Environmental Cadmium Exposure Induces an Increase in Systolic Blood Pressure by Its Effect on GFR

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

To thank the authors for their manuscript on the effects of environmental cadmium exposure on SBP and GFR.

I see the need to make comments to improve the quality of the manuscript in order to assess its publication.

The deleterious effects of cadmium exposure on renal function are already known. Cadmium accumulates in the proximal tubules of the kidney, causing cellular damage and tubular dysfunction. This can result in proteinuria, an alteration in protein reabsorption, and can progress to chronic renal failure. The nephrotoxic effects of cadmium are associated with the induction of oxidative stress, apoptosis, and renal fibrosis, negatively affecting the kidney's ability to filter and excrete metabolic waste. Prolonged exposure can lead to a decrease in the glomerular filtration rate and the development of chronic kidney diseases, highlighting the importance of preventing and controlling environmental and occupational exposure to this metal.

1.- However, both in the introduction and in the discussion there is no paragraph justifying the findings found in the results of this study that explains any mechanisms associated with cadmium-induced renal impairment. Please add it.

 Johri N, Jacquillet G, Unwin R. Heavy metal poisoning: the effects of cadmium on the kidney. Biometals 2010;23:783–92. http://dx.doi.org/10. 1681/ASN.2005121319

2.- In lines 318-319, mention is made of the association in cadmium-induced eGFR reduction and SBP elevation, but the sentence is not completed explaining the reason for this association. Please add a comment on this.

3.- There are known data on the association of proteinuria, hypertension and eGFR drop in relation to cadmium exposure, in the present work there is no mention of proteinuria data of the patients, this is very relevant, please add this data in the results.

4.- There is a recent article published in NDT on cadmium, tobacco and impaired kidney function titled  ¨Cadmium, active smoking and renal function deterioration in patients with type 2 diabetes¨. Please add a paragraph from this paper to the discussion.

Nephrol Dial Transplant (2023) 38: 876–883 https://doi.org/10.1093/ndt/gfac270 Advance Access publication date 17 September 2022

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Reviewer 1.

Comments and Suggestions

To thank the authors for their manuscript on the effects of environmental cadmium exposure on SBP and GFR.

I see the need to make comments to improve the quality of the manuscript in order to assess its publication.

The deleterious effects of cadmium exposure on renal function are already known. Cadmium accumulates in the proximal tubules of the kidney, causing cellular damage and tubular dysfunction. This can result in proteinuria, an alteration in protein reabsorption, and can progress to chronic renal failure. The nephrotoxic effects of cadmium are associated with the induction of oxidative stress, apoptosis, and renal fibrosis, negatively affecting the kidney's ability to filter and excrete metabolic waste. Prolonged exposure can lead to a decrease in the glomerular filtration rate and the development of chronic kidney diseases, highlighting the importance of preventing and controlling environmental and occupational exposure to this metal.

RESPONSE: Thank you for a thorough review of our work, insightful comments, guidance and helpful suggestions to improve the quality of this manuscript. Accordingly, our manuscript has undergone an extensive revision. Changes to the text and new references are in blue.

Comment 1: However, both in the introduction and in the discussion there is no paragraph justifying the findings found in the results of this study that explains any mechanisms associated with cadmium-induced renal impairment. Please add it.

Johri N, Jacquillet G, Unwin R. Heavy metal poisoning: the effects of cadmium on the kidney. Biometals 2010;23:783–92. http://dx.doi.org/10. 1681/ASN.2005121319

RESPONSE:

• The last paragraph of Introduction has been rewritten, as quoted below, to explicitly state our study objectives and explain Cd effects on kidneys and methodology used in this study (lines 75-87). A cadmium review by Johri et al. has been cited as ref. [41].

“The deleterious effects of Cd exposure on kidneys have extensively been investigated in workplace and non-workplace exposure settings [41]. However, a few studies have explored the mechanism by which Cd raises blood pressure. The present study aimed to test the hypothesis that an increase in blood pressure is the result of kidney tubular cell damage by Cd. Because a reduction in the glomerular filtration rate (GFR) is a common sequela of ischemic acute tubular necrosis, acute and chronic tubulointerstitial fibrosis, all of which create impediments to filtration [42,43], we quantified tubular cell damage, tubular proteinuria, systolic and diastolic blood pressures (SBP and DBP) according to the estimated GFR (eGFR) and Cd exposure levels in those diagnosed with and without hypertension. Urinary excretion of Cd (E_Cd) and blood Cd concentrations ([Cd]_b) were used as measures of exposure levels. Urinary excretion of β₂-microglobulin (β₂M) and N-acetyl-β-D-glucosaminidase (NAG) were used to assess tubular proteinuria and damage to the kidney tubular cells, respectively [41].”

