Journal of Feline Medicine and Surgery (2011) --, --e--

doi:10.1016/j.jfms.2011.01.005

Evaluation of intrarenal mesenchymal stem cellinjection for treatment of chronic kidney diseasein cats: a pilot study

Jessica M Quimby DVM, DACVIM1*, Tracy L Webb DVM, PhD

1, Debra S Gibbons DVM, MS,

DACVR3, Steven W Dow DVM, PhD, DACVIM

1,2

1Department of Clinical Sciences,College of Veterinary Medicine andBiomedical Sciences, Colorado StateUniversity, Fort Collins, CO 80523,USA2Department of Microbiology,Immunology and Pathology,College of Veterinary Medicine andBiomedical Sciences, Colorado StateUniversity, Fort Collins, CO 80523,USA3Department of Environmental andRadiological Health Sciences,College of Veterinary Medicine andBiomedical Sciences, Colorado StateUniversity, Fort Collins, CO 80523,USA

*Corresponding author. Tel: þ1-970-2971205. E-mail: jquimby@colostate.edu

1098-612X/11/------+-- $36.00

Please cite this article in press as: Quichronic kidney disease in cats: a pilot

The feasibility of autologous intrarenal mesenchymal stem cell (MSC) therapy incats with chronic kidney disease (CKD) was investigated. Six cats (two healthy,four with CKD) received a single unilateral intrarenal injection of autologousbone marrow-derived or adipose tissue-derived MSC (bmMSC or aMSC) viaultrasound guidance. Minimum database and glomerular filtration rate (GFR)via nuclear scintigraphy were determined pre-injection, at 7 days and at 30 dayspost-injection. Intrarenal injection did not induce immediate or long-termadverse effects. Two cats with CKD that received aMSC experienced modestimprovement in GFR and a mild decrease in serum creatinine concentration.Despite the possible benefits of intrarenal MSC injections for CKD cats, thenumber of sedations and interventions required to implement this approachwould likely preclude widespread clinical application. We concluded that MSCcould be transferred safely by ultrasound-guided intrarenal injection in cats, butthat alternative sources and routes of MSC therapy should be investigated.

Date accepted: 10 January 2011 � 2011 ISFM and AAFP. Published by Elsevier Ltd. All rights reserved.

Chronic kidney disease (CKD) is a major causeof morbidity and mortality in cats. At present,the only definitive treatment option for cats

with CKD is renal transplantation.1,2 However, renaltransplantation is not a viable option for most CKD-affected cats and supportive care, designed to stabilizerenal function and reverse metabolic complications ismost common.3,4 However, these supportive treat-ments do not address the underlying and often pro-gressive disease process.

The effects of mesenchymal stem cell (MSC) ther-apy have been investigated in rodent chronic renalfailure models including genetic disease, glomerulo-nephritis and experimentally-induced CKD.5e18 Inthe majority of experimentally-induced CKD modelsinvestigated, MSC administration has resulted in ben-eficial changes, as evidenced by improvement in renal

-5000; Fax: þ1-970-297-

/0 � 2011 ISFM a

mby JM, et al., Evaluationstudy, J Feline Med Surg (2

functional parameters and reduction of renal fibrosisand glomerulosclerosis.5e8,10 Although the mecha-nisms underlying these effects are not yet fully under-stood, most investigators propose that paracrineeffects from the injected MSC are more importantthan the effects of direct cellular incorporation intofunctional nephrons.19,20

The purpose of this pilot study was to assess thefeasibility of intrarenal MSC transfer in normal catsand in cats with CKD. Unilateral intrarenal transferof MSC was chosen to allow internal comparison be-tween the injected kidney and the non-injectedkidney using glomerular filtration rate (GFR) deter-mined by nuclear scintigraphy. The effects of MSCtransfer on GFR, renal functional parameters, andoverall animal health were assessed. This study wasdesigned to test the hypothesis that MSC could besafely administered to cats with CKD and that MSCinjection would result in improvement in functionof the injected kidney.

nd AAFP. Published by Elsevier Ltd. All rights reserved.

