Abrahamson, Dale R.

Anders, Hans-Joachim

Antignac, Corinne

Borza, Dorin-Bogdan

Cosgrove, Dominic

Ding, Jie

Gregory, Martin

Gross, Oliver

Gubler, Marie-Claire

Harvey, Scott

Heidet, Laurence

Hudson, Billy

Kashtan, Clifford

Lees, George

Licht, Christoph

Miner, Jeffrey H.

Savige, Judy

Segerer, Stephan

Thorner, Paul

Torra, Roser

Tryggvason, Karl

Weber, Manfred

RERO DOC Digital Library

Nephrol Dial Transplant (2009) 24: 731–734doi: 10.1093/ndt/gfn722Advance Access publication 25 December 2008Translational NephrologyStem cell therapy for Alport syndrome: the hope beyond the hype∗Oliver Gross1, Dorin-Bogdan Borza2, Hans-Joachim Anders3, Christoph Licht4, Manfred Weber5,Stephan Segerer6, Roser Torra7, Marie-Claire Gubler8, Laurence Heidet9, Scott Harvey8,Dominic Cosgrove10, George Lees11, Clifford Kashtan12, Martin Gregory13, Judy Savige14, Jie Ding15,Paul Thorner16, Dale R. Abrahamson17, Corinne Antignac8, Karl Tryggvason18, Billy Hudson2 andJeffrey H. Miner191Department of Nephrology & Rheumatology, University of Go¨ttingen, Go¨ttingen, Germany, 2Department of Medicine, Division ofNephrology, Vanderbilt University Medical Center, Nashville, TN, USA, 3Department of Nephrology, Medical Policlinic, Universityof Munich, Munich, Germany, 4Division of Nephrology, The Hospital of Sick Children, University of Toronto, Toronto, Canada,5Merheim Medical Center, University of Witten-Herdecke, Campus Cologne, Cologne, Germany, 6Clinic for Nephrology, USZ andInstitute of Anatomy, University of Zurich, Switzerland, 7Nephrology Department, Fundacio Puigvert, Barcelona, Spain, 8InsermU574, Universite Paris Descartes, 9Centre de Re´fe´rence des Maladies Re´nales He´re´ditaires de l’Enfant et de l’Adulte (MARHEA)Service de Ne´phrologie Pe´diatrique, Hoˆpital Necker-Enfants Malades, Paris, France, 10Boys Town National Research Hospital,Omaha, NE, 11Department of Small Animal Clinical Sciences, Texas A & M University, College Station, TX, 12Division of PediatricNephrology, University of Minnesota, MN, 13Nephrology and Hypertension Division, Department of Internal Medicine, TheUniversity of Utah School of Medicine, UT, USA, 14Department of Medicine, The Northern Hospital, The University of Melbourne,Epping, Australia, 15Department of Pediatrics, Peking University, First Hospital, Beijing, China, 16Department of LaboratoryMedicine, Hospital for Sick Children and University of Toronto, Toronto, Canada, 17Department of Anatomy and Cell Biology,University of Kansas Medical Center, Kansas City, KS, USA, 18Division of Matrix Biology, Department of Medical Biochemistryand Biophysics, Karolinska Institutet, Stockholm, Sweden and 19Renal Division, Washington University School of Medicine, StLouis, MO USAKeywords: alport syndrome; bone marrowtransplantation; renal fibrosis; stem cell therapyAlport syndrome is a hereditary glomerulopathy leading toend-stage renal disease (ESRD), frequently during adoles-cence. It is caused by the absence or abnormal compositionof the type IV collagen α3/4/5 network normally presentin the glomerular basement membrane (GBM) [1]. In theSeptember 2008 issue of the Journal of the American Soci-ety of Nephrology, Katayama and colleagues reported thatbone marrow transplantation (BMT) following lethal irra-diation with either wild-type or Col4a3−/− BM prolongedthe lifespan of Alport mice with similar efficiencies. Sub-lethal irradiation alone also provided significant benefits[2].These results challenge reports fromCook’s andKalluri’sgroups suggesting that wild-type BM-derived cells ame-liorate disease in Alport mice because they can differ-entiate into podocytes and secrete the missing collagenα3/4/5(IV) chains [3,4], which would basically constitute aCorrespondence and offprint requests to: Oliver Gross, Department ofNephrology and Rheumatology, University Hospital Gottingen, Robert-Koch Street 40, 37075 Gottingen, Germany. Tel: +49-551-396331;Fax: +49-551-398906; E-mail: gross.oliver@med.uni-goettingen.de∗Comment on Katayama K et al. Irradiation prolongs survival of alportmice. J Am Soc Nephrol 2008; 19: 1692–1700.curative cell-based therapeutic approach for treating Alportglomerulopathy. Their interpretation that circulating BM-derived cells are recruited to damaged glomeruli where theycan cross the GBM, become podocytes, secrete the miss-ing collagen chains, repair the GBM defects and slow—ifnot reverse [4]—disease progression appeared (and stillappears) incredibly fascinating. However, although theseauthors reported improvements in overall kidney histol-ogy compared with untreated or Col4a3−/− BM-treatedAlport mice, the most meaningful endpoint, i.e. age