Comment 2. In lines 318-319, mention is made of the association in cadmium-induced eGFR reduction and SBP elevation, but the sentence is not completed explaining the reason for this association. Please add a comment on this.

RESPONSE:

• The Discussion has now been organized into three subtitles and the referred paragraph has now been completed and presented in Subtitle 3.3 (lines 329-346), given below.

3.3. A Rise of Blood Pressure Due to Tubular Damage and GFR Loss

To the best of our knowledge, the present study has provided, for the first time, evidence linking Cd-induced eGFR reduction to a rise in blood pressure (Figure 3). SBP was inversely associated with eGFR in women (β = −0.227) and men (β = −0.320) who had medium plus heavy Cd burdens, (E_Cd/C_cr) × 100 ≥ 1 µg/ L filtrate. DBP showed a weak inverse association with eGFR (Figures 3a,3b vs. 3c,3d). By multiple regression analysis (Table 6), an independent effect of eGFR reduction on a rise of SBP was found in the medium plus heavy Cd burden group (β = −0.176). Using the mediation analysis, GFR reduction was a partial mediator of Cd effect on blood pressure increases (Figure 4). Our observation that blood pressure rises as GFR falls helps explain as to why patients with CKD have hypertension almost universally.

Doubling of body burden of Cd was associated, respectively with 41% and 23% and 48% increases in the POR values for low eGFR, moderate and severe tubular proteinuria, evident from increased β₂M excretion levels (Table 4). In Japanese population studies, increased β2M excretion levels were associated with enhanced risks of hypertension and a large decline in eGFR (10 mL/min/1.73 m2) over a five-year observation period [63,64]. Thus, an increased risk of hypertension among study subjects could be attributed to Cd-induced tubular damage and GFR loss. 

Comment 3. There are known data on the association of proteinuria, hypertension and eGFR drop in relation to cadmium exposure, in the present work there is no mention of proteinuria data of the patients, this is very relevant, please add this data in the results.

RESPONSE:

• Data on tubular proteinuria and kidney tubule damage, assessed with urinary excretion of β₂M and NAG have been added to Table 1 (lines 92-98).
• A new Table 4 has been constructed to link Cd exposure to increment of prevalence odds of having low eGFR and tubular proteinuria (lines 149-161).
• The results reported in a new Table 4 are quoted below.

“For every one-year older, the POR for low eGFR, moderate and severe tubular proteinuria rose 14.6%, 3.7% and 6.4%, while doubling of body burden of Cd was associated with 41%, 23% and 48% increases in the POR for low eGFR, moderate and severe tubular proteinuria, respectively. Among diabetics, there were 4.3-fold, 5.5-fold and 13.4-fold increases in the POR for low eGFR, moderate and severe tubular proteinuria, respectively.”

Comment 4. There is a recent article published in NDT on cadmium, tobacco and impaired kidney function titled ¨Cadmium, active smoking and renal function deterioration in patients with type 2 diabetes¨. Please add a paragraph from this paper to the discussion.

Nephrol Dial Transplant (2023) 38: 876–883 https://doi.org/10.1093/ndt/gfac270 Advance Access publication date 17 September 2022

RESPONSE:

• A suggested reference has been cited in the Introduction (lines 64-66).
• Relevant issues are discussed in Section 3.2 (lines 313-329).

“Continued exposure will lead to its accumulation in tissues and organs throughout the body, notably kidneys, where it promoted the progression of kidney disease to kidney failure, especially in diabetics [36].”

[36] Oosterwijk, M.M.; Hagedoorn, I.J.M.; Maatman, R.G.H.J.; Bakker, S.J.L.; Navis, G.; Laverman, G.D. Cadmium, active smoking and renal function deterioration in patients with type 2 diabetes. Nephrol. Dial. Transplant. 2023, 38, 876-883.