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

2 JM Quimby et al

Materials and methods

Healthy specific pathogen-free (SPF) cats

Two healthy SPF purpose-bred domestic shorthaircats (Andrea D Lauerman SPF Cat Colony Resource,Fort Collins, CO) were utilized for the study. Bothcats were 1.5 years of age (one castrated male, one in-tact female). The cats were adopted into privatehomes at the conclusion of the study.

Study population of cats with stable CKD

Cats with stable CKD were recruited from the patientpopulation at the Veterinary Teaching Hospital at Col-orado State University. Cats were determined to have

Table 1. Description of cats included in the study.

Cat Group Signalment IRIS stacreatinine

1 Young healthy 1.5-year MC DSH IRIS: N/ACreatinine: 1UPC: 0.1

2 Young healthy 1.5-year FI DSH IRIS: N/ACreatinine: 1UPC: 0.1

3 CKDþ bmMSC 6-year-old MC DLH IRIS: IVCreatinine: 5UPC: 1.7

4 CKDþ bmMSC 15-year MC Tonkinese IRIS: IIICreatinine: 4UPC: 0.3

5 CKDþ bmMSC 17-year MC Siamese IRIS: IICreatinine: 2UPC: not pe

6 CKDþ bmMSC 14-year MC DSH IRIS: IICreatinine: 2UPC: 0.2

7 CKDþ aMSC 9-year FS Siamese IRIS: IIICreatinine: 3UPC: 0.1

8 CKDþ aMSC 9-year MC DSH IRIS: IICreatinine: 2UPC: 0.2

9 CKDþ aMSC 7-year MC DSH IRIS: IVCreatinine: 6UPC: 0.3

10 CKD no MSC 9-year MC DSH IRIS: IICreatinine: 2UPC: 0.3

11 CKD no MSC 12-year FS DSH IRIS: IIICreatinine: 3UPC: 0.1

12 CKD no MSC 13-year MC DSH IRIS: IICreatinine: 2UPC: 0.2

DSH¼ domestic shorthair, FS¼ female spayed, MC¼ma

Please cite this article in press as: Quimby JM, et al., Evaluationchronic kidney disease in cats: a pilot study, J Feline Med Surg (2

stable CKD based on two repeated biochemical evalu-ations performed at least 2 weeks apart. Pre-treatmentevaluation included complete blood count (CBC), bio-chemistry profile, urinalysis, urine culture, bloodpressure, total T4, urine proteinecreatinine ratio(UPC), feline leukemia virus/feline immunodefi-ciency virus (FeLV/FIV) serology, abdominal radio-graphs, and a renal ultrasound. Cats were excludedfrom the study if they had evidence of ureteroliths,pyelonephritis, uncontrolled hypertension, or concur-rent systemic disease. Administration of concurrentsupportive therapies was allowed provided therewere no changes in therapy during the study period.A summary of the demographics of participatingcats is presented in Table 1. The study was approved

ge and(mg/dl)

Treatment

.71� 105 bmMSC injected into the rightkidney

.31� 105 bmMSC injected into the left kidney

.41� 105 bmMSC injected into the left kidney

.3Unable to expand sufficient bmMSC fortherapy

.6rformed

Unable to expand sufficient bmMSC fortherapy

.0Unable to expand sufficient bmMSC fortherapy

.51� 106 aMSC injected into the left kidney

.62� 106 aMSC injected into the left kidney

.54� 106 aMSC injected into the left kidney

.3GFR repeatability only

.3GFR repeatability only

.6GFR repeatability only

le castrated; IRIS¼ Internation Renal Interest Society.

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

3Mesenchymal stem cell therapy for kidney disease

by the Institutional Animal Care and Use Committeeat Colorado State University, and all owners reviewedand signed consent forms prior to participation in thestudy.

