Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients.

BACKGROUND: Reduced free thiols in plasma are indicative of oxidative stress, which is an important contributor to ischaemia-reperfusion injury (IRI) in kidney transplantation leading to kidney damage and possibly delayed graft function (DGF). In a post-hoc, exploratory analysis of the randomised controlled CONTEXT trial, we investigated whether higher (i.e. less oxidised) plasma levels of free thiols as a biomarker of reduced oxidative stress are associated with a better initial graft function or a higher GFR.
METHODS: Free thiol levels were measured in plasma at baseline, 30 and 90 minutes after reperfusion of the kidney as well as at Day 1, Day 5 and twelve months after kidney transplantation in 217 patients from the CONTEXT study. Free thiol levels were compared to the kidney graft function measured as the estimated time to a 50% reduction in plasma creatinine (tCr50), the risk of DGF and measured GFR (mGFR) at Day 5 and twelve months after transplantation.
RESULTS: Higher levels of free thiols at Day 1 and Day 5 are associated with higher mGFR at Day 5 (p<0.001, r2adj. = 0.16; p<0.001, r2adj. = 0.25), as well as with mGFR at twelve months (p<0.001, r2adj. = 0.20; p<0.001, r2adj. = 0.16). However, plasma levels of free thiols at 30 minutes and 90 minutes, but not Day 1, were significantly higher among patients experiencing DGF.
CONCLUSION: Higher levels of plasma free thiols at Day 1 and Day 5, which are reflective of lower levels of oxidative stress, are associated with better early and late graft function in recipients of a kidney graft from deceased donors. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01395719.

Introduction

Renal transplantation can increase quality and length of life for patients with end-stage renal disease. Delayed graft function (DGF) mediated by ischaemia-reperfusion injury (IRI) is a major challenge particularly in deceased donor kidney transplantation [1], leading to the need for dialysis, higher risk of post-transplant complications, prolonged hospitalisation and inferior long term function in recipients of grafts from brain death donors [2, 3].

Oxidative stress, one of the most important contributors of the IRI process, may be involved in renal damage and DGF [4]. Oxidative stress can be considered as an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity. Plasma thiols are known to scavenge reactive oxygen species, thereby protecting cells against oxidative stress. Although the greater part of plasma thiols is present in oxidized form [5, 6], the level of free thiols bears great relevance in determining the individual redox status. Systemic oxidative stress is associated with reduced levels of these circulating free thiols since these can be rapidly oxidised in conditions of increased production of ROS [7]. Increased ROS production, as reflected by decreased levels of free thiols, is often observed in human diseases in which oxidative stress plays a pathophysiological role, such as diabetes, chronic kidney disease, heart failure, cancer and Crohn’s disease [8-10]. This indicates that the circulating level of free thiols directly reflects the systemic redox status and free thiol groups are assumed to play a protective role against oxidative stress due to their potent ability to scavenge ROS [11]. In addition to low molecular weight molecules, most of redox-active thiol groups (approximately 75% of the total thiol pool) are present in blood proteins, mainly albumin [7]. We have previously demonstrated that higher systemic free thiol levels predict better graft survival and lower mortality in renal transplant patients [12].

In this study, we hypothesised that higher systemic levels of free thiols, representing less oxidative stress, are associated with a better early and late graft function in patients subjected to renal transplantation. Our aim was therefore to examine the changes in systemic free thiols following deceased donor kidney transplantation and to correlate this biomarker with DGF as well as early and late graft function.

Materials and methods

Study design

This present study is a post-hoc analysis including patients from the multinational, double-blinded, randomised, controlled trial CONTEXT (ClinicalTrials.gov Identifier: NCT01395719) [13]. The CONTEXT study investigated the effect of remote ischaemic conditioning (RIC) in the recipient during kidney transplantation with deceased donors. The conditioning was performed by repetitive inflation of a cuff to occlude the blood supply to the leg on the side not used for transplantation before reperfusion of the kidney graft. The study included 225 kidney transplant recipients from June 2011 to December 2014. Three patients were withdrawn from the study leaving 222 patients in the entire cohort. Two hundred patients received a graft from brain death donors whereas 22 received grafts from donors with circulatory death. The primary endpoint of the study was the estimated time to a 50% reduction in P-creatinine (tCr50) as a marker of early graft function [14]. The study was approved by relevant data protection agencies and ethical committees (Denmark: The National Committee on Health Research Ethics; Sweden: Regional Ethical Board; the Netherlands: METCUMCG) and performed in accordance with the Declaration of Helsinki.

Inclusion

Patients receiving kidneys from deceased donors were included after informed concent in Aarhus, Denmark (n = 132); Gothenburg, Sweden (n = 46); and Rotterdam (n = 22) and Groningen (n = 25), the Netherlands. Informed consent was obtained from all patients after submission to hospital prior to transplantation [15]. The donation was organised by ScandiaTransplant or EuroTransplant with no connection between the donation process and transplant recipients.

Plasma free thiols were measured in a total of 217 patients who participated in the CONTEXT trial [15]. Due to graft removal or primary non-function, eleven patients were excluded from the tCr50 analysis. Demographic and clinical information of the transplant recipients (age, gender, plasma (P-) creatinine, need for dialysis) was collected from hospital records.

Ethics statement

None of the transplant donors was from a vulnerable population. No informed consent was obtained from the donors to participate in the CONTEXT study, but the organ donation was organized through ScandiaTransplant or EuroTransplant.

Biochemical analyses

Blood samples were collected at baseline (n = 217), 30 minutes (n = 216) and 90 minutes (n = 206) after reperfusion of the kidney, Day 1 (n = 194) and Day 5 (n = 195) after transplantation. The samples were centrifugated and stored at -80°C.

Plasma free thiols were measured as previously described [12]. Seventy-five μl plasma was diluted 1:4 in 0.1 M Tris buffer (pH 8.2) and transferred to 96-well plates. Using a Sunrise microplate reader (Tecan Trading AG, Männedorf, Switzerland), background absorption was measured at 412 nm with a reference filter at 630 nm. Subsequently, 10μl 3.8 mM 5,5′ -Dithio-bis (2-nitrobenzoic acid) (DTNB; Sigma Aldrich, Zwijndrecht, Netherlands) in 0.1 M phosphate buffer (pH 7) was added to the samples. After 20 min of incubation at room temperature, absorption was read again. The concentration of plasma free thiols in the samples was determined by comparing their absorbance readings to a standard calibration curve of L-cysteine (15–1000 μM; Fluka Biochemika, Buchs, Switzerland) in 0.1 M Tris and 10 mM EDTA (pH 8.2). Using this detection method, which has been thoroughly validated and has proven consistency across different centers, total free thiol content is measured, consisting of the combination of protein-bound free thiols and low-molecular-weight (LMW) free thiols (e.g., cysteine, homocysteine, and glutathione) [7, 12, 16].

P-creatinine was measured as part of the daily routine: twice daily the first week and twice weekly until 30 days after transplantation. If dialysis was needed post-transplant P-creatinine was measured twice a week until 30 days after last dialysis session.