3.2. Different Susceptibility to Cadmium-Induced Hypertension

People with diabetes were more susceptible to adverse kidney effects of Cd than the non-diabetic counterpart. In a Dutch cross-sectional study, including 231 patients with type 2 diabetes, Cd exposure increased the risk of diabetic kidney disease [59]. In a six-year median follow-up of these diabetic patients, a progressive reduction of eGFR was attributable to Cd exposure [36].

In a prospective cohort study, a 49% increase in all-cause mortality among the diabetics was associated with urinary Cd levels > 0.60 μg/L [60]. In the present, the risk of having low eGFR, moderate and severe tubular proteinuria among diabetics rose 4.3-fold, 5.5-fold and 13.4-fold, respectively (Table 4). Both SBP (β = 0.265) and DBP (β = 0.193) rose significantly among diabetics with (ECd/Ccr) × 100 ≥ 1 µg/ L filtrate (Table 6). This Cd-induced SBP and DBP increment may promote kidney disease development in Cd-exposed diabetics given that hypertension is a strong independent risk factor for the development and progression of CKD [3,61]. A 1.76-fold increase in death from CVD among U.S. citizens with hypertension was associated with elevated Cd exposure, indicated by blood Cd levels ≥ 0.80 μg/L [62]. At the same Cd exposure level, the risk of death from CVD rose 2.12-fold among non-smokers who have hypertension [62].

[59] Hagedoorn, I.J.M.; Gant, C.M.; Huizen, S.V.; Maatman, R.G.H.J.; Navis, G.; Bakker, S.J.L.; Laverman, G.D. Lifestyle-related exposure to cadmium and lead is associated with diabetic kidney disease. J. Clin. Med. 2020, 9, 2432.

[60] Liu, Y.; Yang, D.; Shi, F.; Wang, F.; Liu, X.; Wen, H.; Mubarik, S.; Yu, C. Association of serum 25(OH)D, cadmium, CRP with all-cause, cause-specific mortality: A prospective cohort study. Front. Nutr. 2022, 9, 803985.

[61] Mayne, K.J.; Sullivan, M.K.; Lees, J.S. Sex and gender differences in the management of chronic kidney disease and hypertension.  J. Hum. Hypertens. 2023, 37, 649-653.

[62] Chen, S.; Shen, R.; Shen, J.; Lyu, L.; Wei, T. Association of blood cadmium with all-cause and cause-specific mortality in patients with hypertension. Front. Public Health 2023, 11, 1106732.

Reviewer 2 Report

Comments and Suggestions for Authors

The team is a experienced in kidney disease and the nephron-toxicity of several compounds as cadmium. This article is the following of these two of the same group The Effect of Cadmium on GFR Is Clarified by Normalization of Excretion Rates to Creatinine Clearance and Health Risk in a Geographic Area of Thailand with Endemic Cadmium Contamination: Focus on Albuminuria, published in January 2023. This last article aims to determine, to quantify the impact of the presence of cadmium in food in nephro-toxicity in a population in Thailand.

Today, authors are looking at the impact of this contamination in high blood pressure development through a nephron-toxicity. This study is an important one regarding cadmium toxicity research.

Several important parameters as the prevalence of smoking patients are taking in consideration.

Despite several good points of this study, I have several comments regarding this study and the article.

A-     If you agree, I will begin by the study itself.

1/ To my point of view this study is not well-designed regarding the gender parameters. Indeed, on 447 persons included in this study, 333 are women and 114 are men. Thus, around 3-fold more women than men. Nevertheless, it is well documented today that prevalence of hypertension is gender specific (for ex of articles: https://bmjopen.bmj.com/content/11/1/e038326, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239955/, https://www.ahajournals.org/doi/full/10.1161/01.HYP.37.5.1199, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444798/).

Can you explain in the text why you selected more women than men? I did not find an explanation in your manuscript. But it is important to mention it to show us that you are aware of the gender specificity prevalence in hypertension.  

2/ As mentioned in several articles as the first study I mentioned in point 1, the age is also a significant parameter, mainly in women because of menopause, when studying hypertension. Thus, if I appreciate the mean value, I think that the range of the age has to be shown (ex: 18-70; mean x).