Autologous MSC collection and culture

For collection of bone marrow or adipose tissue biop-sies, cats were sedated with ketamine (Fort Dodge)3.3e4.8 mg/kg IV, once (dose repeated once if needed),midazolam (Baxter HealthCare, Deerfield, IL) 0.1 mg/kg IV once and butorphanol (Fort Dodge) 0.1 mg/kgIV once. Intravenous fluids were administered duringsedation at 5 ml/kg/h and blood pressure, pulse andrespiration were monitored. Approximately 1 ml ofbonemarrowwas collected from theproximal humerusand placed into plastic tissue culture flasks (BD Biosci-ences, San Jose, CA) inMSCmedium (low-glucoseDul-becco’s modified Eagle’s medium (DMEM), 100 U/mlpenicillin, 100 mg/ml streptomycin: Invitrogen/Gibco,Carlsbad,CA) plus 15% fetal bovine serum (Cell Gener-ation, Fort Collins, CO). The bone marrow-derivedMSC (bmMSC) were incubated until approximately70% confluent with media changes every 2e3 days.The cells were harvested with trypsin (Invitrogen/Gibco, Carlsbad, CA) and passaged until adequatecell numbers were obtained for injection.

Adipose tissue was obtained from a subcutaneoussite on the ventral abdomen just caudal to the umbili-cus. For preparation of the adipose tissue for culture,the tissue was minced and digested with 1 mg/ml col-lagenase (Sigma Aldrich, St Louis, MO) for 30 min at37�C. The sample was centrifuged and the stromalvascular fraction was washed, plated in MSC mediumand expanded as described above for bmMSC.

Characterization of MSC

Adipose tissue-derived MSC (aMSC) and bmMSCwere characterized by surface marker expression us-ing flow cytometry and a panel of feline-specific andcross-reactive antibodies specific for surface determi-nants expressed by MSC from other species.21�24 Spe-cifically, feline MSC were analyzed for surfaceexpression of CD44 (anti-feline antibody clone:IM7,eBioscience, San Diego, CA), CD105 (antibody clo-ne:SN6, eBioscience, San Diego, CA), and CD90 (anti-body clone:eBio5E10, eBioscience, San Diego, CA).MSC were also assessed for expression of CD4 (anti-feline antibody clone:3-4F4, Southern Biotech, Bir-mingham, AL) and feline MHC class II (antibodyclone: TU39, BD Biosciences, San Jose, CA). Sampleswere analyzed using a Cyan ADP flow cytometer(Beckman Coulter, Brea, CA).

In vitrodifferentiation assayswere conducted to con-firm the multipotency of feline MSC, as assessed bytheir ability to differentiate into three cell lineages (oste-oblasts, chondrocytes, and adipocytes) that are charac-teristic ofMSC.25Assayswereperformedby incubatingconfluent MSC with medium supplemented with

Please cite this article in press as: Quimby JM, et al., Evaluationchronic kidney disease in cats: a pilot study, J Feline Med Surg (2

factors to stimulate differentiation.25 At the end of thedifferentiationperiod, cellswerefixedwith 10%neutralbuffered formalin andstainedwithOil redO (SigmaAl-drich, St Louis,MO) for presenceof lipid,with toluidineblue (Richard-Allan Scientific, Kalamazoo, MI) for car-tilage matrix, or with Alizarin red (Sigma Aldrich, StLouis,MO) for the presence of calcium.25MSC culturedin MSC media alone under identical conditions wereused as differentiation controls.

Intrarenal injection of MSC

Nine cats (two healthy cats, seven cats with CKD)were enrolled in this study. Two healthy cats andfour CKD-affected cats were ultimately treated withMSC in the study. Table 1 summarizes the treatmentsreceived for all cats. Of the seven CKD cats were en-rolled in the study initially, bmMSC could not be cul-tured to produce sufficient treatment quantity in threeof the cats. Increasing doses of MSC were adminis-tered to the enrolled cats. Each recipient cat was se-dated using intravenous administration of ketamine3.3e4.8 mg/kg, once (dose repeated once if needed),midazolam 0.1 mg/kg, and butorphanol 0.1 mg/kg,placed in lateral recumbency, and monitored as previ-ously described. Harvested MSC suspended in PBSwere divided into three 150e200 ml aliquots and in-jected using a 25-gauge needle into three sites in therenal cortex under ultrasound guidance. The corticalinjection sites were assessed for hemorrhage 1 h and24 h after injection using ultrasonography.

