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A study on the protectiveeffect of bone marrow derivedmesenchymal stem cells onchronic renal failure in ratsMohamed Talaat Abd El Aziz,1Hazem Atta,1 Sohair Mahfouz,2Hanaa Mohamed Yassin,3Laila Ahmed Rashed,1 Dina Sabry,1Zienab Abdel Wahab,3 Fatma Taha,1Ghada Abdel Aziz,4Abeer Moustafa El Sayed,5Mohamed Sayed3

1Department of Medical Biochemistry,Unit of Biochemistry and MolecularBiology; 2Department of Pathology;3Department of Physiology Faculty ofMedicine, Cairo University; 4Departmentof Medical Biochemistry, Faculty ofMedicine, Bani Sweif University;5Department of Pathology, NationalCancer Institute, Cairo University, Egypt

Abstract

Chronic renal failure (CRF) is a commondisease with high morbidity and mortality. Theinadequacy of current treatment modalitiesand insufficiency of donor organs for cadaver-ic transplantation have driven a search forimproved methods of dealing with renal fail-ure. This raises the concept of cell-based ther-apy using mesenchymal stem cells (MSCs).The purpose of this work is to evaluate theeffect of MSCs in rats with CRF and to detectthe level of B-cell lymphoma 2 (Bcl-2) andbasic fibroblast growth factor (bFGF) as possi-ble antiapoptotic and anti-inflammatoryparacrine mediators of MSCs action. Thisstudy was carried on female albino rats, whichwere divided into 3 groups, control group, CRFgroup and CRF/MSCs group. MSCs werederived from bone marrow of male albino ratsand were characterized morphologically and byRT-PCR for CD29. Serum urea, creatinine, Na,K, systolic blood pressure and 24-hour urinaryalbumin were estimated for all groups, Y-chro-mosome gene (Sry) was detected by PCR inrenal tissue. Also Bcl-2 and bFGF were exam-ined by RT-PCR in renal tissue of all studiedgroups and the kidney was examined patholog-ically. The results of this work showed that CRFrats receiving MSCs showed significantly low-ered serum urea, creatinine and urinary albu-min levels compared to the CRF group. Alsoimprovement of serum Na and K was observedin CRF/MSCs group compared to CRF group. Asregard Bcl-2 and bFGF they were highlyexpressed in renal tissue of CRF/MSCs group

compared to the CRF group. The (Sry) genewas detected by PCR in the renal tissue ofCRF/MSCs group. These results demonstrate apotential of MSCs to ameliorate the kidneyfunction in rats with CRF possibly through theantiapoptotic and anti-inflammatory paracrinemediators of MSCs.

Introduction

Chronic renal failure (CRF) is a commondisease with high morbidity and mortality.Dysfunction and necrosis of renal tubularepithelial cells is the most common injury inchronic renal failure occurring after ischemicor toxic challenge of the kidney.1

There is growing recognition that the dis-ease state arising from renal failure is theresult of more than just the loss of blood vol-ume regulation, small solute, and toxin clear-ance that are replaced by conventional dialysistherapy.2 The kidney's role in reclamation ofmetabolic substrates, synthesis of glutathione,and free radical scavenging enzymes, gluco-neogenesis, ammoniagenesis, catabolism ofpeptide hormones and growth factors, and theproduction and regulation of multiplecytokines critical to inflammation andimmunological regulation are not addressed bycurrent treatment modalities .3

Thus, there is considerable drive to developimproved therapies for renal failure with thecapacity to replace a wider range of the kid-ney's functions, thereby reducing morbidity,mortality, and the overall economic impactassociated with this condition. Such an ambi-tion lies beyond the reach of conventionalmedicine, with its mainly monofactorialapproach to the treatment of disease. Into thisbreach steps the nascent and expanding fieldof cell therapy, which offers the promise of har-nessing the native abilities of the cell,endowed to it by a billion years of evolution.4

