Spermidine from arginine metabolism activates Nrf2 and inhibits kidney fibrosis

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.

The work by Aihara and colleagues is interesting and technically well done, at least in part. My main problem is that the claims made in the title and in the abstract are far too extensive. All conclusions concerning spermidines involvement in kidney fibrosis are based on in vitro data only. The authors have two possibilities now: 1. repeat the experiments with spermidine supplementation (injection or feeding) in UOO mice (wild type and Arg2 knockout ) plus -Inhibit spermidine production in UOO mice or 2. Strongly reduce the claims (especially in the title) In addition, the sentence: "In human proximal tubule cells, spermidine strongly induced nuclear factor erythroid 2 related factor 2 (Nrf2), and its induction was partly mediated by autophagy. " is an overstatement in the abstract, because the experiments have not been repeated in autophagy deficient cells.
Some Prior works should be cited and disussed: Spermidine is protective against kidney ischemia and reperfusion injury through inhibiting DNA nitration and PARP1 activation Aging Cell. 2021 Jun; 20 (6) This is a well prepared manuscript. The findings are novel and interesting. However, there are two minor points that need to be addressed.
(1)Statistical analysis: The data presented in Fig. 3d and 6b are derived from studies employing two independent variables. The data should be compared statistically by two-way ANOVA, with appropriate post hoc tests for differences among treatment groups. One-way ANOVA is appropriate for multiple levels of a single independent variable and fails to make full use of data derived from two or more independent variables. In general, statistical comparisons should be applied uniformly to the data obtained.
(2)The limitations of the current study should be discussed.
Reviewer #3 (Remarks to the Author): In the present manuscript, Aihara et al. investigated the role of metabolite Spd in chronic renal interstitial fibrosis and the underlying mechanism. They show that arginine metabolism especially Spd level is enhanced in the UUO kidney in mice. Additionally, Spd treatment activates the transcription factor Nrf2 in HK-2 cells. Arg2 knockout reduces Spd levels, Nrf2 and HO-1 expression, and promotes UUO-induced kidney fibrosis. There are some concerns below. 1.Animal number and group information should be included in methods.

2.Please explain why the authors used 20 μM of Spd to treat cells.
3.It is better to list the primer sequences in a table. 4.Molecular weight is missing in WB. 5.The authors should provide the data showing the effect of Spd treatment on chronic renal interstitial fibrosis in UUO mice. 6.Did the authors examine the effect of Spd treatment on oxidative stress in HK-2 cells. 7.There are some typos and grammar mistakes in the manuscript. For example: "Spd expression was remarkably enhanced" should be "Spd level was remarkably enhanced".
We thank the reviewers for their useful comments and suggestions for our manuscript. We appreciate the reviewers' interest in our finding that Spd inhibits fibrosis of the kidneys. The reviewers have been important in shaping and strengthening our arguments during the revision process. We have performed additional experiments, as requested by the reviewers, and revised our manuscript in accordance with their suggestions as described below. Text in italics in this letter indicates the comments from the reviewers. The changes made in response to the reviewers' comments are shown in red in the revised manuscript. Text that has become unnecessary because of revision is shown in blue and has been deleted.
We therefore invite you to revise and resubmit your manuscript, taking into account the points raised. In particular we ask that you address all points raised by the 3 reviewers, specifically we ask that you address the statistical points raised by R2 and add more methodological information, as raised by R3. While the additional in vivo experiments suggested by R1 and R3 would be a plus, they are not mandatory, but, in their absence, please discuss the concomitant limitations of the study.
Response: The comments of the three reviewers are important and have been addressed as much as possible, including animal experiments.

Reviewer #1 (Remarks to the Author):
The work by Aihara and colleagues is interesting and technically well done, at least in part. My main problem is that the claims made in the title and in the abstract are far too extensive. All conclusions concerning spermidines involvement in kidney fibrosis are based on in vitro data only. The authors have two possibilities now: Response: We thank the reviewer for the interest in our research. The title and abstract have been changed in accordance with the reviewer's remarks and the results of additional experiments.

