Author's response to reviews Title: The fate of bone marrow-derived cells carrying a polycystic kidney disease mutation in the genetically normal kidney Authors: Elizabeth Verghese (Elizabeth.Verghese@vu.edu.au) Chad Johnson (Chad.Johnson@monash.edu.au) John F Bertram (John.Bertram@monash.edu.au) Sharon D Ricardo (Sharon.ricardo@monash.edu.au) James A Deane (James.deane@monash.edu.au) Version: 3 Date: 2 April 2012 Author's response to reviews: see over
Re: Manuscript MS 1635539397642485 The fate of bone marrow-derived cells carrying a polycystic kidney disease mutation in the genetically normal kidney Dear Editor We would like to thank the reviewers for taking the time to review our manuscript. Please find attached a revised version of our manuscript (#1635539397642485: The fate of bone marrow-derived cells carrying a polycystic kidney disease mutation in the genetically normal kidney) that has been modified as suggested. Changes to the manuscript are detailed below in a point by point response to the concerns raised by the reviewers. We have also uploaded high resolution versions of the figures. We hope that this revision is satisfactory and look forward to hearing from you. Yours sincerely Dr Elizabeth Verghese PhD, Grad Cert (Higher Ed) Lecturer School of Biomedical and Health Sciences Victoria University PO Box 14428 Melbourne, Vic 8001 AustraliaPhone +61 3 9919 2557 Email: Elizabeth.Verghese@vu.edu.au
Reviewer: Hanna Rennert Major points 1. It is not clear from the results how successful the BMT was. Figure 1 demonstrates results of PCR analysis for the sry gene, but does not provide any quantitative information regarding the success rate. The authors should be more specific regarding the number of animals and the number of cells that successfully engrafted. We used an established protocol that gives approx 80% donor chimerism as measured by GFP in peripheral blood (Li et al JASN 2006), Body weight was also monitored and in the event of weight loss or absent/weak sry detection of engraftment, mice were excluded. Ultimately the detection of BM-derived cells in the kidney is a measure of successful engraftment (eg fig 4c). Numerous BM derived cells were found and analyzed which was the aim of the experiment. Reference to our previous experience with bone marrow transplantation and monitoring body weight is now included in methods and results. 2. In Figure 3 the authors show that the percentage of Y chromosome-positive cells is declining in the kidney. Was that decline also observed in the animals' peripheral blood and how do the authors explain this decline? This is probably due to the resolution of the inflammatory infiltrate. The main point that we are aiming to make in this figure is that BM-derived cells were readily detectable at the each of the time points examined. 3. This sentence in the Results section These frequencies and total numbers of Y chromosome-positive cells observed are consistent with previous reports of BM-derived cells on the tubule [21] should be moved to the Discussion. The authors should also clearly state the mean percentages of Y- chromosome positive cells in the peripheral blood and kidneys as well as the number of animals assessed. We do not have peripheral blood data for these experiments, but when we have monitored engraftment using GFP in the past approximately 80% of leukocytes in peripheral blood are donor derived. This previous work is now cited [24]. Numbers of mice assessed are in the methods and have been added to the fig legend Sections assessed were from 2 mice a 2 weeks, 3 mice at 4 weeks and 2 mice at 12 weeks. 4. The authors note in the Results that BM-derived cells were only found as scattered examples in the renal tubule. The low number of these cells in the
kidney may reflect unsuccessful transplant which would be a limitation of this work, but this is not addressed in the Discussion and needs to be added. Cells were scattered in the kidney, but were not rare indicating robust engraftment (Figure 4c). It was only in the tubule that BM-derived cells were uncommon, but this is as previously reported by other investigators. 5. The authors also indicate that the longer time point (twelve weeks) assessed is sufficient for the post-ischaemic recovery and the development of cystic pathology, however, this may not be sufficient time for complete engraftment, particularly since no information is provided regarding the success of the BM transplant. BM cells contribution to epithelial cells following HSCT is well documented, including in humans. However this may be depended on level of engraftment. This should be discuss in more details. Repopulation by transplanted bone marrow in the irradiated mouse occurs rapidly. Six weeks was allowed for engraftment prior to surgical induction of injury and then a further 12 weeks recovery in our longest time point. Previous studies investigating BM-derived epithelial cells in the kidney have examined mice 4 to 8 weeks after BM transplantation (eg Lin et al 2005 J Clin Invest 115:1756-1764., Duffield et al 2005 J Clin Invest 2005, 115(7):1743-1755.) Minor points 6. Introduction- ADPKD is caused by inherited mutations in one of two genes: PKD1 and PKD2, followed by secondary somatic mutations in the renal epithelial cells leading to cysts development. This is not indicated in the text and should be added. A sentence explaining this has been included in the introduction. In ADPKD, cysts have been reported to originate from the clonal expansion of epithelial cells with an inherited mutation plus an acquired somatic mutation of one of two ADPKD-related genes (PKD1 or PKD2). 7. Results, page 8- Detection rates were not 100% because the Y chromosome is not necessarily be included in a cross-section of every nucleus examined. the Y chromosome is not necessarily included? Text corrected. Detection rates were not 100%, even in male control kidney, because the Y chromosome is not always present in the remaining portion of sectioned nuclei. 8. Results, page 9- From 120 examples of Y chromosome positive cells examined from kidney 2, 4 or 12 wks after injury, only a 10 (2 at 2wk, 5 at 4 and 3 and 12wk) were sufficiently entwined with the tubule that an epithelial