3/ I am sorry but I did not understand the figure 2. This figure is done by average of data or by individual data (as dot-plot)? To my point of view a dot-plot graph is better to show the distribution of the data. The same for figure 4.  

4/ In the methods section you mentioned this one: “The diagnosis of hypertension was based on questionnaires, a physician’s diagnosis or use of antihypertensive medication, and ascertained by a single measurement of systolic and diastolic blood pressures (SBP and DBP).”. Several clinician team, as mine, to be sure of the hypertension diagnosis we do a 24h measurement of the blood pressure or, when time is lacking, 3 measurements of the blood pressure. Can you explain why you did only one measurement?

5/ Can you tell to the reader if a biostatistician review yours statistics? I have no time to check all the statistics models you used.

B-     Comments regarding the text itself

1/ In material and methods, you mentioned this: “The Office of the Human Research Ethics Committee of Walailak University approved the study protocol for the Nakhon Si Thammarat group 342 [46]. The study protocol for the Mae Sot group was approved by the Institutional Ethical Committees of Chiang Mai University and the Mae Sot Hospital [47]”. Can you please give the number of the approval?¨

2/ In the introduction it miss data regarding several prevalence as hypertension or statistics for this line “because of its ubiquitous environmental presence, exposure to the nephrotoxic metal pollutant cadmium (Cd) is also a significant risk factor for CVD [3] and CKD [4]».

3/ “However, the % of hypertension and diabetes in the present study were higher than those recorded in studies from U.S. where % hypertension and diabetes were 39% and 10.3%-13%, respectively [38,39]”. Do you think that it is a limitation to your study?

4/ You have to mention if you followed ethical principles of the declaration of Helsinki. Just a sentence is sufficient. 

Author Response

Reviewer 2.

Comments and Suggestions

The team is an experienced in kidney disease and the nephron-toxicity of several compounds as cadmium. This article is the following of these two of the same group The Effect of Cadmium on GFR Is Clarified by Normalization of Excretion Rates to Creatinine Clearance and Health Risk in a Geographic Area of Thailand with Endemic Cadmium Contamination: Focus on Albuminuria, published in January 2023. This last article aims to determine, to quantify the impact of the presence of cadmium in food in nephron-toxicity in a population in Thailand.

Today, authors are looking at the impact of this contamination in high blood pressure development through a nephron-toxicity. This study is an important one regarding cadmium toxicity research.

Several important parameters as the prevalence of smoking patients are taking in consideration.

Despite several good points of this study, I have several comments regarding this study and the article.

RESPONSES: Thank you for reviewing our work, your comments, suggestions, and guidance for improvement.  Accordingly, our manuscript has been revised extensively. Changes to the text and new references are in blue.

1. Comment on the study: If you agree, I will begin by the study itself.

Comment A1. To my point of view this study is not well-designed regarding the gender parameters. Indeed, on 447 persons included in this study, 333 are women and 114 are men. Thus, around 3-fold more women than men. Nevertheless, it is well documented today that prevalence of hypertension is gender specific (for ex of articles: https://bmjopen.bmj.com/content/11/1/e038326, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239955/, https://www.ahajournals.org/doi/full/10.1161/01.HYP.37.5.1199, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444798/).

Can you explain in the text why you selected more women than men? I did not find an explanation in your manuscript. But it is important to mention it to show us that you are aware of the gender specificity prevalence in hypertension.  

RESPONSE:

• The reasons for oversampling of women, are provided in the Introduction (lines 47-48), Discussion (lines 267-271) and Section 4.1. Participants (lines 355-357).
• Suggested references have been cited in the text where applicable.

Comment A 2. As mentioned in several articles as the first study I mentioned in point 1, the age is also a significant parameter, mainly in women because of menopause, when studying hypertension. Thus, if I appreciate the mean value, I think that the range of the age has to be shown (ex: 18-70; mean x).

RESPONSE:

• We have added the age range in Table 1. Please see also response to comments B3 below.
• An effect of hormonal status among the women and the small number of men included in a study have been declared as limitations (lines 267-271), as given below.

“The limitations of the study are acknowledged. They include only a one-time assessment of Cd exposure and its effects, the heterogeneity in the hormonal status of women; both menopausal and post-menopausal women were included [52-56] and the small number of men (n =114) in the study cohort; this means a definitive conclusion on gender disparity in prevalence, severity and adverse outcomes of hypertension could not be made [53-56].”

