrdquo Heart vol 92 no 6 pp 768ndash774 2006

[72] C Lu W Ren X-M Su J-Q Chen S-H Wu and G-PZhou ldquoEGF-recruited JunDc-fos complexes activate CD2APgene promoter and suppress apoptosis in renal tubular epithelialcellsrdquo Gene vol 433 no 1-2 pp 56ndash64 2009

[73] S Villanueva C Cespedes A Gonzalez and C P VioldquobFGF induces an earlier expression of nephrogenic proteinsafter ischemic acute renal failurerdquo The American Journal ofPhysiologymdashRegulatory Integrative and Comparative Physiologyvol 291 no 6 pp R1677ndashR1687 2006

[74] S Villanueva C Cespedes A A Gonzalez E Roessler and CP Vio ldquoInhibition of bFGF-receptor type 2 increases kidneydamage and suppresses nephrogenic protein expression afterischemic acute renal failurerdquo American Journal of PhysiologymdashRegulatory Integrative and Comparative Physiology vol 294 no3 pp R819ndashR828 2008

[75] W A Silva Jr D T Covas R A Panepucci et al ldquoThe profileof gene expression of human marrow mesenchymal stem cellsrdquoStem Cells vol 21 no 6 pp 661ndash669 2003

[76] 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

[77] S Villanueva E Ewertz F Carrion et al ldquoMesenchymal stemcell injection ameliorates chronic renal failure in a rat modelrdquoClinical Science vol 121 no 11 pp 489ndash499 2011

[78] W Li Q Zhang M Wang et al ldquoMacrophages are involvedin the protective role of human umbilical cord-derived stromalcells in renal ischemia-reperfusion injuryrdquo Stem Cell Researchvol 10 no 3 pp 405ndash416 2013

[79] F Djouad L-M Charbonnier C Bouffi et al ldquoMesenchymalstem cells inhibit the differentiation of dendritic cells throughan interleukin-6-dependentmechanismrdquo StemCells vol 25 no8 pp 2025ndash2032 2007

[80] M E Ezquer F E Ezquer M L Arango-Rodrıguez and P AConget ldquoMSC transplantation a promising therapeutic strategyto manage the onset and progression of diabetic nephropathyrdquoBiological Research vol 45 no 3 pp 289ndash296 2012

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


and is converted into a flatter epithelial cell a process namedas ldquofoot process effacementrdquo In this process the cytoskeletonthat normally supports the delicate architecture of the footprocesses is condensed at the basal side of the flattenedpodocytes altering its functionality [4] Therefore podocyteinjury is associated with the development of albuminuriaand the appearance of more severe glomerular structuralabnormalities [4]

Throughout diabetes onset high levels of blood glucoseinduce both oxidative stress and inflammation which areintimately linked with the development of DN [5ndash7] Oxida-tive stress is caused by an imbalance between the productionof reactive oxygen species (ROS) and the biological systemrsquosability to readily detoxify the reactive intermediates or repairthe resulting damageWhen this occurs oxidation of essentialmacromolecules including proteins lipids carbohydratesand DNA is produced leading to the apoptosis of damagedcells [6]

Higher ROS levels can also induce the production ofinflammatory cytokines which stimulate the production offree radicals exacerbating DN progression [5] Thereforeintensive glycemic control is the main intervention to reduceoxidative stress

Transforming growth factor beta (TGF-120573) is a multifunc-tional cytokine that plays a critical role in the pathogenesisof DN by stimulation of the synthesis of extracellular matrixmolecules including collagen type I fibronectin and laminin1205731 leading to renal fibrosis [8]

Stem cell therapy has been envisioned as a novel strategyfor various diseases since it has the potential to be moreeffective than single-agent drug therapies [9] Multipotentmesenchymal stromal cells also referred as mesenchymalstem cells (MSCs) are a population of self-renewable andundifferentiated cells present in the bone marrow and othermesenchymal tissues of adult individuals [10] MSCs areattractive candidates for renal repair since nephrons are ofmesenchymal origin and because stromal cells are of crucialimportance for signalling leading to differentiation of bothnephrons and collecting ducts [11]