During the past several years, a great deal ofattention has been focused on the plasticity ofbone marrow-derived stem cells. Recent stud-ies have demonstrated that bone marrow-derived stem cells have the ability to cross lin-eage boundaries and to form functional com-ponents of other tissues.5-8

The possibility that bone marrow-derivedstem cells might functionally contribute to therenal tubule regeneration is still a matter ofdebate. Bone marrow progenitor’s cells fromchronic renal failure rats showed no capacityfor in vitro proliferation.9 Three studies havedemonstrated that when female kidneys weretransplanted into male recipients, Y chromo-some-bearing cells were found in the trans-planted kidney;10-12 however, in two of the stud-ies these cells showed only minor contributionin the healing process, as they were found only

to affect 1% of the examined tubules. Themechanism mediating the protective effects ofMSCs might be primarily paracrine, as impliedby their demonstrated expression of severalgrowth factors such as hepatocyte growth fac-tor (HGF), vascular endothelial growth factor(VEGF) and insulin like growth factor-1 (IGF-1), these factors improve the renal functionthrough their antiapoptotic, mitogenic andother cytokine actions. Also MSCs down regu-late the proinflammatory cytokines IL-1-b,TNF-a, and IFN-g, as well as iNOS and up reg-ulate the anti-inflammatory and organ-protec-tive IL-10, as well as basic fibroblast growthfactor (bFGF), transforming growth factoralpha (TGF-a) and antiapoptotic Bc1-2. Thesebeneficial paracrine actions are elicited earlyand late after onset of CRF.13 Based on theabove-mentioned knowledge, the present studyaimed to detect the effect of MSCs in rats withCRF, and to detect the level of Bcl-2 and bFGFas possible antiapoptotic and anti-inflammato-ry paracrine mediators of MSCs action.

Materials and Methods

Preparation of bone marrow-derived mesenchymal stem cell

Bone marrow was harvested by flushing thetibiae and femurs of 6-week-old male whitealbino rats with Dulbecco's modified Eagle'smedium (DMEM, GIBCO/BRL, InvitrogenCorp., Carlsbad, CA, USA) supplemented with10% fetal bovine serum (GIBCO/BRL).Nucleated cells were isolated with a densitygradient [Ficoll/Paque (Pharmacia, GEHealthcare, Pollards Wood, UK)] and resus-pended in complete culture medium supple-mented with 1% penicillin-streptomycin

Stem Cell Studies 2011; volume 1:e11

Correspondence: Dina Sabry, MedicalBiochemistry and Molecular Biology. Faculty ofMedicine, Cairo University, Egypt.Tel. +2.0111200200 - Fax: +2.02.23632297. E-mail: dinnasabry69@yahoo.com

Key words: chronic renal failure, mesenchymalstem cells, experimental rats.

Received for publication: 13 May 2011.Revision received: 24 July 2011.Accepted for publication: 27 July 2011.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright M.T.A. El Aziz et al., 2011Licensee PAGEPress, ItalyStem Cell Studies 2011; 1:e11doi:10.4081/scs.2011.e11

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(GIBCO/BRL). Cells were incubated at 37°C in5% humidified CO2 for 12-14 days as primaryculture or upon formation of large colonies.When large colonies developed (80-90% con-fluence), cultures were washed twice withphosphate buffer saline (PBS) and the cellswere trypsinized with 0.25% trypsin in 1mMEDTA (GIBCO/BRL) for 5 min at 37°C. Aftercentrifugation, cells were resuspended withserum-supplemented medium and incubatedin 50 mL/25 cm2 culture flask (Falcon, BDBiosciences, Franklin Lakes, NJ, USA). Theresulting cultures were referred to as first-pas-sage cultures.14 MSCs in culture were charac-terized by their adhesiveness and fusiformshape Also CD29 gene expression by reversetranscription polymerase chain reaction (RT-PCR) was detected as a marker of MSCs.15

Other methods for MSCs characterizationwere done in previous published studies.16-18

All animal experiments received approval fromthe institutional animal care committee.