Outcome parameters

tCr50 was calculated as previously described [14]. DGF was defined as the need for dialysis within the first post-transplant week. Measured glomerular filtration rate (mGFR) was performed as 51chrome-ethylenediamine tetraacetic acid (51Cr-EDTA) plasma clearance [17] on Day 5 (n = 89) and at twelve months (n = 137) after transplantation in patients included in Aarhus and Gothenburg with clinically detectable kidney graft function. The results were standardised to body surface.

Statistical analyses

Demographic and clinical characteristics of the transplant recipients were presented as proportions n (%), medians (interquartile range, IQR) or means (standard deviation, SD). Assessment of normality of distributions was performed using histograms. Simple linear regression was used to correlate the level of free thiols to kidney graft function while adjusting for recipient age and gender as relevant covariates and r2 was adjusted for the degrees of freedom. mGFR at Day 5 and tCr50 were logarithmically transformed to obtain normal distributions–hence the estimates are presented as a doubling in mGFR Day 5 and tCr50, respectively. Multivariate repeated measurements ANOVA was used to compare the level of systemic free thiols between two groups (e.g. treatment and DGF). Student’s T test was used to compare the individual timepoints in case the ANOVA revealed a difference. Stata® version 16 for Windows (StataCorp LP) was used to perform the statistical analyses.

Results

Recipient and donor characteristics

Table 1 shows the baseline characteristics of the 217 renal transplant recipients. Immunosuppression at discharge, original kidney disease, comorbidity and donor age and gender are all included in the table.

Table 1

Recipient and donor characteristics.

	n = 219
Recipient age (years)	59.0 (49.4–66.0)
Recipient gender, female	83 (38%)
Baseline P-free thiols (μM)	363 (282–449)
Estimated baseline P-creatinine (μmol/l)a	629 (498–762)
Immunosuppression at discharge
Tacrolimusb	200 (94%)
Mycophenolate mofetil	212 (98%)
Corticosteroids	206 (95%)
Original renal disease
Glomerulopathy	50 (23%)
ADPKD	43 (20%)
Diabetes mellitus	25 (12%)
Vascular/hypertension	23 (11%)
Reflux/obstructive	7 (3%)
Other	23 (11%)
Unknown	46 (21%)
Pre-transplant diabetes	42 (19%)
Hypertension	195 (90%)
Donor age (years)	58 (52–65)
Donor gender, female	99 (46%)
Cold ischemia time (hours)a	13.5 (4.6)

Values are presented as proportions n with corresponding percentages (%), medians (interquartile range) or means (standard deviation).

an = 209 due to primary non-function or early graftectomy.

bn = 212 due to missing samples.

Free thiols at all time points followed a normal distribution and no transformation was needed for further analysis. Systemic free thiol levels were in accordance with previous studies that employed the same detection method [8, 9, 12, 16, 18]. In addition, no correlation was found between the time of baseline sampling and the level of free thiols (p = 0.76) (Fig 1), indicating stability of free thiols in the frozen samples. No difference was observed in the level of free thiols between recipients of kidneys from brain death donors and circulatory death donors except at Day 1 where the level of free thiols was higher among patients receiving a graft from a donor with circulatory death (p = 0.04).

Fig 1

Graph showing the level of free thiols (μM) measured at baseline over the total study period (p = 0.76).

We found no difference in plasma free thiol levels depending on treatment (RIC vs sham-RIC) at baseline, 30 minutes, 90 minutes, Day 1 or Day 5 after kidney transplantation (p = 0.25) (S1 Fig), and hence the data from the two groups were pooled for additional analysis.

Free thiols and early graft function

Table 2 shows the associations between free thiols and initial graft function depicted as mGFR Day 5 and tCr50. Higher levels of free thiols measured only 90 mins after reperfusion are weakly correlated to a higher mGFR at Day 5. The association strengthens when free thiols are measured at Day 1 and Day 5 (Table 2 and Fig 2). A higher level of free thiols is weakly correlated to a shorter tCr50. Interestingly, the level of free thiols at baseline, 30 minutes and 90 minutes, but not Day 1, was significantly higher among patients experiencing DGF (Fig 3).

Table 2

The correlations between free thiols at different time points and mGFR on Day 5 or tCr50.

Time point of free thiols sampling				mGFR Day 5				tCr50
	n	βa	95% CIa	p	r2adj.	Pb	r2adj. b	n	βa	95% CIa	p	r2adj.	pb	r2adj. b
30 minutes	89	33	(2;65)	0.04	0.04	0.09	0.02	206	6	(-3;16)	0.20	0.003	0.15	0.004
90 minutes	86	42	(10;75)	0.01	0.06	0.03	0.04	196	1	(-9;11)	0.84	-0.005	0.70	-0.006
Day 1	78	63	(32;94)	<0.001	0.16	<0.001	0.14	188	-11	(-22;-1)	0.03	0.02	0.03	0.02
Day 5	87	73	(46;99)	<0.001	0.25	<0.001	0.24	191	-20	(-29;-11)	<0.001	0.08	<0.001	0.08

astandardised beta coefficients corresponding to a doubling in mGFR or tCr50, respectively.

badjusted for recipient age and sex. CI = confidence interval. r2adj. = correlation coefficient.

Fig 2

Simple linear regression of plasma free thiols and mGFR at Day 5.

A) Free thiols at Day 1. B) Free thiols at Day 5.

Fig 3

Box plot of the level of free thiols (μM) at different time points and delayed graft function (DGF).

Free thiols correlate with graft function at twelve months post-transplant

Higher levels of free thiols measured in the first hours and early days after kidney transplantation correlated with higher mGFR at twelve months (Table 3). Only weak correlations were found for free thiols measured at 30 and 90 minutes after reperfusion, whereas free thiols at Day 1 and Day 5 correlated moderately strong.

Table 3

The correlations between free thiols at early time points and mGFR at twelve months.

Time point of free thiols sampling	mGFR at twelve months
	n	βa	95% CI	p	r2adj.	Pb	r2adj.b
30 minutes	137	1.8	(0.8;2.9)	0.001	0.07	0.03	0.14
90 minutes	130	1.9	(0.8;2.9)	0.001	0.08	0.02	0.14
Day 1	124	2.9	(1.9;3.9)	<0.001	0.20	<0.001	0.23
Day 5	132	2.5	(1.5;3.5)	<0.001	0.16	<0.001	0.20

astandardised beta coefficient.

badjusted for recipient age and sex. CI = confidence interval. r2adj. = correlation coefficient.

Discussion

In the present study a greater increase in plasma free thiols, indicating a favourable redox balance during the first days after renal transplantation, correlated significantly with early kidney graft function (higher mGFR and shorter tCr50) suggesting that less oxidative stress associates with a better early graft function. Conversely, higher levels of free thiols at 30 minutes and 90 minutes after graft reperfusion were observed among patients experiencing DGF. Interestingly, we also identified that higher free thiols early after transplantation were associated with better one-year graft function. Taken together, these data suggest that the level of oxidative stress in the early phase after transplantation relates to graft onset and predicts kidney graft function at twelve months post-transplantation.