Determination of GFR by nuclear scintigraphy

GFR was assessed in each kidney of cats injected withMSC using nuclear scintigraphy and was performedjust prior to injection of MSC, 7 days after injection,and 30 days after injection. Cats were sedated (ket-amine 3.3e4.8 mg/kg and butorphanol 0.1 mg/kg IVonce) at a standard time before the procedure. A tech-nician performed all of the procedures for each partic-ular cat. For each procedure 1.0 mCi of Tc99m-labeledDTPA (Cardinal Health, Dublin, OH) was injected in-travenously via a catheter placed in a standard loca-tion in each cat. Images were obtained using GEMillenium SPECT system (GE Healthcare, Waukesha,WI). Three independent radiologists evaluated theGFR data, and a mean GFR value for each kidney aswell as a global value was determined.

Assessment of GFR variability

To assess the degree of intra-patient variability in re-peated GFR measurements as determined by nuclearscintigraphy, three cats with CKD that did not receiveMSC underwent nuclear scintigraphy GFR assess-ments on two occasions, 1 week apart. Data fromthese cats was used to provide information on inher-ent GFR variability and help interpret the results ofGFR measurements made in cats with CKD treatedwith MSC.

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

4 JM Quimby et al

Clinical monitoring of treated cats

Each treated cat underwent physical examination androutine blood work (CBC, serum biochemistry, urinal-ysis, and determination of UPC) immediately prior toMSC injection and on day 7, day 30, and day 60 afterMSC injection.

Histopathology

Two cats with stage VI CKD were humanely eutha-nased in consultation with their owners due toprogressive renal failure. The tissues of both theMSC-injected and non-injected kidneys were exam-ined histopathologically using hematoxylin and eosinstained sections.

Statistical analysis of data

Changes in GFR, serum creatinine, blood urea nitro-gen (BUN) and packed cell volume (PCV) data overtime in the MSC-injected cats were evaluated by re-peated measures ANOVA, followed by Bonferroni’scorrection. Values were considered statistically differ-ent for P< 0.05. Statistical analyses were done usingPrism5 software (GraphPad, San Diego, CA).

Results

Autologous MSC culture

Feline MSC developed over a 1- to 2-week period intoa relatively homogeneous population of plastic-adher-ent cells with fibroblast-like morphology (Fig 1).BmMSC from CKD cats were difficult to expand to ob-tain sufficient quantities for injection, and for threecats that were initially enrolled in the study, adequate

Fig 1. Phenotype of feline aMSC in culture. Feline adipose-derived MSC were established from adipose tissue biopsies,as described in the materials and methods. Cells assumeda typical elongated morphology, as described previouslyfor feline MSC.26 (Feline adipose MSC, magnification 10�.)

Please cite this article in press as: Quimby JM, et al., Evaluationchronic kidney disease in cats: a pilot study, J Feline Med Surg (2

cells could not be obtained. In contrast, it was ob-served that aMSC were easier to culture and expand,and sufficient cells for treatment were easily obtainedfrom all three CKD cats enrolled.

Characterization of feline MSC

Both bmMSC and aMSC expressed high levels ofCD44, CD90 and CD105 and were negative for expres-sion of CD4 and MHC class II (Fig 2), which was con-sistent with the phenotype described previously forfeline MSC.26 Both bmMSC and aMSC were capableof trilineage differentiation (Fig 3).

Short-term safety of intrarenal injectionof MSC in cats

Cats enrolled in the studywere observed for adverse ef-fects immediately following intrarenal MSC injection,includingphysical examination to assesspulse, respira-tion, mucus membrane color and abdominal or renaldiscomfort. By ultrasound examination, we did not ob-serve hemorrhage 1 hor 24 hpost-injection in anyof thetreated cats. The cats appeared clinically normal, andrenal discomfort was not elicited on abdominal palpa-tion.Oneof the healthyyoung cats developed transient,microscopic hematuria 24 h after MSC injection, butwas not otherwise clinically affected.

Assessment of GFR variability

GFR values were determined on two separate occa-sions for three cats with CKD that did notreceive MSC injection. These studies revealed thatthe mean total GFR for the three untreated cats was1.46� 0.28 ml/kg/min on week 1 and 1.55�0.08 ml/kg/min on week 2. The mean calculated var-iation between repeated GFR values for the three un-treated cats with CKD between week 1 and week 2was 9.6% (data not shown).