Labeling of mesenchymal stem cellsby green fluorescent protein

MSCs were harvested during the 4th passageand were labeled with green fluorescent pro-tein (GFP) using monster green fluorescentprotein vector and transfast tranfectionreagent kit (Promega, Madison, WI, USA). Forimaging GFP of MSCs, unstained slides weredirectly analyzed by confocal laser microscopy(LSM 510, Carl Zeiss, Oberkochen, Germany)incorporating two lasers (Ar and HeNe)equipped with an inverted Axiovert 100 Mmicroscope. Confocal fluorescence imageswere taken using a 20× Plan-Neofluar objec-tive (numerical aperture (NA), 0.5) and a 40×Plan-Neofluar objective (NA 0.75). To visualizeGFP fluorescence, excitation from the 488-nmAr laser line and emission passing through aband-pass 505-530 filter, which passes wave-lengths of 505-530 nm to the detector, wereused.19 Unstained paraffine embedded sec-tions have been examined by anImmunofluorostaining method for detection offluorescence, which served as counterstainingin this method.20

Chronic renal failure model andmesenchymal stem cells adminis-tration

All experimental procedures were performedaccording to the guidelines for the care anduse of animals established by the animal unitin Faculty of Medicine, Cairo University. Thirtyinbred strain of white albino female rats, 8weeks old, weighing between 150 and 200 gwere used in this study. They were inbred andmaintained in an air-conditioned animalhouse with specific pathogen-free conditions,and were subjected to a 12:12-h daylight/dark-ness and allowed unlimited access to chow and

water. The morphological and behavioralchanges of rats were monitored every day.

Rats were divided into the following groups:Control group: involves 10 healthy rats, CRF

group: 20 rats that underwent modified 5/6nephrectomy were anaesthetized with sodiumthiopental by intramuscular injection.Laparotomy was done followed by surgicalexcision of one kidney and cautery of the otherto excise 2/3 of its tissue.21 The wound wasthen sealed by continuous 6/0 stitches in 2 lay-ers. Finally, antibiotic ointment (teramycin)and powder (neomycin) were applied to thewound. CRF was determined by elevated bloodcreatinine then this group was randomly divid-ed into; CRF/MSCs group: 10 rats infused witha single dose of syngenic MSCs 1×106 22 cellsper rat (intravenous route) in rat tail vein; CRFgroup: 10 rats were infused with the same vol-ume of saline and serve as a control. After 4weeks from administration of MSCs, 24 hoursurine samples were collected using specializedwire mesh cages and venous blood was collect-ed from the retro-orbital vein. Arterial bloodpressure was measured by rat-tail cuff methodusing an electronic electro sphygmomanome-ter after the rat was pre warmed to 37°C for 15min. The average of 3 pressure readings wasrecorded for each measurement while the ani-mals were quietly resting. Then all rats weresacrificed with CO2 narcosis, and kidney tissuewas harvested for analysis.

Analysis of kidney histopathologyKidney samples were collected into PBS and

fixed overnight in 40 g/L paraformaldehyde inPBS at 4°C. Serial 5-µm sections the kidneyswere stained with hematoxylin and eosin (HE)and Masson Trichrome (MT) and were scoredhistopathologically according to Lloberas etal.23 Other sections were unstained for fluores-cent examination.

Polymerase chain reaction detec-tion of male-derived mesenchymalstem cells

Genomic DNA was prepared from kidney tis-sues homogenate of the rats in each groupusing Wizard® Genomic DNA purification kit(Promega, Madison, WI, USA). The presenceor absence of the sex determination region onthe Y chromosome male (Sry) gene in recipi-ent female rats was assessed by PCR. Primersequences for Sry gene (forward CATC-GAAGGGTTAAAGTGCCA-3., reverse 5.-ATAGT-GTGTAG-GTTGTTGTCC-3.) were obtained frompublished sequences24,25 and amplified a prod-uct of 104 bp. The PCR conditions were as fol-lows: incubation at 94°C for 4 min; 35 cycles ofincubation at 94°C for 50s, 60°C for 30 s, and72°C for 1 min; with a final incubation at 72°Cfor 10 min. PCR products were separated using2% agarose gel electrophoresis and stained

with ethidium bromide. Positive (male whitealbino rat genomic DNA) negative (femalewhite albino rat genomic DNA) controls wereincluded in each assay.