We did not observe any difference in free thiols as a result of the RIC procedure. The CONTEXT study was designed to test whether remote RIC could improve the outcome after renal transplantation. In line with the absence of detectable effects of RIC on free thiols, no effect of RIC on kidney function and other biomarkers as NGAL was observed in the CONTEXT study [15, 19, 20], therefore the analyses included in this study was performed on pooled data.

We have previously demonstrated that increased systemic levels of free thiols are associated with better graft survival and lower mortality in renal transplant recipients [12]. In addition, other studies have demonstrated that malondialdehyde (MDA), an end product of oxidative lipid peroxidation, predicts allograft survival and the possibility of DGF [21-23], again highlighting the relevance of oxidative stress in the early phase of renal transplantation.

Higher levels of plasma free thiols are a reflection of a beneficial systemic redox status since free thiols play a protective role against oxidative stress and act thereby as an independent marker of ROS levels [24]. Given that free thiol levels correlate with GFR we cannot exclude the possibility, that the association between the early increase in plasma free thiol levels and shorter tCr50 is, at least in part, a reflection of a greater GFR rather than an independent marker of ROS. However, since oxidative stress is closely related to IRI, which is an established complication of renal transplantation, an association between free thiols and kidney damage due to ROS in the context of renal transplantation is conceivable. Furthermore, intrarenal ROS may affect both the glomerular filtration barrier [25] and intrarenal haemodynamics [26] providing additional rationale for the relation between free thiols and mGFR.

We observed higher levels of free thiols at 30 minutes and 90 minutes after graft reperfusion among patients experiencing DGF, which would suggest that the potential detrimental effects of low free thiols and less favourable redox status is not associated with a higher incidence of DGF. The correlation is weak and cannot be observed at Day 1, and thus the significance is uncertain. It is possible that this early increase in free thiols after graft reperfusion is related to an interaction with substances released intraoperatively, such as residual sulfhydryl groups groups from the kidney storage solution, which are not cleared as efficiently in case of DGF.

The study is strengthened by the relatively large number of unselected deceased donor kidney transplant recipients. Moreover, the study is a large multicenter study carried out in Denmark, Sweden and the Netherlands. On the other hand, we recognise certain limitations of the study. Given the fact that the majority of patients were Caucasians, the generalisability of our results in subjects with other ethnicities remains unknown and our study did not prove any causal relationship. Furthermore, the absence of data on plasma albumin or total plasma protein levels refrained us from the possibility to adjust total free thiol levels by calculating the free thiol or total protein/albumin ratio. This protein adjustment would have enabled us to indirectly account for total thiol content as proteins harbour the largest amount of thiols and therefore quantitatively determine the amount of potentially detectable free thiols [16]. However, previous studies from our lab have demonstrated that in most cases, this protein adjustment does not severely affect the eventual results obtained, and thus it does not always lead to different conclusions. Finally, no sufficient biomaterials were available in the present study to perform additional experiments in order to further characterise the thiol redox metabolome in our patients. Measurements of individual thiol species (e.g. cysteine, homocysteine and glutathione) or inclusion of extra indices (e.g., the protein thiolation index, PTI) could have provided us with more in-depth information on extracellular free thiol dynamics [27]. Future studies could be designed to focus on a combination of key components of the thiol redox metabolome resulting in an integrative biomarker approach, representing multiple redox-regulated metabolic pathways. However, such “redox metabolomics” approaches are still under development and are accompanied by various methodological constraints. Similarly, it is as yet unclear what criteria potential thiol redox biomarkers should fulfil to be reliably reflective of the human redox system in a high-throughput setting [28]. Therefore, the single quantification of total free thiol content in serum or plasma is currently considered one of the most useful screening tools for measuring the whole-body redox status in clinical and translational studies.

Conclusion

In conclusion, this study suggests that plasma free thiols correlate with early graft function, and can predict kidney function at twelve months post-transplantation.

The level of plasma free thiols at different time points depending on treatment (Sham-RIC vs. RIC).

RIC = remote ischaemic conditioning.

(DOCX)

Click here for additional data file.

10 Dec 2020

PONE-D-20-32900

Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients

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Reviewer #1: The paper consists of a post hoc exploratory analysis of data generated from the randomized controlled "CONTEXT" trial to understand whether higher levels of free thiols is associated with better graft functions in renal transplants. I have some questions and clarifications, mainly from the statistical analysis side.

1. The statistical analyses plan lacks any power/sample size considerations. The study would improve, if some statements were provided in this regard, like what was the power the authors initially expected, for the samples available. They may use a desired statistical test at 5% significance level.

2. Statistical Analyses subsection written poorly, and analysis conducted is not up to the desired mark.

(a) "Simple linear regression was used to correlate continuous variables" doesn't have a clear meaning. Say something like, "A simple linear regression was used to assess the effect of XX on YYY, controlling for ZZZ (the confounders, etc).

(b) I do not understand the title of the Tables 2 and 3. Linear regression of response (Y) on the desired covariates (X) present the parameter estimates, standard errors, 95% confidence intervals, and p-values. Those need to be presented, if , at all, a linear regression was conducted. What is presented looks like some standard (adjusted) correlations.

(c) Looks like the data collection plan of thiol levels was longitudinal, like baseline, 30 and 90 minutes, etc. So, in addition to analyses at separate points and presenting correlations, I wonder why a formal longitudinal data analyses was not conducted, via linear mixed models, or generalized estimating equations? The time points of thiols sampling could enter the model to model the time trend, or something like that.

Reviewer #2: The manuscript “Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients” by Nielsen et al. fosters the hypothesis that “higher (i.e. less oxidated) levels of free thiols as a biomarker of reduced oxidative stress are associated with a better initial graft function or a higher GFR.” (last sentence of the first paragraph of the abstract).

I have several concerns on this manuscript, the main ones are:

1. the levels of free thiols in plasma reported in this study appear to be much higher than expected (between 100 and 700 uM). The analysis protocol described in the manuscript should lead to measure a minor fraction of these compounds present in this and in other extracellular fluids, i.e. the free (or reduced) form of total thiols only. In fact, such protocol does not include reducing agents to break the disulfide bridges of the oxidized forms, which is a prerequisite for the reaction of thiols with DTND. Therefore the levels expected from this assay should be much lower. Please note that the large majority of total thiols in plasma and other extracellular fluids is present under the oxidized form, which cannot be measured with the proposed assay (i.e. as disulfides or mixed disulfides with Cys34 of Albumin, with an average ratio of reduced to oxidized forms of 0.2) (Giustarini, Dalle-Donne et al. 2006; Jones and Liang 2009; Galli et al. Free Rad Res, 2014; Galli F. et al Kideny Int 2013). Therefore, I do not know what exactly the authors have measured in this study, and this concerns me a lot because the uncertainty on the proposed results invalidates the study hypothesis and aim (i.e. to expect changes in the levels of the reduced form of plasma thiols that may reflect their oxidation state and then the presence of oxidative stress in the transplanted patients).