[52] Vahter, M.; Berglund, M.; Akesson, A. Toxic metals and the menopause. J. Br. Menopause Soc. 2004, 10, 60-64.

[53] Reckelhoff, J.F. Gender differences in the regulation of blood pressure. Hypertension 2001, 37, 1199-1208.

[54] Reckelhoff, J.F. Mechanisms of sex and gender differences in hypertension. J. Hum. Hypertens. 2023, 37, 596-601.

[55] Connelly, P.J.; Currie, G.; Delles, C. Sex differences in the prevalence, outcomes and management of hypertension. Curr. Hypertens. Rep. 2022, 24, 185-192.

[56] Choi, H.M.; Kim, H.C.; Kang, D.R. Sex differences in hypertension prevalence and control: Analysis of the 2010-2014 Korea National Health and Nutrition Examination Survey. PLoS One. 2017, 12, e0178334.

Comment A3. I am sorry but I did not understand the figure 2. This figure is done by average of data or by individual data (as dot-plot)? To my point of view a dot-plot graph is better to show the distribution of the data. The same for figure 4.  

RESPONSE:

• We agree that the data distribution is best presented as a dot-plot.
• Our former figures 2 and 4 were constructed from mean values, adjusted for covariates using univariate analysis. This allows for an independent effect of Cd, gender, diabetes and smoking to be determined.
• In revisions, the results of logistic regression analysis have been provided in a new table 4. Consequently, former figures 2 and 4 have been removed.

Comment A4. In the methods section you mentioned this one: “The diagnosis of hypertension was based on questionnaires, a physician’s diagnosis or use of antihypertensive medication, and ascertained by a single measurement of systolic and diastolic blood pressures (SBP and DBP).”. Several clinician team, as mine, to be sure of the hypertension diagnosis we do a 24h measurement of the blood pressure or, when time is lacking, 3 measurements of the blood pressure. Can you explain why you did only one measurement?

RESPONSE:

• We have addressed issues raised in Section 4.2. Blood Pressure and Cadmium Exposure Ascertainment (lines 379-385) as given below.

4.2. Blood Pressure and Cadmium Exposure Ascertainment

Our study design was population-based that recruited participants from their communities. This precluded a-24 h measurement or more visits to communities for second measurement. One-time measurement of SBP/DBP was the average of 3 repeated measurements. The diagnosis of hypertension relied primarily on the assessment made by the presiding physician and the recorded use of anti-hypertensive medication. Of 233 subjects included as hypertensive cases in our study, 220 (94.4%) were being treated and 13 hypertensive cases were identified during our visit.

Comment A5. Can you tell to the reader if a biostatistician reviewed your statistics? I have no time to check all the statistics models you used.

RESPONSE:

• Our data analysis has been reviewed by a professional biostatistician who advised an application of a simple mediation analysis to show, for the first time, a cause-effect relationship between GFR loss and blood pressure rises (Section 2.7, Figure 4, lines 236-250).
• We confirm that all statistical models have been double checked to ensure consistent coding of categorical variables.

1. Comments regarding the text itself

Comment B1. In material and methods, you mentioned this: “The Office of the Human Research Ethics Committee of Walailak University approved the study protocol for the Nakhon Si Thammarat group [46]. The study protocol for the Mae Sot group was approved by the Institutional Ethical Committees of Chiang Mai University and the Mae Sot Hospital [47]”. Can you please give the number of the approval?¨

RESPONSE:

• Approval numbers have been provided.

Comment B2. In the introduction it missed data regarding several prevalence as hypertension or statistics for this line “because of its ubiquitous environmental presence, exposure to the nephrotoxic metal pollutant cadmium (Cd) is also a significant risk factor for CVD [3] and CKD [4].

RESPONSE: 

• Missing data have been provided in the Introduction (lines 42-54), as given below.

Of concern, mortality from CKD rose from the 13^th leading cause of global death in 2000 to the 7^th in 2021, and it is projected to be the 5^th leading cause of years of life lost by 2040 [4,5].