Polymerase chain reaction detec-tion of bFGF and Bcl-2 geneexpression and quantification

Total RNA was extracted from kidney tissuehomogenate using RNeasy PurificationReagent (Qiagen Inc., Valencia, CA, USA).cDNA was generated from 5 µg of total RNAextracted with 1 µL (20 pmol) antisense primerof each gene and 0.8 µL superscript AMVreverse transcriptase for 60 min at 37°C. ForPCR, 4 µL cDNA were incubated with 30.5 µLwater, 4 µL 25 mM MgCl2, 1 µL dNTPs (10 mM),5 µL 10xPCR buffer, 0.5 µL (2.5 U) Taq poly-merase and 2.5 µL of each primer containing 10pmol. Primer sequences of Bcl-2 and bFGF wereas follows respectively (forward 5'- CTGAGCT-GACCTTGGAGC -3', 5'-GGAGGGCAC TTCCT-GAG-3', reverse 5'- GACTCCAGCCACAAAGATG -3', 5'- GCCTGGCATCACGACT-3'). The reactionmixture was subjected to 40 cycles of PCRamplification as follows: denaturation at 95°Cfor 1 min, annealing at 67°C for 1 min andextension at 72°C for 2 min. UV transillumina-tor visualized the PCR products after separationon 1.5% agarose gel electrophoresis. DNA banddensity quantification was done using gel docu-mentation system (BioDoc Analyze digital - dig-ital gel documentation and professional gelanalysis - Göttingen, Germany). The quality ofRNA and the validity of the amplifications weredetermined by amplification of b-actin for thespecimens.26

Polymerase chain reaction detec-tion of CD29 gene expression

Total RNA was extracted from cells usingRNeasy Purification Reagent (Qiagen,Valencia, CA), and then a sample (1 µg) wasreverse transcribed with AMV reverse tran-scriptase (RT) for 30 minutes at 42°C in thepresence of oligo-dT primer. PCR was per-formed using specific primers (UniGeneRn.25733) forward: 5’-AATGTTTCAGTGCA-GAGC-3’ and reverse: 5’-TTGGGAT GAT-GTCGGGAC-3’. PCR was performed for 35cycles, with each cycle consisting of denatura-tion at 95°C for 30 seconds, annealing at 55°Cto 63°C for 30 seconds, and elongation at 72°Cfor 1 min, with an additional 10-min incuba-tion at 72°C after completion of the last cycle.The PCR product was separated by elec-trophoresis through a 1% agarose gel, stained,and photographed under ultraviolet light.27

Polymerase chain reaction detec-tion of b-actin

The presence of RNA in all tissues was

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assessed by analysis of b-actin, which is ahouse-keeping gene. b-actin primers (forward5'-TGTTGTCCCTGTATGCCTCT-3', reverse 5'-TAATGTCACGCACGATTTCC-3') were designedfrom GenBank (accession no. J00691) andamplified a product of 206 bp.14,15

Biochemical assessmentLevel of 24 hour urinary albumin and serum

creatinine, urea, Na and K were assessedusing conventional available kit.

Statistical analysisData are expressed as mean ± SD.

Significant differences were determined byusing ANOVA and post-hoc tests for multiplecomparisons using SPSS 9.0 computerSoftware. Results were considered significantat P<0.05.