2. To demonstrate the presence of an altered redox of the extracellular environment and to link this alteration with the function of specific organs, more experiments should be performed and other laboratory indices must be investigated.

a. First, the different thiol/disulfide couples of the different low-molecular mass thiols (Cys, Hcy, Cys-Gly, GSH) of extracellular fluids should be investigated together with protein S-thiolation and blood cell thiols. Please note that in healthy subjects these are in the following ranges: 150-300 uM total Cys (including that coming from CySS and Cys-Gly), while Hcy is approx. 5-10 uM and GSH is usually 2-6 uM) and the mean ratio of reduced to oxidized forms is 0.2. In CKD patients on standard hemodialysis the absolute levels of total thiols significantly increase with different extent of modification in the individual thiol species (Galli et al. Free Rad Res, 2014). The identification of these subclasses is much more informative compared with that proposed in the present manuscript.

b. Second, serum albumin and the levels of its thiolation (mixed disulfides) should be considered to explain the interindividual differences observed in this study (see figure 1 and 2). For example, Cys is largely engaged in mixed disulfide formation and Hcy is more than 75 % bound to serum albumin (Galli F. et al Kideny Int 2013; Galli et al. Free Rad Res, 2014).

c. Protein thiolation in plasma is a relevant indicator of oxidative stress in age-related and inflammatory diseases, including CKD (Reggiani et al. 2015, Fanti, Giustarini et al. 2015), and increased levels of biomarker linearly correspond to the decline of thiol to disulfide balance in extracellular fluids. This is relevant biomarker to utilize if one would like to explore the impaired redox of a patient with a systemic (not organ-specific) approach. This biomarker should be investigated together with other indices of damage of plasma proteins and/or polyunsaturated lipids (e.g. protein carbonylation, lipid peroxidation products, etc.). May be the Authors have a bank of samples with aliquots of plasma still available for these determinations.

d. Organ-specific indications cannot be expected from the proposed laboratory strategy to explore plasma thiols. The investigation of individual thiol species would provide much higher chances to obtain some level of information on the transplanted organ (see later in the next point).

3. The changes observed in the levels of free thiols in plasma of this study and their correlation with the success of transplantation and organ function are more than expected if we consider that tubular epithelial cells are very rich in gamma-glutamyl transpeptidase or ��GT (Giustarini, Galvagni et al. 2020). Therefore, reduced or absent function of tubular epithelia cells observed in the late stages of kidney disease, is expected to impair the renal metabolism and extracellular levels of LMW thiols, and especially of Cys. Possibly, what the Authors in this study are measuring with their thiol assay in plasma is the ��GT activity of the transplanted organ that obviously is higher in successfully treated subjects.

4. The redox balance of extracellular thiols declines with the subject’ age (Jones, Mody et al. 2002, Giustarini, Dalle-Donne et al. 2006) and such a decline is even more rapid in case of of premature aging and impaired redox homeostasis, which are characteristic conditions of CKD (Galli et al. Free Rad Res, 2014; Reggiani et al. 2015, Fanti, Giustarini et al. 2015). The results in this study (when the actual reduced form of thiols will be measured) should be corrected for the age of the patients as potential confounding factor.

5. Based on the correlation between thiols and mGFR it could be assumed that GFR could be utilized instead of thiols as a biomarker of a successful transplantation, which I guess is routine in the clinical monitoring of transplanted patients. What plasma thiols (those measured with this study) actually add up to the already available indices of organ function in transplantation protocols?

6. The term “oxidated” should be revised and substituted with oxidized.

7. The Authors have disregarded most of the studies performed so far on plasma thiols in the introduction of their study and in the discussion of the results. This and other aspects discussed earlier in this revision report, demonstrate poor confidence with this topic. I suggest to refer to experts in the field of redox biology and medicine, and especially in thiol analysis, to obtain sufficed advise during the revision of their manuscript.

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Reviewer #2: Yes: Francesco Galli

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11 Feb 2021

Rebuttal letter

Our responses are included in red. Lines numbers refer to Manuscript with Tracked Changes

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The headings and subheadings have been reedited.

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The data is now attached to the manuscript as a supplemental figure.

3. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants. Please ensure you have provided sufficient details to replicate the analyses such as: a) a description of how participants were recruited, and b) descriptions of where participants were recruited and where the research took place.

Participant recruitment as well as number of patients from each center has been added to the revised manuscript in the Materials and Methods section (lines 84-87). For more information see Krogstrup et al, AJT, 2016 (reference no. 15). Age and gender of recipients are presented in Table 1.

Krogstrup N V., Oltean M, Nieuwenhuijs-Moeke GJ, Dor FJMF, Møldrup U, Krag SP, et al. Remote Ischemic Conditioning on Recipients of Deceased Renal Transplants Does Not Improve Early Graft Function: A Multicenter Randomized, Controlled Clinical Trial. Am J Transplant. 2016;1–8. doi: 10.1111/ajt.14075

4. We note that your study involved tissue/organ transplantation. Please provide the following information regarding tissue/organ donors for transplantation cases analyzed in your study.

1. Please provide the source(s) of the transplanted tissue/organs used in the study, including the institution name and a non-identifying description of the donor(s).

The organs were provided from deceased donors (after brain death or circulatory death). The organ donation was organised via ScandiaTransplant or EuroTransplant, this has now been added to the revised manuscript in the Materials and Methods section (lines 87-88).

Donor age and gender are presented in Table 1.

2. Please state in your response letter and ethics statement whether the transplant cases for this study involved any vulnerable populations; for example, tissue/organs from prisoners, subjects with reduced mental capacity due to illness or age, or minors.

Please see the cover letter (ethics statement).

- If a vulnerable population was used, please describe the population, justify the decision to use tissue/organ donations from this group, and clearly describe what measures were taken in the informed consent procedure to assure protection of the vulnerable group and avoid coercion.

- If a vulnerable population was not used, please state in your ethics statement, “None of the transplant donors was from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.”

3. In the Methods, please provide detailed information about the procedure by which informed consent was obtained from organ/tissue donors or their next of kin. In addition, please provide a blank example of the form used to obtain consent from donors, and an English translation if the original is in a different language.

In the Materials and Methods section the following has been added: “Informed consent was obtained from all patients after submission to hospital prior to transplantation (15). The donation was organised by ScandiaTransplant or EuroTransplant with no connection between the donation process and transplant recipients.” (lines 86-88)

The deceased donor donation occurred completely independent of this project that only involved the transplant recipients. Therefore, donor families were only asked whether they would consent that their loved ones could be donors according to the usual routines in the three countries, where donation was organised by ScandiaTransplant or EuroTransplant.

4. Please indicate whether the donors were previously registered as organ donors. If tissues/organs were obtained from deceased donors or cadavers, please provide details as to the donors’ cause(s) of death.

It was not registered in the CONTEXT study whether the organ donors were previously registered as organ donors or whether the decision was made by next of kin. The donor’s cause(s) of death has previously been published (Krogstrup et al, AJT, 2016).

Krogstrup N V., Oltean M, Nieuwenhuijs-Moeke GJ, Dor FJMF, Møldrup U, Krag SP, et al. Remote Ischemic Conditioning on Recipients of Deceased Renal Transplants Does Not Improve Early Graft Function: A Multicenter Randomized, Controlled Clinical Trial. Am J Transplant. 2016;1–8. doi: 10.1111/ajt.14075

5. Please discuss whether medical costs were covered or other cash payments were provided to the family of the donor. If so, please specify the value of this support (in local currency and equivalent to U.S. dollars).

