Cystatin C is a predictor for long-term All-Cause and 1 Cardiovascular Mortality in US Adults with Metabolic 2 Syndrome

This study examined the relationship between Cystatin C (CysC) levels and all-cause, CVD, 5 and cancer mortality in US metabolic syndrome (MetS) patients.


Introduction
Metabolic syndrome (MetS), characterized by central obesity, elevated blood sugar, hypertension, and abnormal lipid levels, has seen a significant uptick in prevalence, currently affecting an estimated 34.7% of US adults [1].This surge raises concerns due to its close link with cardiovascular disease (CVD) and heightened mortality risk.Those diagnosed with MetS not only face an elevated susceptibility to developing CVD, but also bear a risk of reduced life expectancy due to a higher likelihood of experiencing severe CVD events such as heart attacks and strokes [2,3].Additionally, they are at an increased vulnerability to other chronic conditions, including type 2 diabetes, heart disease, and specific types of cancer, all contributing to a decreased overall life expectancy [2][3][4].Furthermore, researches consistently showed that individuals with MetS have an increased likelihood of developing various types of cancer, such as breast, colorectal, endometrial, pancreatic, gastric, and prostate cancer, among others [5,6].Moreover, those with MetS who are diagnosed with cancer tend to experience a higher risk of mortality [5].
Cystatin C (CysC), an endogenous marker of renal function, has garnered attention as a potential prognostic biomarker in various disease states [7].In addition to its role in estimating GFR, CysC has garnered attention for its potential to reflect broader pathophysiological processes beyond kidney function [7,8].Research suggests that elevated levels of CysC are associated with conditions such as inflammation, oxidative stress, and endothelial dysfunction-factors that are not only relevant to kidney disease, but also play crucial roles in the development and progression of various CVD and metabolic disorders, including MetS [9][10][11][12].Despite this mechanistic plausibility, the prognostic significance of CysC in individuals with MetS remains underexplored, presenting a critical knowledge gap in risk stratification and personalized care, particularly regarding long-term mortality prediction.
This study seeks to address this gap by examining the relationship between CysC levels and mortality risks in individuals with MetS, leveraging a well-characterized longitudinal cohort with extended follow-up.By investigating the independent association of CysC with all-cause, CVD, and cancer mortality in this high-risk population, we aim to elucidate its potential as a robust prognostic marker.Furthermore, we endeavor to explore potential effect modifiers and interactions, such as age, gender, and comorbidity burden, to inform tailored risk assessment and targeted interventions.

Data sources and preparation
The data utilized in this study were obtained from the National Health and Nutrition Examination Survey (NHANES), a prospective cohort research initiative.NHANES is a nationally representative survey of non-institutionalized individuals in the United States and involves participants who provided written informed consent following approval by the NCHS institutional review board.Employing a multilevel, stratified probability design, the survey annually samples 5,000 participants who undergo standardized questionnaires and physical examinations.Data collection has been ongoing since 1999, with updated datasets released biennially at https://www.cdc.gov/nchs/nhanes/index.htm.The research protocol was reviewed by the institutional review board at Shenzhen People's Hospital, which determined that the study did not necessitate informed consent as it involved publicly available, de-identified data.

Metabolic syndrome
As per the criteria established by the American Endocrine Association (ACE) and the American Society of Clinical Endocrinology (ACCE), the diagnosis of Metabolic Syndrome (MetS) requires the presence of at least three out of the following five conditions: [14].These criteria encompass an increased abdominal obesity (waist circumference≥88 cm for females and ≥102 cm for males), elevated triglyceride levels(≥150 mg/dL or receiving medication), reduced high-density lipoprotein cholesterol (HDL-C) levels (<40 mg/dL for males and <50 mg/dL for females or receiving medication), heightened blood pressure (SBP ≥130 mm Hg or DBP ≥85 mm Hg or antihypertensive medication), and elevated fasting plasma glucose levels (≥100 mg/dL or receiving medication) [14,15].