Several studies have shown that the administration ofMSCs leads to the amelioration of acute and chronic kidneyinjury [12] In those reports immune modulation and anti-apoptotic effects have been associated with the therapeuticeffect suggesting that MSCs can act through paracrinemechanisms [13 14] AdditionallyMSC-based therapies havebeen considered to be promising strategies to treat patientswith diabetes mellitus and its main complications [15] Weand others have demonstrated that the systemic adminis-tration of MSCs into immunologically induced type 1 dia-betic mice reduced microalbuminuria and preserved normalrenal histology [16ndash20] However animal models used onlydeveloped the earlier harbingers of DN furthermore in thesestudies it is difficult to distinguish whether restoration ofkidney function is based on direct effect of transplantedcells on renal tissue or indirectly through improvement ofpancreatic endocrine function To answer this question andtaking into account that hyperglycaemia is the driving forcefor the development of DN previously we used a severediabetic animal model induced by the administration of

a single high dose (200mgkg) of streptozotocin (STZ) inC57BL6 mice [21] This protocol generates an irreversibleandmassive destruction of insulin-producing cells and severehyperglycaemia leading to a rapid progression of renal failureand the appearance of most of the characteristics DN pathog-nomonic signs [21ndash23] In this model multiple systemicadministrations of MSCs did not reverse hyperglycaemia butprotect kidneys from progression to macroalbuminuria andprevent the development of marked histological alterations[21]

The aim of the present work is to further characterizethe therapeutic effect of MSC administration in an animalmodel of severe diabetesmellitus particularly regarding renalfailure

For this purpose eight weeks after the massive cytotoxicdestruction of insulin-producing cells severe diabetic micewere separated into two groups one received the vehicle (DMmice) and the other 05 times 106 MSCs (DM + MSCs mice)Two and eightweeks later the function histopathology ultra-structure cell proliferation protein profile gene expressionoxidative stress and inflammation of kidneys were examinedDonor MSCs biodistribution was also assessed

2 Material and Methods

21 Animals C57BL6 and C57BL6-Tg (ACTB-EGFP)1Osb J mice (Jackson Laboratory) were housed at constanttemperature and humidity with a 12 12 h light-dark cycleand unrestricted access to a standard diet and waterWhen required animals were slightly anaesthetized withsevoflurane (Abbott) or ketamine (Drag Pharma) plusxylazine (Centrovet) All procedures were in accordancewith institutional and international standards for the humancare and use of laboratory animals [Ethic Committee ofFacultad de Medicina Clinica Alemana Universidad delDesarrollo and Animal Welfare Assurance PublicationsA54427-01 Office for Protection from Research RisksDivision of Animal Welfare NIH (National Institute ofHealth) Bethesda MD USA]

22 Severe Diabetes Induction Eight-week-old male C57BL6 mice were slightly anesthetized and received intraperi-toneally 200mgkg STZ (Calbiochem) immediately after dis-solving in 01M citrate buffer at pH 45 (DM mice) or citratebuffer only (Normal mice) This protocol of STZ admin-istration causes massive cytotoxic destruction of insulin-producing cells generating a severe hyperglycemic conditionwhich accelerates the appearance of the complications asso-ciated to diabetes particularly DN [21ndash23]

23MSC Isolation andExVivo Expansion Eight- to 10-week-old male C57BL6 or C57BL6-Tg (ACTB-EGFP) 1OsbJmice were sacrificed by cervical dislocation Bone marrowcells were obtained by flushing femurs and tibias with sterilephosphate-buffered saline (PBS) After centrifugation cellswere resuspended in 120572-minimum essential medium (120572-MEM) (Gibco) supplemented with 10 selected fetal bovineserum (Gibco) and 016mgmL gentamicin (Sanderson Lab-oratory) and plated at a density of 1 times 106 nucleated cellscm2



















































3 Results






Table2MSC

administratio


Before

MSC

administratio

n2weeks

after

MSC

administratio

n8weeks

after

MSC

administratio

nNormal

T1DM

Normal

T1DM

T1DM

+MSC

Normal

T1DM

T1DM

+MSC

Urin

aryalbu

min

excretion(U

-Alb120583gU-C

ream

g)64plusmn14

350plusmn31lowast

67plusmn11

402plusmn43lowast

274plusmn4lowast

72plusmn08

622plusmn95lowast

309plusmn25lowast

Bloo

durea

nitro

gen(m

gdL

)301plusmn2

402plusmn33lowast

32plusmn18

421plusmn22lowast

455plusmn47lowast

319plusmn25

673plusmn38lowast

465plusmn34lowast

Plasmac

ystatin

C(m

gL)