No medical cost or cash payments were provided to the family of the donor.

5. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere.

"Table 1 presenting recipient and donor characteristics (except free thiols level) have been published in previous CONTEXT study papers."

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5. Review Comments to the Author

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Reviewer #1: The paper consists of a post hoc exploratory analysis of data generated from the randomized controlled "CONTEXT" trial to understand whether higher levels of free thiols is associated with better graft functions in renal transplants. I have some questions and clarifications, mainly from the statistical analysis side.

1. The statistical analyses plan lacks any power/sample size considerations. The study would improve, if some statements were provided in this regard, like what was the power the authors initially expected, for the samples available. They may use a desired statistical test at 5% significance level.

We thank the reviewer for this important comment. Before executing the CONTEXT study, a power calculation on the primary endpoint was performed in order to find the number of patients to include in the treatment arms (Krogstrup et al., BMJ Open, 2015 – reference no. 13). However, as this study is a post hoc analysis, no power calculation was performed.

Krogstrup N V, Oltean M, Bibby BM, Nieuwenhuijs-Moeke GJ, Dor FJMF, Birn H, et al. Remote ischaemic conditioning on recipients of deceased renal transplants, effect on immediate and extended kidney graft function: a multicentre, randomised controlled trial protocol (CONTEXT). BMJ Open. 2015;5(8):e007941.

2. Statistical Analyses subsection written poorly, and analysis conducted is not up to the desired mark.

(a) "Simple linear regression was used to correlate continuous variables" doesn't have a clear meaning. Say something like, "A simple linear regression was used to assess the effect of XX on YYY, controlling for ZZZ (the confounders, etc).

Thank you for the suggestion to improve the statistical subsection. It has now been rewritten in the revised manuscript in the Materials and Methods section:

“Simple linear regression was used to correlate the level of free thiols to kidney graft function while adjusting for recipient age and gender as relevant covariates and r2 was adjusted for the degrees of freedom.” (lines 118-120)

(b) I do not understand the title of the Tables 2 and 3. Linear regression of response (Y) on the desired covariates (X) present the parameter estimates, standard errors, 95% confidence intervals, and p-values. Those need to be presented, if , at all, a linear regression was conducted. What is presented looks like some standard (adjusted) correlations.

We agree that the titles may cause some confusion. For that purpose we have now changed the titles of Table 2 and 3 as well as added the standardised beta coefficient with 95% confidence intervals of the linear regression analyses (Table 2 and 3).

(c) Looks like the data collection plan of thiol levels was longitudinal, like baseline, 30 and 90 minutes, etc. So, in addition to analyses at separate points and presenting correlations, I wonder why a formal longitudinal data analyses was not conducted, via linear mixed models, or generalized estimating equations? The time points of thiols sampling could enter the model to model the time trend, or something like that.

The data collection plan was indeed longitudinal of origin. We appreciate the reviewer’s comment since it touches upon a fair argument that it is appropriate to use a formal longitudinal data analysis method. Therefore, the comparison between the two groups (RIC vs. sham-RIC as well as DGF vs. no DGF) has now been performed using multivariate repeated measurements ANOVA. The method is described in the statistics section in the Material and Methods (lines 122-124) and the result of the RIC vs sham-RIC is included in line 148.

Reviewer #2: The manuscript “Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients” by Nielsen et al. fosters the hypothesis that “higher (i.e. less oxidated) levels of free thiols as a biomarker of reduced oxidative stress are associated with a better initial graft function or a higher GFR.” (last sentence of the first paragraph of the abstract).

I have several concerns on this manuscript, the main ones are:

1. the levels of free thiols in plasma reported in this study appear to be much higher than expected (between 100 and 700 uM). The analysis protocol described in the manuscript should lead to measure a minor fraction of these compounds present in this and in other extracellular fluids, i.e. the free (or reduced) form of total thiols only. In fact, such protocol does not include reducing agents to break the disulfide bridges of the oxidized forms, which is a prerequisite for the reaction of thiols with DTND. Therefore the levels expected from this assay should be much lower. Please note that the large majority of total thiols in plasma and other extracellular fluids is present under the oxidized form, which cannot be measured with the proposed assay (i.e. as disulfides or mixed disulfides with Cys34 of Albumin, with an average ratio of reduced to oxidized forms of 0.2) (Giustarini, Dalle-Donne et al. 2006; Jones and Liang 2009; Galli et al. Free Rad Res, 2014; Galli F. et al Kideny Int 2013). Therefore, I do not know what exactly the authors have measured in this study, and this concerns me a lot because the uncertainty on the proposed results invalidates the study hypothesis and aim (i.e. to expect changes in the levels of the reduced form of plasma thiols that may reflect their oxidation state and then the presence of oxidative stress in the transplanted patients).

We thank the reviewer for outlining the above concerns. It is correct that the true redox potential of plasma thiols is difficult to address, but we are more than happy to further clarify the measurement method that was used in this study. Here, we have determined total free thiol content in plasma by derivatization with DTNB as thiol-reactive agent. This is based on the standardised Ellman reaction. Using this method, we actually measure the combination of protein-bound free thiols (since proteins were not removed from the samples) and low-molecular-weight (LMW) free thiols, e.g. cysteine, glutathione and homocysteine. The reviewer is correct that total thiol content (including oxidized thiols with disulphide bridges) was not measured here. In that case, we would have required to use a stronger reducing agent like dithiothreitol (DTT), which is able to additionally reduce disulphide bonds and oxidized protein-bound thiols. (Sutton TR et al., Redox Biol 2018).

In the present study, we determined total free thiol content since this method has been well-developed and optimized in the literature (Ellman reaction) and in our center. Our measurement method has been well validated. We did extensive tests to show that the samples are in optimal conditions. E.g. repeated freezing and thawing had no effect on the level of free thiols measured. The method has also proven its power to predict clinical parameters in many previous studies, in which free thiol levels were also in agreement with the range within the present study. (Frenay AS et al., Free Radic Biol Med 2016; Koning AM et al., Pharmacol Res 2016; Bourgonje AR et al., Front Physiol 2019; Abdulle AE et al., Physiol Rep 2019) In these studies, free thiols were also considered a reliable reflection of the systemic redox status, whereas total thiol content is usually referred to as being reflective of the systemic redox reserve. (Cortese-Krott MM et al., Antioxid Redox Signal 2017) Alternatively, one could define systemic redox status by measuring the ratio of reduced over oxidized thiols or by adjusting free thiol to total plasma protein content. In the latter way, you could indirectly account for total thiol content as proteins constitute by far the largest pool of redox-active thiols both in blood and in cells. Unfortunately, however, protein levels were not available for the present study and we do not have sufficient plasma left to add these measurements, but we can reassure the reviewer about this point as previous studies from our center also demonstrated no differences in results and conclusions with or without adjustment to total protein levels.

Sutton TR, Minnion M, Barbarino F, et al. A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome. Redox Biol 2018;16:359-380.

Frenay AS, de Borst MH, Bachtler M, Tschopp N, Keyzer CA, van den Berg E, et al. Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients. Free Radic Biol Med 2016;99:345-351.

