Title: Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction

Transcription

1 Author's response to reviews Title: Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction Authors: Xi Qiao Lihua Wang Yanhong Wang Xiaole Su Yufeng Qiao Yun Fan Zhiqiang Peng Version: 1 Date: 18 Jun 2017 Author s response to reviews: Answers to the Reviewers' comments Sonia Saad (Reviewer 1): In this manuscript, Qiao et al. aimed to determine whether Intermedin's (IMD's) effect on renal fibrosis is through heme oxygenase-1. The authors used a UUO model of renal fibrosis and a rat proximal tubular epithelial cell line exposed to TGFb to determine the mechanistic effect of IMD. Both models are relevant for the study. The manuscript is well written and the authors are well experienced in the research area. However there is a concern that the experiments performed do not support the conclusion drawn. This is mainly due to the following major points: 1- ZnPP is not a specific inhibitor for HO-1 and is known to have different effects due to light sensitivity (Ny et al. 1995). ZnPP can for instance, abolish acetylcholine-induced vasodilatation in a HO independent manner (zygmunt et al 1994) and is shown to inhibit soluble guanylate cyclase in tissue homogenates (Ignano et al. 1984). Hence in order to determine the specific role of HO-1, mice deficient for HO-1 are required. Such mice were shown to promote renal fibrosis in UUO model (relevant for the current study) (Kie et al. JASN 2008). Answer: Thank you very much for the positive comments, which provided ideas in the further study. As you mentioned, using HO-1 deficient mice is much more specific than HO-1 inhibitor ZnPP to determine the role of HO-1 in renal fibrosis. We used ZnPP in this study as a HO-1

2 inhibitor based on previous reports (see the following references). We have discussed this limitation in the newly revised manuscript (Line 420, Page 21). 1. Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, Zhao D, Zaky J, Melinek J, Lassman CR, Kolls JK, Alam J, Ritter T, Volk HD, Farmer DG, Ghobrial RM, Busuttil RW, Kupiec-Weglinski JW. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest. 1999;104(11): Kim JH, Yang JI, Jung MH, Hwa JS, Kang KR, Park DJ, Roh GS, Cho GJ, Choi WS, Chang SH. Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway. J Am Soc Nephrol. 2006;17(5): the authors did not perform a HO-1 activity assay in the animal tissues (in the right and left kidneys to determine specificity for the IMD effect) and cells to confirm that the inhibitor reduces HO-1 activity as described. Answer: Thank you. We detected HO activity and added these results in the newly revised manuscript (Figure 2d, Figure 3 and supplemental Figure 1). Minor points/questions: -Was ZnPP prepared in dark/light conditions? can the authors comment? Answer: ZnPP was prepared in dark condition, and we have commented this in the newly revised manuscript (Line 127, Page 7 and Line 147, Page 8). -page 14 lane 259: delete "in the" Answer: We have corrected this error in the newly revised manuscript. -page 14 lane 270, delete "of" Answer: We have corrected this error in the newly revised manuscript. -page 16 lane 302, consider using "against" renal fibrosis instead of "including" Answer: Thank you. According to your suggestion, we have replaced against with "including" in the newly revised manuscript. -page 16 lane 311, consider revising the sentence: "we showed that inhibition of HO-1 enzyme activity " since the HO-1 activity was not assessed in this study especially in the UUO kidneys which express increased levels of IMD.

3 Answer: We detected HO activity and added these results in the newly revised manuscript (Figure 2d and supplemental Figure 1). -Can the authors determine the protein levels and activity of HO-1 in Cells treated with ZnPP? Answer: We detected HO-1 protein levels and HO activity in the cells, and added these results in the newly revised manuscript (Figure 3). -Can the authors discuss the renal specific effect of ZnPP and systemic effects? Answer: We have discussed this point in the newly revised manuscript (Line 383, Page 19). - It will be beneficial to the clinical implication of the research outcomes. Answer: The clinical implication of the research outcomes has been included in the newly revised manuscript (Line 441, Page 22). Wai han Yiu (Reviewer 2): The findings of this study provide a protective mechanism of IMD on renal fibrosis via upregulation of HO-1 expression. Authors demonstrated the importance of HO- 1 in preventing inflammation, fibrosis and EMT processes of UUO rat by administrating HO-1 inhibitor, ZnPP. However, the data of the study in incomplete to clearly show the association between IMD/HO-1/renal fibrosis. 1. Although ZnPP is known as HO-1 inhibitor, authors should provide further evidence on the inhibition of HO-1 activity or increased oxidative stress in ZnPP-treated UUO rats. Answer: We detected HO activity and added these results in the newly revised manuscript (Figure 2d). 2. Additional in vitro experiment with the absence of HO-1 may confirm the role of this protein in TGF beta-mediated EMT. Answer: Thank you very much for the positive comments, which provided ideas in the further study. As you mentioned, using cells absence of HO-1 is much more specific than HO-1 inhibitor ZnPP to determine the role IMD plays in TGF-beta-mediated EMT. We used ZnPP in this study as an inhibitor for HO-1 based on previous reports (see the following references). We have discussed this limitation in the newly revised manuscript (Line 422, Page 21). 1. Gong X, Ivanov VN, Hei TK. 2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells. Arch Toxicol. 2016;90(9):