atESRD, was not tested in either study for reasons not ex-plained [3,4]. This issue is critical, because a previousstudy of Alport mice that received BM-derived mesenchy-mal stem cells also improved renal histology, but there wasno delay of death from renal failure [5].One potentially important difference between the stud-ies of Katayama et al. and of Prodromidi et al. as well asSugimoto et al. is the genetic background of theAlportmiceused. Progression of Alport’s disease in mice is influencedby the genetic background [6]. Amixed genetic backgroundcarries the risk of spoiling the most meaningful endpoint,i.e. the age at ESRD (Figure 1). The increased lifespanin C57BL/6J Alport mice may be explained in part by an‘escape phenomenon’ characterized by an alternative colla-gen switch (Figure 2), whereas the GBM of the C57BL/6JAlport mice used by Prodromidi et al. and Sugimoto et al.[3,4] may be stabilized by incorporation of α5/6(IV) chains,C© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.For Permissions, please e-mail: journals.permissions@oxfordjournals.org732 O. Gross et al.Fig. 1. Age at death from renal failure in Col4a3−/− mice. Lifespan de-pends on the genetic background. Furthermore, the standard deviation inlifespan strongly depends on a clean genetic background (see box plot inthe middle, crossbreeding of C57BL/6J and 129 × 1/SvJ-backgrounds).The immense standard deviation in an unclean background might con-found interpretation of experiments, including histological evaluations.Fig. 2. Immunostaining of different α(IV) collagen chains in Alportmouse kidneys. The GBM deposition of the α3/4/5(IV) chains dependson the gene defect (heterozygous or homozygous state) as well as on thegenetic background. Note the ‘escape phenomenon’ within the GBM ofC57BL/6J Col4a3−/− mice: these mice incorporate significant amountsof α5/6(IV) chains into their GBM. This phenomenon might contributeto the improved lifespan of Alport mice on this background (Figure 1) ascompared to the 129× 1/SvJ Alport mice, which showmuch less α5/6(IV)incorporation.possibly increasing the lifespans of the mice. The GBM ofthe 129 × 1/SvJ Alport mice used by Katayama et al. [2]exhibits far lower (though still detectable) incorporationof the α5/6(IV) chains, which correlates with their rapidprogression to ESRD [7].Cell-based therapies aim towards repairing the underly-ing defect: here, the defective assembly of α3/4/5(IV) col-lagen. Katayama et al. certainly shared this aim, but theyfound no deposition of α3/4/5(IV) collagen in their WTBM-transplanted Alport mice, despite the extended lifes-pan [2]. We disagree with the claim in LeBleu and Kalluri’srecent editorial [8] that ‘careful examination of immuno-histochemistry and Western blot images in the study byKatayama et al. reveals a likely faint labelling for α3 chain’.Although both Prodromidi et al. and Sugimoto et al. con-cluded that α3(IV) was present in the GBM of their BM-transplanted Alport mice [3,4], neither presented definitivesupporting evidence. Focal staining for α3(IV) [3,4] doesnot prove true GBM deposition; patients with X-linked Al-port syndrome show intracellular α3(IV) mRNA and pro-tein in podocytes that is unaccompanied by α4 or α5(IV)[9], and similar findings have been reported in Alport dogs[10]. Furthermore, widespread linear staining for α5(IV) inthe GBM of Sugimoto et al.’s treated Alport mice, whichthey presented as evidence for α3/4/5(IV) deposition [4],might then be irrelevant due to the alternative switch [7]discussed above. Surprisingly, Sugimoto et al.’s untreatedCol4a3−/− mice did not show GBM staining for α5(IV)[4]. This might be explained by an impure genetic back-ground, with the untreated Col4a3−/− mice having somecontribution from a 129 × 1/SvJ (129) or another back-ground. This could also explain their worse renal functionas compared to the BMTgroup, which shows the alternativeswitch.A convincing demonstration of assembly of the correctGBM collagen IV network requires biochemical evidenceof α3/4/5(IV) chain co-assembly, such as by assaying thatchains are co-immunoprecipitated by monoclonal antibod-ies with well-defined α(IV) chain specificities. Even as-suming that small amounts of α3/4/5(IV) were depositedin the Col4a3−/− mouse GBM after BMT [3,4], it is un-likely that such a focal restoration of the correct networkwould improve renal survival. Indeed, insufficient incor-poration of α3/4/5(IV) into the GBM can lead to severekidney involvement in Col4a5+/− female mice, in which∼50% of podocytes are fully capable of producing thegenuine α3/4/5(IV) collagen network [11]. Nevertheless,Sugimoto et al. did detect