Serum laboratory parameters
The Dade Behring N Latex CysC assay was used to measure CysC levels in blood samples [16].
This assessment was conducted on the Dade Behring Nephelometer II.According to Newman's evaluation of various assay methodologies, this current assay is deemed the most accurate and precise among automated assays across the clinical concentration range, demonstrating an intraassay imprecision range between 2.0 and 3.0% coefficient of variation.The inter-assay imprecision range falls between 3.2 and 4.4% coefficient of variation.Furthermore, the assay range spans from 0.23 to 7.25 mg/dl.Newman also observed that this assay exhibited superior sensitivity and lacked analytical interference when compared to other automated assays [16].
Serum albumin, globulin, fast glucose, fast insulin, creatinine, uric acid, blood urea nitrogen (BUN), triglyceride, total cholesterol, HDL, low-density lipoprotein cholesterol (LDL), and Creactive protein (CRP) were analyzed by laboratory methods reported elsewhere [17] Serum creatinine levels were measured using the kinetic Jaffe rate method [18].Albumin levels were detected using the Bromcresol Purple method within the Boehringer Mannheim Diagnostics albumin system [17].Cholesterol levels were detected through an enzymatic reaction coupled with photometry [17].Glucose levels were assessed using the glucose hexokinase method, while insulin levels were quantified using the insulin radioimmunoassay (RIA) [17].BUN levels were determined using the enzymatic kinetic method, while uric acid levels were assessed through the colorimetric method.CRP levels were quantified using latex-enhanced nephelometry [17].

All-cause, CVD and cancer mortality
The study focused on all-cause mortality, CVD mortality and cancer mortality as the primary outcomes.All-cause mortality referred to the number of participants who passed away due to any cause after completing their baseline survey but before December 31, 2018.Mortality follow-up data was obtained from NHANES Public-use Linked Mortality Files (https://www.cdc.gov/nchs/data-linkage/mortality.htm) using the International Classification of Diseases, Tenth Revision (ICD-10) codes to identify causes of death.These alphanumeric codes are used to track diseases and health-related issues, enabling researchers to categorize deaths based on the leading causes of death.Specifically, CVD deaths were identified using specific ICD codes (054-068), encompassing coronary artery disease, heart failure, stroke, and peripheral artery disease.This method ensured accurate tracking of deaths related to CVD by utilizing precise ICD codes for categorization [19,20].

Covariates
Our study encompassed a range of clinical and demographic factors as covariates to address potential sources of confounding.These covariate variables included age, sex, body mass index (BMI), race and ethnicity, educational level, marital status, smoking status, alcohol drinking status, annual family income, and chronic diseases.Information regarding these covariates was derived from survey responses in NHANES.Participants were classified into five racial/ethnic groups: Mexican American, other Hispanic, non-Hispanic White, Black, or Other (including multiracial).Educational classifications comprised less than high school, high school graduate or equivalent, Some College or AA degree, and college graduate or above [21].Marital status was delineated using seven categories: Never married, Married, Widowed, or Divorced or Separated.
Medical conditions such as anemia, angina, heart attack, congestive heart failure, coronary heart disease, chronic kidney disease, asthma, chronic obstructive pulmonary disease (COPD), diabetes mellitus (DM), hypertension, hyperlipidemia [22], cancer, and stroke were diagnosed by a physician or other qualified healthcare professional.Smoking and drinking behaviors were grouped into three categories: never, past, and current use.BMI was calculated using the standard formula: weight (kg)/[height (m2) × height (m2)].The calculation of eGFR was performed using the CKD-EPI equation [23].

Statistical Analysis
Continuous variables were accompanied by 95% confidence intervals (CIs), while categorical variables were presented as percentage frequencies.Comparison of continuous and categorical data was performed using T-tests and χ2 tests.Given the low rates of missing data for all variables, no imputation approach was employed.Mortality risk was assessed using Cox proportional hazards regression models.We employed Cox regression models with the following adjustments: no adjustment (Model 1); adjusted for age, sex, and body mass index (Model 2); Model 2 with additional adjustments for race, marital status, education, alcohol use, smoking status, annual family income, and eGFR (Model 3); and Model 3 with further adjustments for asthma, congestive heart failure, coronary heart disease, chronic kidney disease, COPD, DM, hypertension, hyperlipidemia, stroke, and cancer (Model 4).Visual representation was provided through curve fitting and Kaplan-Meier curves.The statistical analyses were conducted using the R software package (http://www.R-project.org,The R Foundation), the nhanesR package, and Free Statistics software version 1.9.Statistical significance was determined by a two-sided P value <0.05.

Demographics
Table 1 presents the characteristics of 1,980 individuals with MetS, divided into three CysC tertiles.The average age was 58.0 ± 16.5 years, with significant age group variations.Gender distribution showed a 51% female and 49% male split.Race composition varied significantly.