Results

Isolated and cultured undifferentiated MSCsreached 70–80% confluence at 14 days. Theywere identified by their fusiform fibroblast like-structure and gene expression of CD29 (Figure1). MSCs improve the kidney function: Asshown in Table 1 results of the present studyshowed that mesenchymal stem cells have a sig-nificant improvement in kidney function,serum Na concentration increased significantlyin CRF group (P<0.001) in comparison to con-trol group. However after MSC injection, serumNa concentration significantly decreased(P<0.001). Regarding serum K, urea and 24-hour urinary albumin, their concentration wassignificantly elevated in CRF group (P=0.002).However, following MSC injection, their levelsstarted to decrease significantly (P=0.03) com-pared to the CRF group. However serum creati-nine showed significant elevation in CRF groupcompared to the control group. MSCs infusiondid not affect significantly serum creatinine.MSCs ameliorate the systolic blood pressure(SBP): As regards the systolic blood pressure(Table 1), there was a significant decrease aswell as normalization of its level in CRF/MSCsgroup as compared to CRF group and to controlgroup respectively.

Homing of labeled mesenchymalstem cells in renal tissues

Labeled MSCs with GFP were detected usingfluorescent microscope in unstained renal tis-sues in CRF/MSCs group only, confirming thatthese cells were actually seeded into the renaltissues as shown in (Figure 2).

Mesenchymal stem cells affect thekidney morphologically

Histopathological examination of kidney tis-

sue following MSC injection showed, denseinterstitial, periglomerular, perivascular anddiffuse interstitial tissue infiltrates (renal cor-tex), renal medulla shows medullary tubuleswith dilated lumina and focal hydropic degen-erative changes. MT shows dense fibrosisinvaded by a dense collection of MSC andendarteritis obliterans. (Figures 3 and 4).

Mesenchymal stem cells and geneexpression of anti-inflammatoryand antiapopotic markers

As regards gene expression, Bcl-2 and b-FGFgenes were decreased in CRF group, whereas,its band density was significantly increased inCRF/MSCs rat groups. Bcl-2 and b-FGF DNA

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Table 1. Serum Na, K, urea, creatinine, 24 h urinary albumin and SBP (mean ± SD)among the studied groups.

Measured parameters Control CRF CRF/MSCMean ± SD Mean ± SD Mean ± SD

Na (mEq/L) 134±4.28 152±5.69* 142±4.78°K (mEq/L) 3.7±0.44 5.5±0.68* 4.8±0.57*°Urea mg/dL) 42±4.74 68.8±13.83* 49.1±5.11°Creatinine (mg/dL) 0.198±0.08 1.02±0.55* 0.43±0.1*Urinary albumin (mg/24 h) 0.08±0.02 29.4±6.8* 5.4±0.63*°SBP (mm Hg) 100.8±4.21 141.6±4.45* 118±4.05°*Significant P as compared to group 1(P<0.05); °significant P as compared to group 2 (P<0.05).

Figure 1. UV transilluminated agarose gel of polymerase chain reaction (PCR) product ofCD29 gene in mesenchymal stem cells culture. Lane1, PCR marker; Lane 2, CD29 genein MSCs culture.

Figure 2. Confocal laser imaging showed strong green auto fluorescence appear in kidneytissue after mesenchymal stem cells (MSCs) injection. Arrows indicate positive signals forfluorescence of labeled MSCs in renal tissues.

Figure 3. A) Photomicrographs of kidney tissue (cortex) from chronic renal failure (CRF)rat group stained with Hematoxylin and Eiosin renal cortex, B) renal medulla and C)masson trichrome cortex, showing increased cellularity and congestion of the glomeruliin renal cortex, medulla shows atrophic tubules and hyaline degenerative changes.

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band density quantification were demonstratedat Table 2. On the other hand, Sry gene that wasused as Y chromosome marker was expressedin CRF/MSCs rat group only (Figure 5).

Discussion

Bone marrow-derived stem cells contributeto cell turnover and repair in various tissuetypes, including the kidneys.11,28 MSCs areattractive candidates for renal repair, becausenephrons are of mesenchymal origin andbecause stromal cells are of crucial importancefor signaling, leading to differentiation of bothnephrons and collecting ducts.29 MSCs com-monly are defined as bone marrow-derivedfibroblast-like cells, which despite the lack ofspecific surface markers can be selected bytheir adherence characteristics in vitro andwhich have the ability to differentiate alongthe three principal mesenchymal lineages:osteoblastic, adipocytic, and chondrocytic.30,31

In the present study, bone marrow derivedmesenchymal stem cells were isolated frommale rats, grown and characterized by theiradhesiveness and fusiform shape and by detec-tion of CD 29, one of surface marker of ratmesenchymal stem cells and were used todetect their possible antiinflammaory andantiiapoptotic role in amelioration of renalfunction in experimental model of chronicrenal failure.