Effects of MSC injection on GFR

In the two healthy, young cats that received intrarenalMSC injection, the mean pre-treatment global GFR(sum of both kidneys) as determined by nuclear scin-tigraphy was 3.3� 0.57 ml/kg/min (mean and SD),the mean 7-day GFR was 2.7� 0.86 ml/kg/min, andthe mean 30-day GFR was 3.6� 1.5 ml/kg/min.

Renal function in three cats with CKD that receivedunilateral intrarenal injection of MSC (one bmMSC,two aMSC) was evaluated immediately prior to MSCinjection, onday 7post-injection, and onday 30 post-in-jection. A fourth cat was unable to undergo GFRmoni-toring via nuclear scintigraphy due to fractious nature.The mean pre-treatment global GFR for the three catswith CKD was 0.88� 0.53 ml/kg/min, (median0.72 ml/kg/min) which was 35% of the reported nor-mal global GFR value for healthy adult cats (2.5 ml/kg/min).27 Following MSC transfer, mean globalGFR value for the three MSC-injected cats was

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

Fig 2. Expression of cell surface markers by feline adipose-derived MSC. Primary cultures of feline MSC obtained from cats3, 7 and 8 were passaged three to five times in culture, then collected by trypsinization and immunostained for assessment ofcell surface marker expression by flow cytometry, as described in the materials and methods. Feline adipose MSC expressedhigh surface levels of CD44, CD90, and CD105 (panel A), but did not express CD4 or MHC class II (panel B). Isotype controlsare represented in red and unstained MSC are represented in blue. Similar results were obtained with adipose MSC from twoadditional young healthy research cats (not described in this study).

5Mesenchymal stem cell therapy for kidney disease

1.1� 0.59 ml/kg/min (median 0.74 ml/kg/min) at 30days, which though numerically increasedwas not sig-nificantly different from the pre-treatment global GFRvalue.WhileGFRvariability in non-treated catswas as-sessed to be approximately 9.6%, CKD cats 7 and 8 hadglobal GFR increases of 16% and 55% from baseline, re-spectively. In cat 3, who received bmMSC, no changefrom baseline was observed.

GFR values were also determined for individualkidneys of cats with CKD injected with MSC (Fig 4).In the MSC-injected kidney of all three cats, themean pre-treatment GFR value was 0.45� 0.39 ml/kg/min, (median 0.26 ml/kg/min) compared witha mean value of 0.58� 0.54 ml/kg/min (median0.28 ml/kg/min) determined on day 30. For thenon-injected kidney, the mean pre-treatment valuewas 0.42� 0.14 ml/kg/min, (median 0.46 ml/kg/min) compared to 0.47� 0.05 ml/kg/min (median0.46 ml/kg/min) on day 30. These data suggesteda trend towards increasing GFR values for the injectedkidney.

Effects of MSC injection on clinical parametersof renal function and outcome

In the two healthy cats that received intrarenal injec-tions with bmMSC, we did not observe substantial

Please cite this article in press as: Quimby JM, et al., Evaluationchronic kidney disease in cats: a pilot study, J Feline Med Surg (2

changes in relevant laboratory values (serum creati-nine, BUN, PCV, UPC ratio) measured before and af-ter MSC injection (data not shown).

In CKD cats 7 and 8, that received aMSC, a modestthough statistically insignificant improvement in se-rum creatinine concentration was noted, particularlyat the 60-day time point (Fig 5). The PCV, BUN, andUPC ratio values were unchanged following treat-ment. Cat 7 was euthanased for acute lymphocyticleukemia 4 months after completing the MSC study,an outcome which was considered unrelated to thestudy itself. Cat 8 was still alive with stable CKD 10months after MSC injection.

In CKD cat 3 (stage IV: creatinine 5.4 mg/dl) thatreceived bmMSC, a small improvement in serum cre-atinine was observed on day 7 (creatinine 5.1 mg/dl)and day 30 (creatinine 5.2 mg/dl), and the UPC valueincreased over the same time period (initial UPC 1.1;30-day UPC 3.3). This cat traveled some distance forits 60-day recheck (800 miles) and although theowners perceived the cat was doing well at home be-fore travel, renal function in this cat had declined sub-stantially upon arrival (creatinine 8.2 mg/dl), and thecat was subsequently euthanased at day 100.