Koning AM, Meijers WC, Pasch A, Leuvenink HGD, Frenay AS, Dekker MM, et al. Serum free thiols in chronic heart failure. Pharmacol Res 2016;111:452-458.Koning AM et al., Pharmacol Res 2016

Bourgonje AR, Gabriëls RY, de Borst MH, Bulthuis MLC, Faber KN, van Goor H, et al. Serum Free Thiols Are Superior to Fecal Calprotectin in Reflecting Endoscopic Disease Activity in Inflammatory Bowel Disease. Antioxidants (Basel) (2019) 8(9):351.Bourgonje AR et al., Front Physiol 2019;

Abdulle AE, Bourgonje AR, Kieneker LM, Koning AM, la Bastide-van Gemert S, Bulthuis MLC, et al. Serum free thiols predict cardiovascular events and all-cause mortality in the general population: a prospective cohort study. BMC Med (2020) 18(1):130.

Damba T, Bourgonje AR, Abdulle AE, Pasch A, Sydor S, van den Berg EH, et al. Oxidative stress is associated with suspected non-alcoholic fatty liver disease and all-cause mortality in the general population. Liver Int 2020;40(9):2148-2159.

Cortese-Krott MM, Koning A, Kuhnle GGC, Nagy P, Bianco CL, Pasch A, et al. The Reactive Species Interactome: Evolutionary Emergence, Biological Significance, and Opportunities for Redox Metabolomics and Personalized Medicine. Antioxid Redox Signal (2017) 27(10) :684-712.

2. To demonstrate the presence of an altered redox of the extracellular environment and to link this alteration with the function of specific organs, more experiments should be performed and other laboratory indices must be investigated.

a. First, the different thiol/disulfide couples of the different low-molecular mass thiols (Cys, Hcy, Cys-Gly, GSH) of extracellular fluids should be investigated together with protein S-thiolation and blood cell thiols. Please note that in healthy subjects these are in the following ranges: 150-300 uM total Cys (including that coming from CySS and Cys-Gly), while Hcy is approx. 5-10 uM and GSH is usually 2-6 uM) and the mean ratio of reduced to oxidized forms is 0.2. In CKD patients on standard hemodialysis the absolute levels of total thiols significantly increase with different extent of modification in the individual thiol species (Galli et al. Free Rad Res, 2014). The identification of these subclasses is much more informative compared with that proposed in the present manuscript.

We agree with the reviewer that from a redox biology perspective, it would be highly interesting to look into changes in different thiol/disulphide couples of different LMW thiols. Indeed, when investigating changes in free thiol levels, individual thiol species may be differentially affected by a certain clinical event and this may become clearer when performing the above suggested measurements. Although we would have strongly considered to perform these experiments when we had sufficient biomaterials left, this is currently outside the scope of the present manuscript. Our aim was to associate total free thiol content with early and one-year graft function in patients subjected to renal transplantation. Therefore, more in-depth analysis of thiol subclasses should be strongly considered for future studies to gather more information about the dynamics of thiol changes. (Discussion, lines 226-238)

b. Second, serum albumin and the levels of its thiolation (mixed disulfides) should be considered to explain the interindividual differences observed in this study (see figure 1 and 2). For example, Cys is largely engaged in mixed disulfide formation and Hcy is more than 75 % bound to serum albumin (Galli F. et al Kideny Int 2013; Galli et al. Free Rad Res, 2014).

Of course, we acknowledge the role of serum albumin in measuring total free thiol content. Ideally, we would have performed an adjustment to total protein or albumin level as albumin is quantitatively the most important human plasma protein, and plasma proteins harbour the largest amount of extracellular free thiols and therefore greatly determine the quantity of potentially detectable free thiol groups (Turell et al., Free Radic Biol Med, 2013). Unfortunately, in this study we had no albumin measurements available to be included in the analysis. Although we may be forced to accept this is a limitation of our study, we previously showed that adjustment had no effect on outcome (Abdulle et al., BMC Med, 2020; Bourgonje et al., Antioxidants, 2019). (Discussion, line 218-222)

Turell L, Radi R, Alvarez B. The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radic Biol Med. 2013 Dec;65:244–53.

Abdulle AE, Bourgonje AR, Kieneker LM, Koning AM, la Bastide-van Gemert S, Bulthuis MLC, et al. Serum free thiols predict cardiovascular events and all-cause mortality in the general population: a prospective cohort study. BMC Med (2020) 18(1):130.

Bourgonje AR, Gabriëls RY, de Borst MH, Bulthuis MLC, Faber KN, van Goor H, et al. Serum Free Thiols Are Superior to Fecal Calprotectin in Reflecting Endoscopic Disease Activity in Inflammatory Bowel Disease. Antioxidants (Basel) (2019) 8(9):351.

c. Protein thiolation in plasma is a relevant indicator of oxidative stress in age-related and inflammatory diseases, including CKD (Reggiani et al. 2015, Fanti, Giustarini et al. 2015), and increased levels of biomarker linearly correspond to the decline of thiol to disulfide balance in extracellular fluids. This is relevant biomarker to utilize if one would like to explore the impaired redox of a patient with a systemic (not organ-specific) approach. This biomarker should be investigated together with other indices of damage of plasma proteins and/or polyunsaturated lipids (e.g. protein carbonylation, lipid peroxidation products, etc.).

First of all, we agree with the reviewer that protein thiolation is also a relevant indicator of oxidative stress in both healthy and diseased conditions and that additional measurements of other oxidative damage markers (e.g., protein carbonyls, lipid peroxidation products) would deliver us a more accurate reflection of the whole-body redox status. Of course, a combination of markers or combination of multiple read-outs may enhance reliability and applicability of the results. However, the measurement of total free thiols in plasma/serum actually behaves very similar as what the reviewer describes about protein thiolation: it is a relevant indicator of oxidative stress in age-related and inflammatory diseases and increased levels correspond to the decline of the thiol to disulphide balance in extracellular fluids as has been described in previous clinical studies that measured total free thiol content (Cortese-Krott MM et al., Antioxid Redox Signal 2017; Santolini et al., Curr Opin Physiol 2019; Cortese-Krott MM et al., 2020; Abdulle AE et al., BMC Med 2020; Damba T et al., Liver Int 2020; Frenay AS et al., Free Radic Bio Med 2016). All these studies supported the notion that one single quantification of total free thiols may be a robust method to evaluate the global redox state and accurately represent levels of systemic oxidative stress in a variety of conditions; for example, a previous study demonstrated an improved patient and graft survival in renal transplant recipients over several years of follow-up (Frenay AS et al, Free Radic Biol Med 2016).

Cortese-Krott MM, Koning A, Kuhnle GGC, Nagy P, Bianco CL, Pasch A, et al. The Reactive Species Interactome: Evolutionary Emergence, Biological Significance, and Opportunities for Redox Metabolomics and Personalized Medicine. Antioxid Redox Signal (2017) 27(10) :684-712.

Santolini J, Wootton SA, Jackson A, Feelisch M. The Redox Architecture of Physiological Function. Curr Opin Physiol 2019;9:34-47.