In the current study 5/6 nephrectomymethod was applied to induce CRF in the stud-ied rats, the assessment of kidney functionswhich showed significant elevation of urea,creatinine and K levels occurred as well as sig-nificant increase in serum Na level.

Significant elevation of systolic blood pres-sure (which can be either a cause or a conse-quence of chronic kidney disease (CKD) rapid-ly developed compared to the control group.These results are in agreement with these ofFleck et al.18 who found that the functionalparameters clearly indicated the developmentof CRF following 5/6 nephrectomy.

The results of the present work showed thatmost of the examined parameters had signifi-cant improvement following MSCs injectioncompared to the renal failure group. Na, K,urea, Bcl-2, bFGF, SBP and urinary albuminshowed statistically significant improvementfollowing MSC injection. However, creatininewas the sole examined parameter that failed toreach statistical significance throughout theexperiment duration, with, probably needingmore time to do. Similar result was obtained byKunter et al.;32 in their study, creatinine alsofailed to reach statistical significance with.Improvement of those parameters goes inaccordance with Caldas et al.,33 whose studyshowed in addition significant reduction in

creatinine level. Histopathological examination of renal tis-

sue samples showed dense interstitial,periglomerular, perivascular and diffuse inter-stitial tissue infiltrates. MT showed densefibrosis invaded by a dense collection of MSCsas indicated by autofluorescence appears inkidney tissue after MSCs injection. Those wereexamined with H&E stain, that is a more sen-sitive detector regarding cellular infiltration,finally with Mason Dichromate, which is a bet-ter detector of collagen fibers hence, fibrosisand scaring. These findings agreed with thoseof Li et al.,34 who recorded similar perivascular

and periglomerular infiltration, in addition,they reported cell fusion, with occurrence ofbinucleated cells. Similar findings wereobtained by Hauger et al.,35 who used MRI todetect homing of donors' cells into the injuredkidneys.

Following stem cell injection, those donors'cells could be detected in recipient failing kid-neys by using PCR technique as evident bydetection of Sry gene in female rat thatreceived MSCs derived from male rats and byautofluorescence that appeared in kidney tis-sue after MSCs injection. Fusion or transdif-ferentiation, this could not be answered in this

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Table 2. Band density quantification for Bcl-2 and b-FGF genes expression.

Group Bcl-2 (mean ± SD) b-FGF (mean ± SD)

Control 0.472±0.019° 0.549±0.029°CRF 0.128±0.003 0.283±0.008CRF/MSCs 0.462±0.021° 0.621±0.032°°significant P as compared chronic renal failure (CRF) group to other groups (P<0.05).

Figure 4. A) Photomicrographs of kidney tissue (cortex) from chronic renal failure/mes-enchymal stem cell (CRF/MSC) rat group stained with Hematoxylin and Eiosin cortex, B)medulla and C) and masson trichrome cortex, showing dense interstitial tissue infiltrateby periglomerular, perivascular and diffuse interstiial tissue infiltrates.

Figure 5. A) UV transilluminated agarose gel of polymerase chain reaction (PCR) prod-ucts of Bcl-2, B) b-FGF, C) b-actin D) and Sry genes in different studied groups.

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study. However, both techniques definitelyproved that those cells were able to maintainhigh population all through the study, in otherwords, for 4 weeks following MSC injection.These results agree with those of Li et al.34 whoshowed 50% replacement of proximal tubularcells with donor cells. These results also agreewith Rookmaaker et al.36 who declared thatbone-marrow-derived cells might home toinjured glomerular endothelium, differentiateinto endothelial cells, and participate in regen-eration of the highly specialized glomerularmicrovasculature. In addition, they confirmedprevious observations that bone-marrow-derived cells can replace injured mesangialcells.37 Also Tögel et al.38 stated that infusedMSC were detected in the kidney only earlyafter administration and were predominantlyin glomeruli and attached in peritubular capil-laries.