4 2. Yu X, Song N, Guo X, Jiang H, Zhang H, Xie J. Differences in vulnerability of neurons and astrocytes to heme oxygenase-1 modulation: Implications for mitochondrial ferritin. Sci Rep. 2016;6: More inflammatory chemokine or cytokine and fibrotic markers should be demonstrated for the importance of HO-1 in IMD-mediated anti-inflammatory and anti-fibrotic pathway in UUO. Answer: Thank you. We examined inflammatory cytokine IL-6 expression by immunohistochemical assay and added these results in the newly revised manuscript (Figure 5c and 5d). As for the fibrotic markers, we have investigated and reported the influence of IMD on their alterations in the previous study (Qiao X, et al. Nephrology (Carlton). 2015;20: PMID: ). Based on these results, the present study aimed to explain the anti-fibrotic mechanism of IMD, so we didn t repeat the results. We stated the related results in the background section of the newly revised manuscript. (Line 73, Page 5). Abolfazl Zarjou (Reviewer 3): In this manuscript Xi Qiao and colleagues describe anti-fibrotic properties of Intermedin in a UUO model. Importantly, authors provide evidence that such inhibitory properties are mediated via induction of HO-1 which in turn is associated with a decrement in inflammatory cell infiltration. The studies are interesting with potential therapeutic applications and experiments are well designed. A few additions would strengthen the manuscript. Major comments: 1. While the dose of zinc protoporhyrin that is used in this study has been previously shown to inhibit HO activity, this important information is missing from the current manuscript. Another concern is that one dose of zin protoporphyrin may not be sufficient to provide 7 days of inhibition. To clarify this issue authors should measure HO activity in both in vitro and in vivo settings. Answer: Thank you very much for the positive comments. We gave one dose of zin protoporphyrin according to previous report (see the following references). To clarify this issue, we detected HO activity and added these results in the newly revised manuscript (Figure 2d, Figure 3 and supplemental Figure 1). 1. Kim JH, Yang JI, Jung MH, Hwa JS, Kang KR, Park DJ, Roh GS, Cho GJ, Choi WS, Chang SH. Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway. J Am Soc Nephrol. 2006;17(5): It would be interesting to examine some of the transcription factors that are involved in HO-1 modulation to provide further mechanistic insight into the mechanism of Intermedin induced HO-1 activation pathway.

5 Answer: Thank you very much for the positive comments, which provided ideas in the further study. As you mentioned, examining some of the transcription factors that are involved in HO-1 modulation will provide further mechanistic insight into the mechanism of intermedin induced HO-1 activation pathway. We discussed this limitation in the newly revised manuscript (Line 429, Page 21). Minor comments: 1. Discussion should include the limitations of this study. For instance, using a HO-1 KO mouse model would have been preferable as the pharmacological inhibitors are known to have pleiotropic effects. UUO is increasingly criticized as a model of CKD and increasingly other models are replacing UUO such as ischemia-reperfusion, repeated cisplatin nephrotoxicity. Answer: Thank you. We discussed these limitations in the newly revised manuscript (Line 423, Page 21). 2. Please note that zinc protoporphyrin also inhibits HO-2 activity and hence text should reflect this property as well. Answer: Thank you. We discussed this issue in the newly revised manuscript (Line 380, Page 19). 3. A few spelling and grammatical errors are present. Please review manuscript carefully prior to resubmission. For instance page 5, line 77 change "resent" to "recent" Answer: Thank you. We have double checked our manuscript and corrected the spelling and grammatical errors in the newly revised manuscript. Elangovan Vellaichamy (Reviewer 4): Please include all comments for the authors in this box rather than uploading your report as an attachment. Please only upload as attachments annotated versions of manuscripts, graphs, supporting materials or other aspects of your report which cannot be included in a text format. Please overwrite this text when adding your comments to the authors. Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction. Although it is an interesting study, the data presented are too preliminary and no new mechanism is established/ proposed. Answer: Thank you. We enriched the study by detecting HO activity in the obstructed kidneys and contralateral kidneys of UUO rats (Figure 2d and supplemental Figure 1), HO-1 protein level and HO activity of NRK-52E (figure 3), inflammatory cytokine IL-6 expression in the obstructed

6 kidneys of UUO rats (Figure 5c and 5d). We hope our revised manuscript is enough to clarify the mechanism IMD plays in renal fibrosis and suitable for publication. Minor: 1. difference among the immuno- histo- slides, between groups? Answer: Kidney slices for histological examinations and immunohistochemical analysis were sectioned at 4 μm, and there is no difference among groups. 2. How do achieve the uniform transfection of IMD? Answer: Ultrasound-induced microbubble destruction has been proposed as a new technique for local delivery of genes to specific target tissues (see the following references). Our previous studies have demonstrated that its uniform, efficiency and safety (Qiao X, et al. Am J Physiol Renal Physiol. 2013;304:F112- F119. PMID: Qiao X, et al. Nephrology (Carlton). 2015;20: PMID: ). 1. Lan HY, Mu W, Tomita N, Huang XR, Li JH, Zhu HJ, Morishita R, Johnson RJ. Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol Jun;14(6): it is very clear in the histology slides that dilation of blood vessels and tubules are evident in the in IMD transfected kidneys, this issue is neglected. Answer: We have replaced the histology slide of IMD transfected kidney with more representative one (Figure 4) 4. HO-1 expression is increased, what is the status of enzyme activity? why the activity of this enzyme is not included in the study Answer: We detected HO activity and added these results in the newly revised manuscript (Figure 2d, Figure 3 and supplemental Figure 1). 5. HO-1 enzyme activity is directly related to antioxidant activity! Answer: As you mentioned, HO-1 enzyme activity is directly related to antioxidant activity. We detected HO activity and added these results in the newly revised manuscript (Figure 2d, Figure 3 and supplemental Figure 1).