Cortese-Krott MM, Santolini J, Wootton SA, Jackson A, Feelisch M. The reactive species interactome. In: Sies H. Oxidative Stress: Eustress and Distress. Academic Press, 2020: 51-64.

Abdulle AE, Bourgonje AR, Kieneker LM, Koning AM, la Bastide-van Gemert S, Bulthuis MLC, et al. Serum free thiols predict cardiovascular events and all-cause mortality in the general population: a prospective cohort study. BMC Med (2020) 18(1):130.

Damba T, Bourgonje AR, Abdulle AE, Pasch A, Sydor S, van den Berg EH, et al. Oxidative stress is associated with suspected non-alcoholic fatty liver disease and all-cause mortality in the general population. Liver Int 2020;40(9):2148-2159.

Bourgonje AR, Gabriëls RY, de Borst MH, Bulthuis MLC, Faber KN, van Goor H, et al. Serum Free Thiols Are Superior to Fecal Calprotectin in Reflecting Endoscopic Disease Activity in Inflammatory Bowel Disease. Antioxidants (Basel) (2019) 8(9):351.

Frenay AS, de Borst MH, Bachtler M, Tschopp N, Keyzer CA, van den Berg E, et al. Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients. Free Radic Biol Med 2016;99:345-351.

Maybe the Authors have a bank of samples with aliquots of plasma still available for these determinations.

Unfortunately, we do not have a bank of samples with plasma aliquots left in order to perform the suggested determinations. The samples have already been allocated to other substudies (e.g. Thorne et al., Clin Proteomics, 2020; Nielsen et al., PloS One, 2019).

Thorne AM, Huang H, Eijken M, Norregaard R, Ploeg RJ, Jespersen B, et al. Subclinical effects of remote ischaemic conditioning in human kidney transplants revealed by quantitative proteomics. Clin Proteomics. 2020;1–13.

Nielsen MB, Krogstrup N V., Nieuwenhuijs-Moekeid GJ, Oltean M, Dor FJMF, Jespersen B, et al. P-NGAL Day 1 predicts early but not one year graft function following deceased donor kidney transplantation - The CONTEXT study. PLoS One. 2019;14(2):1–14.

d. Organ-specific indications cannot be expected from the proposed laboratory strategy to explore plasma thiols. The investigation of individual thiol species would provide much higher chances to obtain some level of information on the transplanted organ (see later in the next point).

We agree with the reviewer that the measurement of individual thiol species would provide us with an extra level of biological information compared to the single quantification of total free thiols. However, as mentioned above, we were not able to perform these measurements. In addition, the reviewer touches upon a fair argument here that organ-specific information cannot be expected from the measurement of total free thiols. Although determination of total free thiols is rather unspecific in relation to individual oxidative stress-mediated diseases, they are very specific to systemic oxidative stress and that makes it one of the most advocated, easy and accurate ways of oxidative stress quantification in vivo. Thus, by performing this measurement, our study does show a clear association between systemic oxidative stress and early and late graft function in recipients of kidney grafts from deceased donors.

3. The changes observed in the levels of free thiols in plasma of this study and their correlation with the success of transplantation and organ function are more than expected if we consider that tubular epithelial cells are very rich in gamma-glutamyl transpeptidase or ��GT (Giustarini, Galvagni et al. 2020). Therefore, reduced or absent function of tubular epithelia cells observed in the late stages of kidney disease, is expected to impair the renal metabolism and extracellular levels of LMW thiols, and especially of Cys. Possibly, what the Authors in this study are measuring with their thiol assay in plasma is the ��GT activity of the transplanted organ that obviously is higher in successfully treated subjects.

We thank the reviewer for bringing up this interesting hypothesis. We agree that this could very well be one of the contributing mechanisms to the changes we observed in the levels of plasma free thiols and their correlations with early and one-year graft function in renal transplant recipients. Unfortunately, we don’t have gamma-glutamyl transpeptidase measured in our study, but if we would have it available, this would have been an interesting and potentially valuable secondary analysis to further complement the presented results. We have previously published results on P-NGAL, a tubular function marker, which predicted the early kidney graft function (mGFR Day 5 and the estimated time to a 50% reduction in P-creatinine) well, but failed to be able to predict graft function at three and twelve months after kidney transplantation (Nielsen et al., PlosOne, 2019).

Nielsen MB, Krogstrup N V., Nieuwenhuijs-Moekeid GJ, Oltean M, Dor FJMF, Jespersen B, et al. P-NGAL Day 1 predicts early but not one year graft function following deceased donor kidney transplantation - The CONTEXT study. PLoS One. 2019;14(2):1–14.

4. The redox balance of extracellular thiols declines with the subject’ age (Jones, Mody et al. 2002, Giustarini, Dalle-Donne et al. 2006) and such a decline is even more rapid in case of premature aging and impaired redox homeostasis, which are characteristic conditions of CKD (Galli et al. Free Rad Res, 2014; Reggiani et al. 2015, Fanti, Giustarini et al. 2015). The results in this study (when the actual reduced form of thiols will be measured) should be corrected for the age of the patients as potential confounding factor.

We agree with the reviewer that age / aging strongly influence the levels of extracellular free thiols. In the previous studies cited in our above replies, the effect of age on thiol levels is very clear and has been replicated many times. Therefore, we agree that we should take into account the confounding effect of age. Age was therefore already included as a covariate in our linear regression models (statistics, lines 120, as well as Table 2 and 3).

5. Based on the correlation between thiols and mGFR it could be assumed that GFR could be utilized instead of thiols as a biomarker of a successful transplantation, which I guess is routine in the clinical monitoring of transplanted patients. What plasma thiols (those measured with this study) actually add up to the already available indices of organ function in transplantation protocols?

Our findings that plasma free thiols are associated with early and one-year graft function indeed raise the question whether and how this could be applied in clinical practice. The reviewer is correct that the GFR is routinely monitored in transplanted patients. However, in most cases, this will be the estimated GFR (eGFR) instead of the mGFR, which is the gold standard for measuring kidney function, but not frequently used in clinical practice as the mGFR is very time-consuming and costly to determine and is accompanied by a high patient burden as the transplant recipient needs to attend the nuclear medicine department for several hours in order to complete the determination of the mGFR. Furthermore, especially in the early days after transplantation, the kidney is not in a steady state condition and this affects both of these estimates. However, the measurement of plasma free thiols as a reflection of systemic oxidative stress may serve as an attractive screening tool as it is largely non-invasive and can be performed at relatively low costs (estimated at less than 2 dollars per assay). Nevertheless, it should be noted that further study is required to determine if plasma free thiols could be used as a real screening tool as this would depend on several circumstances, which can also change over time. For example, if a renal transplant recipient is suspected of having graft failure, a result of a relatively low concentration of plasma free thiols might be a screening result that could help to determine whether there is a necessity for further (invasive and/or costly) testing, e.g. by taking a renal biopsy.

6. The term “oxidated” should be revised and substituted with oxidized.

Thank you for this correction (line 22).

7. The Authors have disregarded most of the studies performed so far on plasma thiols in the introduction of their study and in the discussion of the results. This and other aspects discussed earlier in this revision report, demonstrate poor confidence with this topic. I suggest to refer to experts in the field of redox biology and medicine, and especially in thiol analysis, to obtain sufficed advise during the revision of their manuscript.