007plusmn001

011plusmn001lowast

006plusmn001

011plusmn002lowast

008plusmn001

006plusmn002

013plusmn002lowast

008plusmn001

Plasmac

reatinine(mgL)

226plusmn006

229plusmn008

230plusmn007

227plusmn006

230plusmn013

228plusmn007

260plusmn005lowast

229plusmn005

Markersof

renalfun

ctionweree

valuated

before

MSC

administratio

n(eight

weeks

after

diabetes

indu

ction)

andaft

ertwoandeightw

eeks

after

MSC

administratio

nAllanim

alsw

ereh

ousedin

metaboliccagesto

collecturinefor

albu

minuriadeterm

inationsblood

wascollected

andused


inea

ndurea

nitro

genlevels

Dataa



Normal119901lt005

versus

DM

forthe

sametim

epoint












Normal DM2

wee

ks

after

MSC

adm

inist

ratio

n8

wee

ks

after

MSC

adm

inist

ratio

n

VP

VP VP

VP

VPVP

DM + MSCs

(a)

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



NormalDM

DM + MSCs

lowast

lowast

Rena

l cor

pusc

le ar

ea (120583

m2)

(b)

2000

2500

3000

3500

4000

4500

5000



NormalDM

DM + MSCs

lowast

lowast

Glo

mer

ular

tuft

area

(120583m

2)

(c)






Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

Glo

mer

ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

NormalDM

DM + MSCs

lowastlowast

lowast

lowast



(b)

00

04

08

12

16

20

24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































































3 Results






Table2MSC

administratio


Before

MSC

administratio

n2weeks

after

MSC

administratio

n8weeks

after

MSC

administratio

nNormal

T1DM

Normal

T1DM

T1DM

+MSC

Normal

T1DM

T1DM

+MSC

Urin

aryalbu

min

excretion(U

-Alb120583gU-C

ream

g)64plusmn14

350plusmn31lowast

67plusmn11

402plusmn43lowast

274plusmn4lowast

72plusmn08

622plusmn95lowast

309plusmn25lowast

Bloo

durea

nitro

gen(m

gdL

)301plusmn2

402plusmn33lowast

32plusmn18

421plusmn22lowast

455plusmn47lowast

319plusmn25

673plusmn38lowast

465plusmn34lowast

Plasmac

ystatin

C(m

gL)

007plusmn001

011plusmn001lowast

006plusmn001

011plusmn002lowast

008plusmn001

006plusmn002

013plusmn002lowast

008plusmn001

Plasmac

reatinine(mgL)

226plusmn006

229plusmn008

230plusmn007

227plusmn006

230plusmn013

228plusmn007

260plusmn005lowast

229plusmn005

Markersof

renalfun

ctionweree

valuated

before

MSC

administratio

n(eight

weeks

after

diabetes

indu

ction)

andaft

ertwoandeightw

eeks

after

MSC

administratio

nAllanim

alsw

ereh

ousedin

metaboliccagesto

collecturinefor

albu

minuriadeterm

inationsblood

wascollected

andused


inea

ndurea

nitro

genlevels

Dataa



Normal119901lt005

versus

DM

forthe

sametim

epoint












Normal DM2

wee

ks

after

MSC

adm

inist

ratio

n8

wee

ks

after

MSC

adm

inist

ratio

n

VP

VP VP

VP

VPVP

DM + MSCs

(a)

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



NormalDM

DM + MSCs

lowast

lowast

Rena

l cor

pusc

le ar

ea (120583

m2)

(b)

2000

2500

3000

3500

4000

4500

5000



NormalDM

DM + MSCs

lowast

lowast

Glo

mer

ular

tuft

area

(120583m

2)

(c)






Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

Glo

mer

ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

NormalDM

DM + MSCs

lowastlowast

lowast

lowast



(b)