Participants with MetS
The fitted curves indicated that lower CysC levels in participants with MetS decreased all-cause mortality (Figure 1A), CVD mortality (Figure 1B), and cancer mortality (Figure 1C).Conversely, as CysC levels increased, the risk of all-cause, CVD mortality, and cancer mortality increased significantly (Figure 1A-Figure 1C).Kaplan-Meier survival curves also suggested that higher CysC levels were associated with increased all-cause mortality, CVD mortality, and cancer mortality risk (P<0.05)(Figure 2A-C).After adjusted for various covariates in multiple models, the HR of total CysC level with all-cause, CVD, and cancer mortality were 1.63 (95% CI 1.42-1.88,P < 0.001), 1.53 (95% CI 1.19-1.95,P = 0.001), and 1.53 (95% CI 1~2.32,P = 0.048), respectively (Table 2).Hazard Ratios (HRs) stratified by CysC levels for all-cause, CVD, and cancer mortality in participants with MetS were adjusted for various covariates in multiple models.

Covariate Adjustment:
In Models 2, 3, and 4, various covariates were systematically adjusted, progressively refining the analysis.Model 4, which comprehensively accounted for demographic factors, lifestyle variables, estimated glomerular filtration rate, and a spectrum of comorbidities, underscored the robust, independent, and dose-dependent association between elevated CysC levels and increased allcause, CVD, and cancer mortality in individuals with MetS.These results provided compelling evidence for the significant impact of CysC levels on mortality outcomes, even after meticulous adjustments for multiple confounding factors in Model 4, further emphasizing the critical role of CysC as a prognostic marker in individuals with MetS.

Subgroup analyses
To investigate the robustness of the association between CysC and all-cause mortality, CVD mortality, and cancer mortality in individuals with MetS, stratified analyses were conducted across various subgroups.The examined variables included gender, asthma, congestive heart failure, coronary heart disease, chronic kidney disease, chronic obstructive pulmonary disease (COPD), DM, hypertension, hyperlipidemia, stroke, and cancer.However, none of these variables demonstrated a significant modification of the relationship between CysC and the risk of all-cause mortality, CVD mortality, or cancer mortality in individuals with MetS (all p-values for interaction > 0.05), indicating an absence of substantive impact on the association between CysC levels and mortality risks by these factors (Table 3).The ROC curves for serum biomarkers and age in predicting All-Cause, CVD, and Cancer