α3/4/5(IV) in whole kidneylysates. Western analyses show up to half as much α3/4/5noncollagenous (NC1) domain monomers in the trans-planted mutant kidneys as compared to the wild-type con-trol kidneys. Given that the α3/4/5(IV) network is foundthroughout the cortex in proximal tubules and in collectingducts, as well as in the GBM, widespread incorporationof collagen IV-secreting transplanted cells throughout theparenchyma would be needed to achieve such levels. Anal-ysis of isolated glomeruli would be necessary to provideconvincing evidence of incorporation of the α3/4/5(IV) net-work in GBM and this was not performed [4].Katayama et al. show that irradiation alone providesbenefits without BMT [2], implying that a significantStem cell therapy for Alport syndrome 733portion of the amelioration of disease reported by Cook’sand Kalluri’s groups [3,4] might have little to do with thetransplanted cells. Some statements (one of them quotedabove) in LeBleu and Kalluri’s recent editorial [8] onKatayama’s paper appear to be designed to weaken its im-pact. As the data of Katayama et al.’s study challenge theproposed concept/mechanism of BMT therapy for Alportsyndrome, a careful analysis of their data and their approachis necessary to avoid further controversy with regard to theimportance of these studies:1. Katayama’s study was performed in Col4a3−/− miceon the 129× 1/SvJ background,which renders compar-isons to data generated on the C57BL/6J backgroundpotentially problematic. The age at the time of BMTalso differed among the studies. The different back-grounds and ages at BMT could be responsible, in part,for the different outcomes, given differences in timingand severity of GBM defects and their potential effectson the behaviour of the transplanted BM cells. Indeed,LeBleu and Kalluri suggest that Katayama’s 129 ×1/SvJ Alport mice, transplanted at 3 weeks, would nothave the GBM defects that are ‘a likely prerequisitefor successful cell-based therapy’ [8]. Yet results in thetwo references cited [12,13] and others [6] indicate thatit is highly unlikely that at 3 weeks of age the AlportGBM on the 129 background is normal. More impor-tantly, there is no evidence to support the assumptionthat GBM damage is a pre-requisite for a stem cellrepopulation.2. Katayama et al. did not directly compare treatmentwithand without BMT at a given irradiation dose. How-ever, the finding that Col4a3−/− mice transplantedwith Col4a3−/− BM after irradiation with 8 Gy(stands for Gray, irradiation dosage) live longer thannon-transplanted Col4a3−/− mice irradiated with 3,6 or 7 Gy (stands for Gray, irradiation dosage) doessupport the author’s interpretation of a dose-dependenteffect of irradiation.3. Katayama et al.’s analysis of α(IV) chain expressionwas limited to 7- and 11-week-old mice, only 4 or 8weeks after BMT. The study lacks an additional latertime point such as 15 weeks of age (12 weeks afterBMT). This later time point would have allowed usto exclude the existence of incomplete BM reconstitu-tion and later progenitor cell migration into the kidney,whichmight have beenmissed at the earlier time points.4. Katayama et al. do not provide data on albuminuria,but the BUN and serum creatinine levels (being highlycorrelated with albuminuria in the Sugimoto study)convincingly demonstrate improvement of renal func-tion.5. Finally, Le Bleu and Kalluri raised doubts [8] aboutKatayama’s finding that irradiated mice lived longerindependent of a significant effect on interstitial fi-brosis. However, previous studies in Col4a3−/− miceon the 129 background have already demonstrated thatthe extent of interstitial fibrosis does not correlate withsurvival time [5].In summary, despite some limitations underscoring ourlack of basic understanding of this emergent area of re-search, the study by Katayama clearly demonstrates thatBMT as a collagen IV replacement therapy does not pro-vide additional benefits beyond irradiation to prolong thesurvival of Col4a3−/− mice on the 129 × 1/SvJ geneticbackground. In agreement with a previous editorial [8], fu-ture studies in this field should use Alport mice with a cleangenetic background. Most importantly, histological studiesshould be supported by showing the numbers of animalstreated and survival curves, including standard deviationsin both, with treated and untreated mice being littermatesof each other.Taking the above conditio sine qua non into account,Col4a3−/− mice have been particularly useful for testingpotential therapies. As a potentially life-threatening proce-dure, BMT will need to be demonstrated to be superior toless dangerous drug-based therapies for Alport