00

04

08

12

16

20

24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2MSC

administratio


Before

MSC

administratio

n2weeks

after

MSC

administratio

n8weeks

after

MSC

administratio

nNormal

T1DM

Normal

T1DM

T1DM

+MSC

Normal

T1DM

T1DM

+MSC

Urin

aryalbu

min

excretion(U

-Alb120583gU-C

ream

g)64plusmn14

350plusmn31lowast

67plusmn11

402plusmn43lowast

274plusmn4lowast

72plusmn08

622plusmn95lowast

309plusmn25lowast

Bloo

durea

nitro

gen(m

gdL

)301plusmn2

402plusmn33lowast

32plusmn18

421plusmn22lowast

455plusmn47lowast

319plusmn25

673plusmn38lowast

465plusmn34lowast

Plasmac

ystatin

C(m

gL)

007plusmn001

011plusmn001lowast

006plusmn001

011plusmn002lowast

008plusmn001

006plusmn002

013plusmn002lowast

008plusmn001

Plasmac

reatinine(mgL)

226plusmn006

229plusmn008

230plusmn007

227plusmn006

230plusmn013

228plusmn007

260plusmn005lowast

229plusmn005

Markersof

renalfun

ctionweree

valuated

before

MSC

administratio

n(eight

weeks

after

diabetes

indu

ction)

andaft

ertwoandeightw

eeks

after

MSC

administratio

nAllanim

alsw

ereh

ousedin

metaboliccagesto

collecturinefor

albu

minuriadeterm

inationsblood

wascollected

andused


inea

ndurea

nitro

genlevels

Dataa



Normal119901lt005

versus

DM

forthe

sametim

epoint












Normal DM2

wee

ks

after

MSC

adm

inist

ratio

n8

wee

ks

after

MSC

adm

inist

ratio

n

VP

VP VP

VP

VPVP

DM + MSCs

(a)

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



NormalDM

DM + MSCs

lowast

lowast

Rena

l cor

pusc

le ar

ea (120583

m2)

(b)

2000

2500

3000

3500

4000

4500

5000



NormalDM

DM + MSCs

lowast

lowast

Glo

mer

ular

tuft

area

(120583m

2)

(c)






Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

Glo

mer

ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

NormalDM

DM + MSCs

lowastlowast

lowast

lowast



(b)

00

04

08

12

16

20

24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Normal DM2

wee

ks

after

MSC

adm

inist

ratio

n8

wee

ks

after

MSC

adm

inist

ratio

n

VP

VP VP

VP

VPVP

DM + MSCs

(a)

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000



NormalDM

DM + MSCs

lowast

lowast

Rena

l cor

pusc

le ar

ea (120583

m2)

(b)

2000

2500

3000

3500

4000

4500

5000



NormalDM

DM + MSCs

lowast

lowast

Glo

mer

ular

tuft

area

(120583m

2)

(c)






Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

Glo

mer

ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

NormalDM

DM + MSCs

lowastlowast

lowast

lowast



(b)

00

04

08

12

16

20

24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal DM

2 w

eeks

aft

er M

SC ad

min

istra

tion

8 w

eeks

aft

er M

SC ad

min

istra

tion

DM + MSCs

(a)

Glo

mer

ulos

clero

sis in

dex

(fold

of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

NormalDM

DM + MSCs

lowastlowast

lowast

lowast



(b)

00

04

08

12

16

20

24

NormalDM

DM + MSCs

mRN

A T

GF-120573

mRN

A G

APD

H

lowast

lowastlowast



(c)

Normal DM DM + MSCs

(d)

Figure 2 Continued


Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Col

lage

n I (

fold

of c

hang

e ver

sus n

orm

al)

0

05

10

15

20

25

30

35

NormalDM

DM + MSCs

lowast

(e)

00

05

10

15

20

25

30

35

40

45

50

mRN

A co

llage

n Im

RNA

GA

PDH

mRN

A fi

bron

ectin

mRN

A G

APD

H

00

05

10

15

20

25

30

35

40

45

00

05

10

15

NormalDM

DM + MSCs NormalDM

DM + MSCs NormalDM

DM + MSCs

lowast

lowast

lowast

lowast

mRN

A la

min

in1205731

mRN

A G

APD

H

(f)






Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal DM

(a)

(b)

(c)

(d)

(e)

(f)

DM + MSCs










Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal DM DM + MSCs

(a)

(b)

(c)

00

01

02

03

04

05

06

Glo

mer

ular

capi

llary

lum

en ar

eatu

ft ar

ea

NormalDM

DM + MSCs

lowast

(d)

Glo

mer

ular

pod

opla

nin

area

tuft

area

006

008

010

012

014

016

018

NormalDM

DM + MSCs

lowast

(e)