We regret to hear that our manuscript demonstrated poor confidence with the topic. It has never been our intention to ignore the important work of others. We have now added references on the subject to the Introduction section in the revised manuscript. Furthermore, we have had extensive discussion with redox biology experts from the University of Groningen and decided to include Arno R. Bourgonje as co-author to further improve the manuscript. Their advices are integrated into our rebuttal letter as well as the revised manuscript.

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Reviewer #1: No

Reviewer #2: Yes: Francesco Galli

Submitted filename: Response to Reviewers.docx

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26 Mar 2021

PONE-D-20-32900R1

Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients

PLOS ONE

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Partly

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Reviewer #1: (No Response)

Reviewer #2: I Don't Know

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20 Apr 2021

Dear reviewer #2

First of all, we would to express our gratitude again for your previous comments and suggestions on our manuscript, which were highly appreciated by all authors. We regret to hear that you were not fully satisfied with our revised manuscript. As we considered your suggestions and critiques to be very relevant, we have done our utmost best to address them carefully in order to optimise our manuscript and submit a successful revision. However, you have outlined several concerns about our study, mainly from a redox biological perspective, which we have all extensively addressed in our first rebuttal letter. Unfortunately, however, we were not able to completely resolve all of them, mainly due to the lack of available resources (i.e. sample volumes for measuring additional thiol compounds or laboratory indices). For the current revision, we were a bit unsure which main limits you are precisely referring to in your above statement, but we assume that they relate to the following two issues:

1. Lack of additional experiments and laboratory indices:

We agree with the reviewer that from a redox biology perspective, it would be highly interesting and relevant to perform more granular analyses by focusing on individual thiol species and include extra indices, which may be differentially affected by a certain clinical event and provide a deeper understanding of redox dynamics. Although we would have strongly considered to perform these experiments when we had sufficient biomaterials left, we are forced to accept this as a limitation of our study. Unfortunately, we do not have a bank of samples with aliquots left as remaining ones have already been allocated to other studies. This led us to decide to devote a substantial part of the Discussion section to your suggestions, so we could provide that as an inspiration for future studies. For further details see our previous replies to comments 2a, 2b, 2c, and 2d.

2. The measurement method of free thiols:

In our present study, we have determined total free thiol content in plasma by derivatization with DTNB as thiol-reactive agent. This is based on the standardised Ellman reaction. Using this method, we measure the combination of protein-bound free thiols (since proteins were not removed from the samples) and low-molecular-weight (LMW) free thiols, e.g. cysteine, glutathione and homocysteine. You are fully correct that total thiol content (including oxidized thiols with disulphide bridges) was not measured in our study. In that case, we should have used a stronger reducing agent like dithiothreitol (DTT), which is able to additionally reduce disulphide bonds and oxidized protein-bound thiols(1).However, we can reassure you that our measurement method has been well-developed, optimized and validated in our center and in literature. Over the past couple of years, extensive quality control experiments have been performed to ensure that samples are in optimal conditions and the generated results are trustworthy, e.g. the method is accompanied by high stability as repeated freeze-thaw cycles confer no major effects on free thiol levels of individual samples. Furthermore, our method has proven its power to predict clinical parameters in many previous studies(2–6), in which the levels of free thiols were in accordance with the concentration ranges as observed within the present study, using the exact same detection method. Finally, our method has been employed in different centers (Switzerland e.g.), while measurement results remain consistent, ruling out a potential location/environment-specific variation as well. Usually, mean total free thiols in serum/plasma add up to 400-600 μM(7), but this may vary by disease condition, ranging from 100 to 1000 μM (as can be detected by the L-cysteine calibration curve, see Materials & Methods).

Although you are absolutely correct that some major limitations will remain there, we have made every possible effort and used all available resources to achieve the best outcome for our study. Accordingly, we have improved the manuscript as carefully as possible based on your comments and suggestions. Additionally, as you suggested, we included additional expertise in redox biology and medicine, which also led to an updated list of authors. Again, we regret to be informed that you were not fully satisfied with our rebuttal. However, in light of all these considerations, we sincerely hope for your understanding and that you will re-consider your evaluation of our manuscript in light of PLoS ONE’s publication criteria.

Thank you for taking the time and effort to evaluate our manuscript. We look forward to receive your response.

1. Sutton TR, Minnion M, Barbarino F, Koster G, Fernandez BO, Cumpstey AF, et al. A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome. Redox Biol. 2018 Jun 1;16:359–80.

2. Abdulle AE, Bourgonje AR, Kieneker LM, Koning AM, La Bastide-Van Gemert S, Bulthuis MLC, et al. Serum free thiols predict cardiovascular events and all-cause mortality in the general population: A prospective cohort study. BMC Med. 2020 May 27;18(1).

3. Bourgonje AR, von Martels JZH, Bulthuis MLC, van Londen M, Faber KN, Dijkstra G, et al. Crohn’s Disease in Clinical Remission Is Marked by Systemic Oxidative Stress. Front Physiol. 2019 Apr 26;10:499.

4. Koning AM, Meijers WC, Pasch A, Leuvenink HGD, Frenay A-RS, Dekker MM, et al. Serum free thiols in chronic heart failure. Pharmacol Res. 2016 Sep;111:452–8.

5. Frenay A-RS, de Borst MH, Bachtler M, Tschopp N, Keyzer CA, van den Berg E, et al. Serum free sulfhydryl status is associated with patient and graft survival in renal transplant recipients. Free Radic Biol Med. 2016;99:345–51.

6. Damba T, Bourgonje AR, Abdulle AE, Pasch A, Sydor S, van den Berg EH, et al. Oxidative stress is associated with suspected non-alcoholic fatty liver disease and all-cause mortality in the general population. Liver Int. 2020 Sep 1;40(9):2148–59.

7. Turell L, Radi R, Alvarez B. The thiol pool in human plasma: the central contribution of albumin to redox processes. Free Radic Biol Med. 2013 Dec;65:244–53.

Submitted filename: Response to Reviewer 2 (2nd revision).docx

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28 Jul 2021

Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients

PONE-D-20-32900R2

Dear Dr. Nielsen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Gianpaolo Reboldi, MD, MSc, PhD

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PLOS ONE

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Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #2: No

Reviewer #3: Yes

**********

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Reviewer #2: N/A

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: it is my opinion that the limits of this study, addressed since the previous round of revision, do not allow the publication in this brad-spectrum Journal as regular original article. By admission of the Authors, these limits derive from the available resources and competencies that are obviously insufficient to reach original and relevant data. Also, the association between thiols and GFR is not such surprising and however the prediction power of measuring thiols on the graft function has not sufficiently been demonstrated being the study design and statistic power other limits that have not been addressed.

May be the Authors could convert this study in a different format; for example a letter to the Editor in a nephrology journal could be an option.

Reviewer #3: The subject of this study is interesting and in line with currently literature. In general, this study is well conducted, and the paper is very well written.

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Reviewer #2: No

Reviewer #3: No

2 Aug 2021

PONE-D-20-32900R2

Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients

Dear Dr. Nielsen:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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PLOS ONE