In CKD cat 9 (stage IV: creatinine 6.5 mg/dl) thatreceived aMSC, a small change in creatinine wasseen at day 7 (creatinine 6.1 mg/dl) but by day 30

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

Fig 3. Trilineage differentiation of feline aMSC. (A) Control aMSC incubated in standard media stained with Oil red O. (B)aMSC produced intracellular lipid vacuoles when incubated in adipocytic differentiation media for 21 days. (C) ControlaMSC incubated in standard media stained with Alizarin red. (D) aMSC stained positive for calcium with Alizarin redfollowing differentiation into osteocytic phenotype after 21 days of incubation in differentiation media. (E) Cryosection ofpellets of cartilage matrix (stained with toluidine blue) formed by aMSC when exposed to chondrocytic differentiation mediafor 21 days.

6 JM Quimby et al

this cat was clinically less stable (creatinine 8.4) andwas subsequently euthanased at day 42.

Histopathology

Histopathologic examination of the kidneys was per-formed in both CKD cats 3 and 9. In cat 3, therewere abundant numbers of lymphocytes and plasmacells found multifocally, expanding the interstitiumas well as extensive interstitial fibrosis. Rare subcap-sular cysts were noted. Tubules were frequently lost,atrophied and ectatic with some evidence of regener-ation and frequently contained luminal protein castsand scattered calcium oxalate crystals. There was min-eralization of tubules within the cortex and medulla.Periglomerular fibrosis, glomerulosclerosis, minerali-zation and atrophy were present in 50% of glomeruli.In cat 9 there were lymphocytes, plasma cells andlesser neutrophils multifocally throughout the intersti-tium. There was tubular loss, atrophy and regenera-tion often associated with fibrosis. Numeroustubules contained sloughed epithelial cells, as wellas, cellular, protein and waxy casts. Multifocally, tu-bules contained intraluminal crystals that were

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crescent or circular with radiating spokes and polar-ize. There were crystals present within the pelvis,which was lined by hyperplastic epithelium. Periglo-merular fibrosis, glomerulosclerosis and a thickenedbasement membrane were present in 95% of glomer-uli. Examination of the MSC-injected kidney of eithercat did not show evidence of pathologic changes (vas-cular anomalies, tissue disruption, abnormal cell type)at the presumed injection sites.

DiscussionCKD is a major cause of morbidity and mortality incats. In this study, intrarenal injection of autologousMSC was well tolerated in IRIS stages II and IIICKD cats and may have induced mild improvementin renal function. Two cats with CKD exhibited mildimprovement in creatinine and GFR values after intra-renal injection of MSC, though the differences werenot statistically significant, which can be attributedin part to the small numbers of animals enrolled inthis study. As this was a pilot feasibility and safetystudy, an untreated group was not included. Twocats with IRIS stage VI did not experience

of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005

Fig 4. Changes in GFR over time in cats with CKD that re-ceived unilateral intrarenal MSC injections. GFR via nuclearscintigraphy was evaluated prior to treatment, on day 7 andday 30 in three cats for the injected kidney (A) and non-in-jected kidney (B). Modest improvement in GFR in the in-jected kidney was seen in one cat. Mild improvement inGFR of both kidneys was seen in one cat. While GFR vari-ability in non-treated cats was assessed to be approximately9.6%, CKD cats 7 and 8 had global GFR increases of 16% and55% from baseline, respectively.

Fig 5. Changes in serum creatinine concentration over timein cats with CKD that received unilateral, intrarenal MSC in-jections. Serum creatinine concentrations were evaluatedprior to treatment, on day 7, day 30 and on day 60 in twocats that completed a 60-day trial of MSC injection therapy.There was a 9.8% overall decline in mean serum creatinineat day 60 compared to pre-treatment value in the two cats.