However, those results disagree with someother studies; Li et al.34 and Duffield et al.39

who disagreed on whether there was evidencefor transdifferentiation, but both concludedthat while bone marrow derived cells (BMDC)recruitment occurs, repair is predominantlyelicited via proliferation of endogenous renalcells.

Duffield et al.39 state that BDMC contributea regenerative cytokine environment that maybe important in the resulting functional repair.

Similarly, it was found that bone marrow-derived stem cells seemed to contribute rela-tively small numbers of cells (3-22%) to regen-erating renal tubular40 and glomerular cell pop-ulations11 that is, the majority of reparativecells were derived from intrinsic kidney cells.

Regardless the cause, whether it's MSC dif-ferentiation, fusion or merely cytokineinduced renal improvement; following MSCinjection, the results of the present workshowed increase in bFGF and Bcl-2 geneexpression in renal tissues. Several studiesstated that After 24 h of MSCs infusion, onlyexceptionally scarce numbers of MSCs werefound in the kidney, a pattern that essentiallyrules out the possibility that significant num-bers of infused MSC are retained in the kidneywhere they could physically replace lost kidneycells by transdifferentiation. This conclusion isfurthermore supported by the fact that therewere no intrarenal transdifferentiation eventsof MSC within 3 days of administration,whereas occasional MSC-derived capillaryendothelial cells were identified only after 5-7days. From this, it could be deduce that themechanisms that mediate the protectiveeffects of MSC must be primarily paracrine, asimplied by their expression of several growthfactors such as HGF, VEGF, and IGF-I, all knownto improve renal function in ARF, mediated bytheir antiapoptotic, mitogenic and othercytokine actions. Collectively, these as yetincompletely defined paracrine actions of MSC

result in the renal down-regulation of proin-flammatory cytokines IL-1-b, TNF-a, and IFN-g, as well as iNOS, and up-regulation of anti-inflammatory and organ-protective IL-10,41 aswell as bFGF, TGF- a, and Bcl-2. The lack ofrenoprotection obtained by infused fibroblastsmay be due, at least in part, to the fact thatMSC exhibit a comparatively higher expres-sion of VEGF, HGF, and IGF-I, therefore sug-gesting that the combined delivery, by MSC, ofthese factors appears to result in superiorrenoprotection than that obtained with thegrowth factors that are more highly expressedby fibroblasts (EGF, HB-EGF, BMP-7, bFGF).

In consideration of the role of endogenousbone marrow MSCs, a possible approach couldalso be the stem cells mobilization. However,the possible effects of BM-recruited cells andof inflammatory cells in this experimental set-ting require further investigation. Currently,the most promising approach is considered tobe the administration of in vitro expandedMSC applied to acute tubular and glomerularinjury. Injected MSCs were shown to home tothe injured kidney and to accelerate morpho-logical and functional regeneration, possiblyby a paracrine or even endocrine mechanisms,although their engraftment and transdifferen-tiation was not observed in the majority of thestudies. A major role in the effect of MSCs hasbeen attributed to the production of growthfactors and cytokines with immunosuppres-sive, antiinflammatory, anti-apoptotic and pro-liferative effects. Several clinical trials havebeen designed or are in progress to evaluatethe effect of MSCs administration in renaltransplantation, acute renal injury or chronicallograph nephropathy (www.clinicaltrial.gov).The effect of MSC administration in chronicrenal damage still deserves investigation.42

To conclude, this study proved that theimpairment in renal functions that associatesCRF could be significantly corrected by singleintravenous injection of donor's stem cells.Stem cells reach the injured kidney through itsarterial supply and start infiltration within fewdays and sooner, improvement of renal func-tions start. The mechanism of action of thosecells might be not only through improvementof healing process but also through anti-inflammatory and antiapoptotic paracrineactions.

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