nephropa-thy, some of them being broadly used as off-label therapiesthroughout the world. European and American Alport reg-istries currently investigate the long-term outcome and thesafety of these medications. Unfortunately, scientific ar-ticles on the preliminary and controversial basic researchin Alport-mice may be misinterpreted as ‘a cure has beenfound’ and editorial headlines that try to turn doubts aboutthe concept into a proof of the desired truth are mislead-ing and have had unfavourable secondary effects [3,4,8].In fact, the lay press turned the hope of a BMT cure forAlport glomerulopathy into hype. This creates a difficultsituation for clinicians taking care of Alport patients: hopesthat clinicians cannot fulfil put an unfortunate burden onnephrologists dealing with parents who might be desperatebecause of their child’s impaired renal function and whomight be ready to take every chance to help their child.Such hopes might turn into unjustified risks!Our current knowledge about Alport syndrome and itspossible therapy by BMT is as yet incomplete and revealsmany open questions that need to be addressed experimen-tally before clinical BMT studies should be considered inAlport patients. Due to the associated risks of the BMTprocedure, it is currently only being performed in poten-tially lethal diseases. In fact, given the perspective of renaltransplantation, Alport syndrome may not generally qual-ify for BMT in terms of improving patient survival. Thus,any experimental doubt that the effects of BMT are supe-rior to those of other treatments (like drugs or irradiation)increase the need for open-minded discussions and moreresearch before taking inappropriate risks that could even-tually harm our young patients.Conflict of interest statement. C.K. is the executive director of the AlportSyndrome Treatments andOutcomes Registry (ASTOR), USA. O.G. is theprincipal investigator of the European Alport therapy registry, supportedby the Association pour l`Information et la Recherche sur les maladiesre´nale Ge´ne´tiques France and the KfH-Foundation Preventive Medicine.References1. Hudson B, Tryggvason K, Sundaramoorthy M et al. Alport’s syn-drome, goodpasture’s syndrome, and type IV collagen. N Engl J Med2003; 348: 25734 O. Gross et al.2. Katayama K, Kawano M, Naito I et al. Irradiation prolongs survivalof Alport mice. J Am Soc Nephrol 2008; 19: 1692–17003. Prodromidi EI, Poulsom R, Jeffery R et al. Bone marrow-derivedcells contribute to podocyte regeneration and amelioration of renaldisease in a mouse model of Alport syndrome. Stem Cells 2006; 24:2448–24554. Sugimoto H, Mundel TM, Sund M et al. Bone-marrow-derived stemcells repair basement membrane collagen defects and reverse genetickidney disease. Proc Natl Acad Sci USA 2006; 103: 7321–73265. Ninichuk V, Gross O, Segerer S et al. Multipotent mesenchymal stemcells reduce interstitial fibrosis but do not delay progression of chronickidney disease in collagen 4A3-deficient mice. Kidney Int 2006; 70:121–1296. Andrews KL, Mudd JL, Li C et al. Quantitative trait loci influencerenal disease progression in a mouse model of Alport syndrome. AmJ Pathol 2002; 160: 721–7307. Kang JS, Wang XP, Miner JH et al. Loss of alpha3/alpha4(IV) col-lagen from the glomerular basement membrane induces a strain-dependent isoform switch to alpha 5 alpha 6(IV) collagen associ-ated with longer renal survival in Col4a3−/− Alport mice. J Am SocNephrol 2006; 17: 1962–19698. LeBleu VS, Kalluri R. Stem cell-based therapy for glomerular dis-eases: an evolving concept. J Am Soc Nephrol 2008; 19: 1621–16239. Heidet L, Cai Y, Guicharnaud L et al. Glomerular expression of typeIV collagen chains in normal and X-linked Alport syndrome kidneys.Am J Pathol 2000; 156: 1901–191010. Harvey SJ, Zheng K, Sado Y et al. Role of distinct type IV collagennetworks in glomerular development and function. Kidney Int 1998;54: 1857–186611. Rheault MN, Kren SM, Thielen BK et al. Mouse model of X-linkedAlport syndrome. J Am Soc Nephrol 2004; 15: 1466–147412. Cosgrove D, Rodgers K, Meehan D et al. Integrin alpha 1 beta 1and transforming growth factor-beta1 play distinct roles in Alportglomerular pathogenesis and serve as dual targets for metabolic ther-apy. Am J Pathol 2000; 157: 1649–165913. Hamano Y, Grunkemeyer JA, Sudhakar A et al. Determinants ofvascular permeability in the kidney glomerulus. J Biol Chem 2002;277: 31154–31162Received for publication: 28.11.08Accepted in revised form: 1.12.08

Stem cell therapy for Alport syndrome: the hope beyond the hype

Stem cell therapy for Alport syndrome: the hope beyond the hype

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