Figure 4 Continued


0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

500

1000

1500

2000

2500

3000

3500

4000

4500

NormalDM

DM + MSCs

lowast

Glo

mer

ular

vol

ume (

120583m

3) p

er p

odoc

yte

(f)

0

02

04

06

08

10

12

mRN

A n

ephr

inm

RNA

GA

PDH

NormalDM

DM + MSCs

lowast

(g)



(a)


(b)







00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

04

08

12

16

20

Glo

mer

ular

Ki-6

7 pr

olife

ratio

n in

dex

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(a)

Tubu

lar K

i-67

prol

ifera

tion

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

00

04

08

12

16

20

24

28

32

lowast

lowast

lowast



(b)

NormalDM

DM + MSCs

Glo

mer

ular

apop

totic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)

0

1

2

3

4

5

6

lowast

lowast



(c)

NormalDM

DM + MSCs

Tubu

lar a

popt

otic

inde

x(fo

ld o

f cha

nge v

ersu

s nor

mal

)



0

1

2

3

4

5

6

7

lowast

lowast

lowast

(d)






4 Discussion



lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















lowast

lowastlowast



Plas

ma b

FGF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

18

(a)



Plas

ma E

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

02

04

06

08

10

12

14

16

(b)



lowast

lowastlowast

lowast

Plas

ma H

GF

(leve

l of c

hang

e ver

sus n

orm

al)

00

05

10

15

20

25

30

35

40

45

(c)

lowast

mRN

A b

FGF

mRN

A G

APD

H

lowastlowast

lowast

0

1

2

3

4

5

6



NormalDM

DM + MSCs

(d)

0

025

050

075

100

125

lowast

lowast

mRN

A E

GF

mRN

A G

APD

H



NormalDM

DM + MSCs

(e)

0

05

10

15

20

25 lowast

mRN

A H

GF

mRN

A G

APD

H

lowast



NormalDM

DM + MSCs

(f)







0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5000

10000

15000

20000

25000

30000

ROS

tota

l (U

Ap

rote

in m

g)

lowastlowastlowast



(a)

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

lowast




Lipi

d pe

roxi

datio

n (U

-8-is

o-PF

G2120572

pgU

-Cre

a mg)

(b)

004

006

008

010

012

014

016



Oxi

dativ

e pro

tein

dam

age

(3-n

itrot

irosy

ne n

gpr

otei

n120583

g)

(c)

lowast

lowastO

xida

tive D

NA

dam

age

(U-8

-OH

dG n

gU

-Cre

a mg) lowast

lowast

0

5

10

15

20

25

30

35

40



(d)

NormalDM

DM + MSCs

lowast

lowast

Tota

l ant

ioxi

dant

capa

city

(mM

Tro

lox

eqiv

pro

tein

mg)

lowast

lowast

0150

0175

0200

0225

0250

0275



(e)



Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Normal

DM


DM + MSCs

Normal DM DM + MSCs

09 plusmn 03

23 plusmn 02lowast

05 plusmn 01

(a)

Normal DM DM + MSCs

(b)

16

14

12

10

8

6

4

2

0

F48

0 po

sitiv

e cel

ls pe

r tiss

ue se

ctio

ns

lowast

NormalDMDM + MSCs

(c)

NormalDMDM + MSCs

00

02

04

06

08

10

12

14

lowast

Plas

ma I

L-6

(fold

of c

hang

e ver

sus n

orm

al)

lowast



(d)

Figure 9 Continued


Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Plas

ma I

L-10

(fol

d of

chan

ge v

ersu

s nor

mal

)

lowast

00

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(e)

Plas

ma I

L-4

(fold

of c

hang

e ver

sus n

orm

al)

lowast

lowast

00

05

10

15

20

25

30



NormalDMDM + MSCs

(f)

0

1

2

3

4

5

6

7

8

mRN

A IL

-10

mRN

A G

APD

H

lowast

lowast



NormalDMDM + MSCs

(g)

lowast

mRN

A IL

-4m

RNA

GA

PDH

0

02

04

06

08

10

12

14

16

18

20

22



NormalDMDM + MSCs

(h)



















5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































5 Conclusions



Abbreviations




Acknowledgments


References


























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article proregenerative microenvironment...

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