[4] Foreman, K.J.; Marquez, N.; Dolgert, A.; Fukutaki, K.; Fullman, N.; McGaughey, M.; Pletcher, M.A.; Smith, A.E.; Tang, K.; Yuan, C.W.; et al. Forecasting life expectancy, years of life lost, and all-cause and cause-specific mortality for 250 causes of death: Reference and alternative scenarios for 2016-40 for 195 countries and territories. Lancet 2018, 392, 2052-2090.

[5] GBD, 2021. Forecasting Collaborators. Burden of disease scenarios for 204 countries and territories, 2022-2050: a forecasting analysis for the Global Burden of Disease Study 2021. Lancet 2024, 403, 2204-2256.

“Because of its ubiquitous environmental presence, exposure to the nephrotoxic metal pollutant cadmium (Cd) is also a significant risk factor for CVD [6] and CKD [7]. A 75% increase in deaths from any cause among those with CKD was associated with Cd exposure even at low levels, reflected by urinary Cd levels ≥ 0.60 μg/g creatinine [8]. The risk of having CVD rose 2.58-fold and 2.79-fold at blood Cd level of 1 μg/L and urinary Cd excretion rate of 0.5 μg/g creatinine, respectively [6]. Notably, Cd exposure appeared to adversely affect females and males differently [9-12].

In the U.S. general population, an overall mean urinary Cd excretion was 0.5 µg/g creatinine, and 2.5%, 7.1%, and 16% of non-smoking women (aged ≥ 20 years) had urinary Cd levels > 1, > 0.7, and > 0.5 μg/g creatinine, respectively [13]. In comparison, a study from Thailand reported a 22.5% prevalence of Cd excretion ≥ 1 µg/g creatinine in non-smoking women who had low body iron stores [14]. Thus, the proportions of at-risk subpopulations are concerning.”

[8] Zhang, J.; Wang, X.; Ma, Z.; Dang, Y.; Yang, Y.; Cao, S.; Ouyang, C.; Shi, X.; Pan, J.; Hu, X. Associations of urinary and blood cadmium concentrations with all-cause mortality in US adults with chronic kidney disease: A prospective cohort study. Environ. Sci. Pollut. Res. Int. 2023, 30, 61659-61671.

[9] Nishijo, M.; Satarug, S.; Honda, R.; Tsuritani, I.; Aoshima, K. The gender differences in health effects of environmental cadmium exposure and potential mechanisms. Mol. Cell. Biochem. 2004, 255, 87-92.

[10] Trzcinka-Ochocka, M.; Jakubowski, M.; Szymczak, W.; Janasik, B.; Brodzka, R. The effects of low environmental cadmium exposure on bone density. Environ. Res. 2010, 110, 286-293.

[11] Nishijo, M.; Nambunmee, K.; Suvagandha, D.; Swaddiwudhipong, W.; Ruangyuttikarn, W.; Nishino, Y. Gender-specific impact of cadmium exposure on bone metabolism in older people living in a cadmium-polluted area in Thailand. Int. J. Environ. Res. Public Health 2017, 14, 401.

[12] Madrigal, J.M.; Ricardo, A.C.; Persky, V.; Turyk, M. Associations between blood cadmium concentration and kidney function in the U.S. population: Impact of sex, diabetes and hypertension. Environ. Res. 2018, 169, 180-188.

[13] Mortensen, M.E.; Wong, L.Y.; Osterloh, J.D. Smoking status and urine cadmium above levels associated with subclinical renal effects in U.S. adults without chronic kidney disease. Int. J. Hyg. Environ. Health 2011, 214, 305-310.

[14] Satarug, S.; Ujjin, P.; Vanavanitkun, Y.; Baker, J.R.; Moore, M.R. Influence of body iron store status and cigarette smoking on cadmium body burden of healthy Thai women and men. Toxicol. Lett. 2004, 148, 177-185.

Comment B3. “However, the % of hypertension and diabetes in the present study were higher than those recorded in studies from U.S. where % hypertension and diabetes were 39% and 10.3%-13%, respectively [38,39]”. Do you think that it is a limitation to your study?

RESPONSE:

• We have added statements, quoted below (lines 261-273) to clarify this point, and to address on age and gender differences raised in comment A2 above.