7Mesenchymal stem cell therapy for kidney disease

improvement in renal values and both cats were sub-sequently euthanased for progression of disease. Bothcats had end-stage disease at enrollment, with a pre-dicted median survival time of 44 days.28 Histopathol-ogy in cat 9 revealed the presence of crystals,consistent with melamineecyanuric acid, and thiscould have affected the cat’s ability to respond toMSC therapy. Although it is possible the intrarenalMSC injections may have precipitated decline in thesecats, we believe this unlikely as the decompensationoccurred sometime after intrarenal injection.

The difficulty experienced in expanding autologousbmMSC caused us to abandon these cells in favor ofaMSC for the remainder of the study. The use ofaMSC in lieu of bmMSC is advantageous for severalreasons. Collection of aMSC was less technically chal-lenging and larger numbers of MSC could be obtainedfrom the fat biopsy. Age and disease status may have

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also reduced the ability to establish and expandbmMSC cultures to a greater degree than with aMSCcultures, a phenomenon that has been observed inother species.29

As this was a pilot study, with no previous safetydata for MSC intrarenal injection to rely on, a dose-es-calation study was conducted. Potentially, the use ofdose-escalation as opposed to a single dose of MSCis a limitation of this study and could affect the abilityto compare results between enrolled cats. Though theintrarenal injection method for MSC treatment of CKDin cats was feasible and appeared safe, our experiencewith this study leads us to believe that this approachis unlikely to be readily applicable clinically. Duringthe study we observed that the multiple sedations re-quired for obtaining bone marrow or adipose tissuesamples, injections, and monitoring of renal functionall contributed to substantial stress for the animalsthat over time could have adversely affected renalfunction. For example, the owners of all three catswith CKD noted that the hospital visits and sedationevents affected the normal behavior of the cats andthat it was generally 1e2 days before the cats resumedtheir normal behavior. Therefore, despite the potentialbenefits of intrarenal MSC injections for CKD in cats,we believe the large number of sedations and inter-ventions required to implement this approach wouldpreclude widespread clinical application.

Additionally, recent literature suggests that the in-trarenal injection model utilized in this project maynot be necessary, and this is a potential limitation of

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8 JM Quimby et al

this pilot study. It is now generally thought that para-crine mechanisms are responsible for the therapeuticbenefit seen in renal disease and that MSC do notneed to be injected into the site of interest due to theirmigratory capabilities.7,19,20 This is a potential expla-nation for the mild bilateral increase in GFR in cat 7;MSC could have also affected the non-injected kidney.

Thus, it may be preferable to consider alternativeMSCsources and routes of administration for treatmentof cats with CKD. Compelling support for intravenousadministration of MSC comes from a recent study bySemedo et al.7 In this study, it was reported in a rodentmodel of CKD that repeated intravenous delivery ofrelatively small numbers of MSC could elicit a signifi-cant positive impact on renal function and renal inflam-mation. Additionally, use of allogeneic instead ofautologous MSC would eliminate the need to sedatethe animals to obtain tissue samples and acceleratetreatment administration. Allogeneic MSC have beenwidely used in experimental stem cell transfer investi-gations, including clinical trials in humans, and thuswould not be expected to elicit unexpected adverse ef-fects relative to autologous MSC therapy.30�32

In summary, the results of the pilot study reportedhere lead us to conclude that MSC transfer by the in-trarenal route is feasible in cats with CKD. Unilateralinjection of the MSC did not adversely affect renalfunction in cats with CKD and may have elicited mod-est improvement in renal function in two of the fourtreated cats. Despite the possible benefits of intrarenalMSC injections for CKD in cats, we believe the largenumber of sedations and interventions required to im-plement this approach preclude widespread clinicalapplication. Continued evaluation of the efficacy ofMSC therapy in cats with CKD is warranted, but alter-native sources and routes should be explored.

AcknowledgementsThe authors wish to acknowledge the assistance of DrsA Marolf, D Gall and A Valdez for analyzing nuclearscintigraphy results andDrMichael Lappin for provid-ing SPF cats used in this study. This study was sup-ported in part by grants from the Winn FelineFoundation and the Morris Animal Foundation.

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of intrarenal mesenchymal stem cell injection for treatment of011), doi:10.1016/j.jfms.2011.01.005