plus -Inhibit spermidine production in UOO mice
Response: We agree with the reviewer that we should investigate whether administration of Spd can reduce fibrosis in the kidney. We injected Spd (10 mg/kg) intraperitoneally to UUO mice for 2 weeks as described in previous reports (PMID: 30241944, Gao M. et al, Biochm. Biophys. Res. Comm. 2018). Kidney fibrosis, which was significantly higher in Arg2 KO mice than in WT mice, was suppressed by Spd, and this significant difference between WT and Arg2 KO mice disappeared ( Fig. 7m  and n, Supplementary Fig. S9a). In the comparison between vehicle and Spd treatment, no significant difference in fibrosis was found in WT or Arg2KO mice, although the effect of Spd was greater in Arg2 KO mice (p = 0.38 in WT mice and p = 0.07 in Arg2 KO mice). We believe that the effects of Spd treatment were stronger in Arg2 KO mice in which Spd levels were reduced. An analysis of Smox KO mice would be informative in a mouse model that inhibits Spd production, but we have not performed this experiment. Therefore  In addition, the sentence: "In human proximal tubule cells, spermidine strongly induced nuclear factor erythroid 2 related factor 2 (Nrf2), and its induction was partly mediated by autophagy. "is an overstatement in the abstract, because the experiments have not been repeated in autophagy deficient cells.
Response: The part of the title "…protects kidney from fibrosis" was replaced with "…inhibits kidney fibrosis". HCQ alone could not explain the activation of Nrf2 from Spd via autophagy. Therefore, Atg5 knockdown tubular cells and Atg5 KO mouse embryonic fibroblasts (MEFs) were examined. Nrf2 activation by Spd was also attenuated in Atg5 knockdown cells ( Supplementary Fig. S4b−d). In MEFs, unlike tubular cells, the activation of Nrf2 by Spd was not strong. However, Nrf2 activation by Spd was partially suppressed in Atg5 KO MEFs. Autophagy is partly involved in Nrf2 activation, but it does not make a major contribution. Therefore, in the Abstract, we have removed our results indicating that Spd activates Nrf2 partly via autophagy. (1) Statistical analysis: The data presented in Fig. 3d and 6b are derived from studies employing two independent variables. The data should be compared statistically by twoway ANOVA, with appropriate post hoc tests for differences among treatment groups. One-way ANOVA is appropriate for multiple levels of a single independent variable and fails to make full use of data derived from two or more independent variables. In general, statistical comparisons should be applied uniformly to the data obtained.
Response: Data with two independent variables, including those shown in Fig. 3d and 6b, were re-analyzed using two-way ANOVA. Additionally, post hoc tests of Tukey's multiple comparisons were conducted.
Changes have been made to the following parts of the manuscript: Methods: page 30, 533-534 Figs. 3d, 6b, 6c, 6e, and 7n Supplementary Figs. S4c, d, f, and g, and 9a The above-mentioned figures show data that were analyzed using a two-way ANOVA.
(2) The limitations of the current study should be discussed.
Response: We have mentioned that the renoprotective effects of Spd are diverse and cannot be explained by our results alone. We have also added a sentence stating that the exacerbation of fibrosis in Arg2 KO mice may not be due to a reduction in Spd alone (Discussion; page 19, lines 334-338).

Reviewer #3 (Remarks to the Author):
In the present manuscript, Aihara et al. investigated the role of metabolite Spd in chronic renal interstitial fibrosis and the underlying mechanism. They show that arginine metabolism especially Spd level is enhanced in the UUO kidney in mice.
Additionally, Spd treatment activates the transcription factor Nrf2 in HK-2 cells. Arg2 knockout reduces Spd levels, Nrf2 and HO-1 expression, and promotes UUO-induced kidney fibrosis. There are some concerns below.

Animal number and group information should be included in methods.
Response: Information on the number of mice and experimental conditions has been added to the subsection "Experimental procedures for UUO in mice" in the Methods section (page 21, line 364 to 368). The conditions and numbers of mice used for metabolomic analysis are already described in the subsection "Metabolomic analysis of UUO kidney tissue" in the Methods section.

Please explain why the authors used 20 μM of Spd to treat cells.
Response: The Spd concentration used in experiments is important. In previous reports, Spd was added to cultured cells, and concentrations varied from 0.1-200 μM. We investigated cell viability at concentrations in this range in tubular cells ( Supplementary  Fig. S3a). On the basis of these results, 20 μM Spd was selected as the concentration at which cell death does not occur. This information has also been added to the Results section.