“The high prevalence of hypertension in this cohort was the strength as it means that even a modest sample size (n < 1000) could give enough number of cases from which a reliable conclusion could be drawn. Previously, an effect of smoking on the risk of CVD has been found to be partially mediated by Cd [50,51]. An inclusion of smokers and diabetics was additional strength as they enabled adjustment for their effects in realistic population situations. Another strength was that Cd exposure was assessed with blood and urinary Cd levels.

The limitations of the study are acknowledged. They include only a one-time assessment of Cd exposure and its effects, the heterogeneity in the hormonal status of women; both menopausal and post-menopausal women were included [52-56] and the small number of men (n =114) in the study cohort; this means a definitive conclusion on gender disparity in prevalence, severity and adverse outcomes of Cd-induced hypertension could not be made [53-56].”

[52] Vahter, M.; Berglund, M.; Akesson, A. Toxic metals and the menopause. J. Br. Menopause Soc. 2004, 10, 60-64.

[53] Reckelhoff, J.F. Gender differences in the regulation of blood pressure. Hypertension 2001, 37, 1199-1208.

[54] Reckelhoff, J.F. Mechanisms of sex and gender differences in hypertension. J. Hum. Hypertens. 2023, 37, 596-601.

[55] Connelly, P.J.; Currie, G.; Delles, C. Sex differences in the prevalence, outcomes and management of hypertension. Curr. Hypertens. Rep. 2022, 24, 185-192.

[56] Choi, H.M.; Kim, H.C.; Kang, D.R. Sex differences in hypertension prevalence and control: Analysis of the 2010-2014 Korea National Health and Nutrition Examination Survey. PLoS One. 2017, 12, e0178334.

Comment B4. You have to mention if you followed ethical principles of the declaration of Helsinki. Just a sentence is sufficient. 

RESPONSE:

• The Helsinki statement has been added in Section 4.1 Participants (lines 355-356).

“This study was conducted following the principles outlined in the Declaration of Helsinki.”

Reviewer 3 Report

Comments and Suggestions for Authors

The authors evaluated the effects of environmental cadmium exposure on systolic blood pressure and eGFR and concluded, by mediation analysis that a 22.7% increase in SBP and a 5% increase in DBP were attributable to Cd-induced loss of GFR. Besides this, a two-fold increased risk of hypertension was associated with urinary Cd excretion of 0.98 µg/g creatinine and a blood Cd level of 0.61 µg/L.

Overall, there are many studies exploring toxic effects of Cd, but the advantage of this research is related to the fact that the authors evaluated the effects of Cd in doses that can be measured in blood and urine in population that is not professionally exposed to Cd. One of the limitations of the study is also that it is only descriptive research, with no potential mechanisms that are in the basis of observed effects.

In Introduction, the authors hypothesized……. an increase in blood pressure is the result of a declining GFR due to kidney damage from toxic Cd accumulation…But levels of Cd in blood and urine evaluated in present study, are not toxic doses, these levels of Cd are reported widely in studies of non-occupationally exposed populations. Also, these concentrations are measured only once in included subjects, so it cannot be claimed that it was the result of …toxic Cd accumulation…My suggestion is to rephrase the hypothesis.

Table 1. There is no statistical difference in % Hypertension between different groups according to Cd exposure levels. How these results can be explained?

More information about subjects involved in the study are also needed, detailed inclusion and exclusion criteria. Why were subjects with diabetes and increased BMI (overweight) included in the study? 6.9% had low eGFR, what were the reasons for low eGFR?

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer 3.

Comments and Suggestions

The authors evaluated the effects of environmental cadmium exposure on systolic blood pressure and eGFR and concluded, by mediation analysis that a 22.7% increase in SBP and a 5% increase in DBP were attributable to Cd-induced loss of GFR. Besides this, a two-fold increased risk of hypertension was associated with urinary Cd excretion of 0.98 µg/g creatinine and a blood Cd level of 0.61 µg/L.

Overall, there are many studies exploring toxic effects of Cd, but the advantage of this research is related to the fact that the authors evaluated the effects of Cd in doses that can be measured in blood and urine in population that is not professionally exposed to Cd. One of the limitations of the study is also that it is only descriptive research, with no potential mechanisms that are in the basis of observed effects.

RESPONSE: Thank you for evaluating for our work, knowledgeable comments, and suggestions for improvement.  We have accordingly revised our paper to respectfully address all issues and concerns raised. Changes to the text are in blue. 