phenotype might be considered. Check sentence. only 10 examples? Text corrected. From 120 examples of Y chromosome positive cells examined, only 10 cells were sufficiently entwined with the tubule that an epithelial phenotype might be considered (2 cells at 2 weeks, 5 cells at 4 weeks and 3 cells at 12 weeks). 9. Discussion, page 12- Many of the initial studies in this area appear to have overestimated the contribution of BM-derived cells to renal epithelia due to unreliable tracing and phenotype determination techniques.. Provide references. Duffield et al 2005 JCI [19] is now cited.. 10. Manuscript should be check carefully for typos and grammar. There are numerous typos throughout the text: Introduction-2nd paragraph change donour to donor. Figure legend 3: change inury to injury ; Figure legend 6: change modelling to modeling, epithithelial to epithelial. Typos have been corrected and spelling Americanized. Reviewer: Anna Paola Paola Mitterhofer Major Compulsory Revisions 1. In the paper the aim of the study is not clearly expressed and the assumptions described in the background don t bind together. Moreover the key points are not correct. Authors describe the study but not the objectives of the study. Authors assess that In light of reports cells derived from hematopoietic stem cells in the bone marrow (BM) can integrate into the kidney and form epithelial cells referring to papers published in 2001. They don t consider that it has been widely demonstrated in subsequent studies that the role of bone marrow-derived cells to epithelial integrity restoration after an ischemic insult is very poor and not significant. (Reference 15.,16., Kidney tubular epithelium is restored without replacement with bone marrowderived cells during repair after ischemic injury Duffield JS, Bonventre JV. Kidney Int. 2005 Nov;68(5):1956-61). Authors assess that the reoccurrence of PKD has not been reported in a genetically normal renal graft but nevertheless they decide to use PKD (Polycystic Kidney Disease) mutant bone marrow-derived cells to evaluate PKD cell capability of expressing the disease phenotype in ischemic genetically normal kidney. This to explain a phenomenon which notoriously doesn t happen.
While there is a consensus that BM-derived cells are not major contributors to epithelial repair in the kidney, low level contributions have been reported in several recent studies. More recent reports of BM-derived epithelial cells include (Lin et al 2005 JCI; Yang et al J Am Soc Nephrol 22: 2028 2036, 2011, Syres et al 2009 Blood 114: 2542-2552, Li et al 2012 Am J Phys Renal 302:F9-F19). These studies are now cited in the manuscript. To our knowledge, there has been no specific examination of bone marrow-derived cells carrying a PKD causing in the transplanted human kidney. While macroscopic manifestations of engrafted PKD cells can presumably be excluded, the existence and phenotype of rare tubular cells could be informative with regard to the process of cystogenesis. We reasoned that it would be worthwhile to investigate this question in a controlled mouse model where tissue preparation and cell detection techniques could be optimized. 2. methods applied seem to be well conduct referring to a previous study of the same authors on post- ischemic acute tubular necrosis. Authors show many nice figures, well described. 3. part of methods are described in background and should be shifted. (from We used a mouse model to test whether mutant. to to induce transplanted BM cells to home to the kidney and give rise to renal epithelial cells. We feel that this needs to stay in the introduction as it gives an overview of our experimental approach. Specific details are in the methods section. 4. the data reported are well described but the research doesn t constitute a useful contribution to the field The examination of putative BM-derived epithelial cells in the kidney has been complicated by a lack of unambiguous markers. We have use bone marrow with a mutation causing epithelial overproliferation, a phenotype that has the potential to identify a genuine epithelial contribution. We also address the issue of the stage of repair by examining three time points. Additionally we note a qualitative difference in the Y-chromosome signal from epithelial and interstitial cells that is informative. 5. data consist in the careful description of pathology results of the experiment conducted obtained step by step, with three-dimensional reconstruction and description 6. most conclusions are foregone Authors discuss data already known in literature, related to the role of BM-derived cells in ischemia restoration, but they refer to these reports only in the discussion and conclusion. This means that study design is completely unfounded References 9, 10, 11, 12, 13, 16, 18, 19, 20 refer to the BM-derived cells and
ischemia (or acute renal failure) and are included in introduction. Reference 20 has been added to the introduction and is a review reinforcing the opinions (that BM does not give rise to epithelium) expressed in reference 19. 7. limitations of the work are not clearly stated We discuss the rarity of BM-derived cells in the renal tubule, the difficulty of unambiguously determining the identity of BM-derived cells in the renal tubule, and the possibility of BM-derived cells fusing with the renal tubule. 8. title and abstract accurately convey what has been found. Authors avoid the ischemic field already explored with BM-derived cells This field is discussed and referenced throughout (References 9, 10, 11, 12, 13, 16, 18, 19, 20). Additional relevant references have been added including some more recent reports (references 14, 15, 16) 9. the writing is acceptable