Comment 1.

In Introduction, the authors hypothesized……. an increase in blood pressure is the result of a declining GFR due to kidney damage from toxic Cd accumulation…But levels of Cd in blood and urine evaluated in present study, are not toxic doses, these levels of Cd are reported widely in studies of non-occupationally exposed populations. Also, these concentrations are measured only once in included subjects, so it cannot be claimed that it was the result of …toxic Cd accumulation…My suggestion is to rephrase the hypothesis.

RESPONSE:

• An entire last paragraph of the Introduction has been improved to address the potential mechanisms underlying our hypothesis that blood pressure rises as GFR falls (lines75-87), given below.

“The deleterious effects of Cd exposure on kidneys have extensively been investigated in workplace and non-workplace exposure settings [41]. However, a few studies have explored the mechanism by which Cd raises blood pressure. The present study aimed to test the hypothesis that an increase in blood pressure is the result of kidney tubular cell damage by Cd. Because a reduction in the glomerular filtration rate (GFR) is a common sequela of ischemic acute tubular necrosis, acute and chronic tubulointerstitial fibrosis, all of which create impediments to filtration [42,43], we quantified tubular cell damage, tubular proteinuria, systolic and diastolic blood pressures (SBP and DBP) according to the estimated GFR (eGFR) and Cd exposure levels in those diagnosed with and without hypertension. Urinary excretion of Cd (E_Cd) and blood Cd concentrations ([Cd]_b) were used as measures of exposure levels. Urinary excretion of β2-microglobulin (β₂M) and N-acetyl-β-D-glucosaminidase (NAG) were used to assess tubular proteinuria and damage to the kidney tubular cells, respectively [41].”

Comment 2.

Table 1. There is no statistical difference in % Hypertension between different groups according to Cd exposure levels. How these results can be explained?

RESPONSE:

• Older age, higher BMI, more prevalent of diabetics in the low-Cd burden group mean that % hypertension cannot be attributed to differences in Cd burden (Table 1).
• However, after adjustment for these confounding factors (age, BMI and diabetes), a two-fold increased risk of hypertension was associated with urinary Cd excretion of 0.98 µg/g creatinine and a blood Cd level of 0.61 µg/L.

Comment 3.

More information about subjects involved in the study are also needed, detailed inclusion and exclusion criteria. Why were subjects with diabetes and increased BMI (overweight) included in the study? 6.9% had low eGFR, what were the reasons for low eGFR?

RESPONSE:

• We have now provided inclusion and exclusion criteria in Section 4.1. Participants (lines 368-373).
• We included diabetes and obese subjects because these conditions are common causes of low eGFR worldwide. We have stated these inclusion as the strength of our study (lines 261-273), quoted below.

“The high prevalence of hypertension in this cohort was the strength as it means that even a modest sample size (n < 1000) could give enough number of cases from which a reliable conclusion could be drawn. Previously, an effect of smoking on the risk of CVD has been found to be partially mediated by Cd [50,51]. An inclusion of smokers and diabetics was additional strength as they enabled adjustment for their effects in realistic population situations. Another strength was that Cd exposure was assessed with blood and urinary Cd levels.

The limitations of the study are acknowledged. They include only a one-time assessment of Cd exposure and its effects, the heterogeneity in the hormonal status of women; both menopausal and post-menopausal women were included [52-56] and the small number of men (n =114) in the study cohort; this means a definitive conclusion on gender disparity in prevalence, severity and adverse outcomes of Cd-induced hypertension could not be made [53-56].”

• We provided the reasons for low eGFR among study subjects in new Table 4 (lines 149-161), summarized below.

For every one-year older, the risk for low eGFR rose 14.6%. Doubling of body burden of Cd increased risk for low eGFR 41%. Among diabetics, the risk of low eGFR rose 4.3-fold, compared to non-diabetics with the same overall Cd burden.

• Given that low eGFR is a diagnostic criterion for CKD our observation that blood pressure rises as GFR falls thus help explain why patients with CKD have hypertension almost universally.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

We would like to thank the authors for having accepted the recommendations made in this review, which has greatly improved the quality of the manuscript. For my part it can be published. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors responded to all comments.

Comments on the Quality of English Language

MInor editing of English language required.