Discussion
Our study has unearthed a compelling association between CysC levels and mortality risks in individuals with MetS.The significance of our findings lies in the revelation of CysC's broader prognostic implications beyond its traditional role in renal function assessment.Notably, the observed dose-dependent relationships between elevated CysC levels and heightened risks of allcause, CVD, and cancer mortality underscore the multi-dimensional nature of CysC as a prognostic indicator in the context of MetS.The study's results address the unmet need for robust prognostic markers in this at-risk population, underscoring the clinical significance of CysC in risk stratification and prognostication for individuals with MetS.
Our results shed light on the demographic and clinical landscape of individuals with elevated CysC levels within the MetS population.We observed distinct differences in age, gender distribution, race composition, and the prevalence of comorbidities across CysC tertiles.Notably, Higher CysC Tertile associated with altered metabolic parameters, including lower albumin, higher globulin levels, and elevated markers of renal dysfunction (creatinine, uric acid, and blood urea nitrogen).Inconsistent lipid profile trends and marginally significant CRP levels suggest renal impact on lipid metabolism and potential systemic inflammation.Higher CysC Tertile also exhibited a higher burden of chronic conditions, emphasizing the intricate interplay between CysC levels, aging, and the complexity of multimorbidity in this population.
CysC is implicated in the adverse prognosis of several diseases.A cohort study involving 1502 individuals suggests that CysC may exert detrimental effects on metabolism, particularly in the context of abdominal obesity, thereby potentially promoting and predicting the onset and progression of MetS [25].Furthermore, an 8-year follow-up study of 7027 individuals indicates that serum CysC levels are associated with an increased incidence of diabetes and elevated mortality risk in middle-aged and elderly populations [26].Notably, CysC is proposed as a potential biomarker for heightened risks of MetS, CVDs, and renal impairment [27].Additionally, it serves as a marker for muscle wasting [28,29], a condition linked to increased all-cause mortality, with a causal relationship observed between muscle wasting and overall mortality [30].Our study found a clear dose-response relationship between CysC levels and mortality risk, with higher CysC levels consistently associated with elevated risks of all-cause, CVD, and cancer mortality.The robustness of these associations was further validated through comprehensive covariate adjustments, underscoring the independent prognostic value of CysC as a biomarker for adverse mortality outcomes in individuals with MetS.
This study offering valuable insights into the underlying pathophysiological mechanisms contributing to heightened mortality risks in MetS population.CysC, known to be involved in processes related to inflammation and endothelial dysfunction, serves as a crucial link to the development and progression of CVD and cancer [31,32].Elevated levels of CysC reflect increased inflammatory burden, insulin resistance, and endothelial dysfunction, providing a mechanistic explanation for the elevated risk of adverse CVD and oncological outcomes [10,11,33,34].Furthermore, impaired renal function, as indicated by elevated CysC levels, mirrors the systemic impact of MetS and contributes to the amplified risk of mortality.Additionally, the close association of CysC with atherosclerosis, a primary pathological process underlying CVD events, enhances its predictive value for CVD mortality [12,31].These mechanistic insights provide a deeper understanding of the complex interplay between CysC and mortality risk in this population, emphasizing the pivotal role of this biomarker in clinical prognosis and risk assessment.
We found that CysC emerged as the most robust predictor across all-cause and CVD mortality outcomes in patients with MetS, demonstrating superior predictive efficacy compared to eGFR, Urea nitrogen, Creatinine, Uric acid, and CRP.Notably, our analysis also identified eGFR as a significant predictor, albeit exhibiting slightly lower predictive efficacy compared to CysC.The combination of CysC with age substantially enhanced the predictive accuracy for mortality outcomes, particularly for all-cause and CVD mortality.In contrast, Urea nitrogen, Creatinine, Uric acid, and CRP exhibited relatively lower predictive performance across all mortality outcomes.While these biomarkers may provide valuable insights into metabolic and inflammatory processes, their predictive utility for mortality risk assessment in MetS patients appears limited compared to CysC and eGFR.These findings strongly suggest that CysC may emerge as a promising biomarker for assessing the risk of all-cause mortality and CVD death in MetS patients, offering invaluable insights for clinical practice.Integrating CysC assessment into risk stratification protocols may empower clinicians to pinpoint high-risk individuals who stand to benefit from targeted While our study benefits from a large sample size, a prolonged follow-up duration, and meticulous adjustments for potential confounders, it is important to acknowledge the observational nature of our study, which introduces inherent limitations.Although our study rigorously adjusted for various covariates, residual confounding cannot be entirely excluded.Therefore, future prospective studies and randomized controlled trials are warranted to further validate our findings and investigate the potential therapeutic implications of modulating CysC levels.Additionally, mechanistic studies are needed to unravel the biological underpinnings of the observed associations and to identify potential targets for intervention or risk modification.
personalized interventions in individuals with MetS.Moving forward, further research endeavors are essential to harness the full potential of CysC as a prognostic tool and to develop targeted interventions aimed at improving the outcomes of individuals with MetS and elevated CysC levels.Declarations States, 2011-2016.JAMA 2020, 323(24):2526-2528.2. Li D, Wang L, Zhou Z, Song L, Chen S, Yang Y, Hu Y, Wang Y, Wu S, Tian Y: Lifetime risk of cardiovascular disease and life expectancy with and without cardiovascular disease according to changes in metabolic syndrome status.Nutr Metab Cardiovasc Dis 2022, M, Foster MC, Anderson C, Burke GL, Haq N, Kalyani RR, Ouyang P, Sibley CT, Tracy R, Woodward M et al: Metabolically Healthy Obesity, Transition to Metabolic Syndrome, and Cardiovascular Risk.J AM COLL CARDIOL 2018, 71(17):1857-1865.4. Pammer LM, Lamina C, Schultheiss UT, Kotsis F, Kollerits B, Stockmann H, Lipovsek J, Meiselbach H, Busch M, Eckardt KU et al: Association of the metabolic syndrome with mortality and major adverse cardiac events: A large chronic kidney disease cohort.J INTERN MED 2021, 290(6):1219-1232. 5. Karra P, Winn M, Pauleck S, Bulsiewicz-Jacobsen A, Peterson L, Coletta A, Doherty J, Ulrich CM, Summers SA, Gunter M et al: Metabolic dysfunction and obesity-related cancer: Beyond Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgae225/7643125 by guest on 13 April 2024 mortality in cancer patients: a retrospective cohort study .J Cachexia Sarcopenia Muscle 2022, Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgae225/7643125 by guest on 13 April 2024 33.Balta S, Demirkol S, Ay SA, Cakar M, Sarlak H, Celik T: Serum cystatin-C levels correlate with endothelial dysfunction in patients with the metabolic syndrome.J INTERN MED 2013, Park SA, Ko SH, Yim HW, Ahn YB, Yoon KH, Cha BY, Son HY, Kwon HS: Insulin resistance and inflammation may have an additional role in the link between cystatin C and cardiovascular disease in type 2 diabetes mellitus patients.METABOLISM 2010, 59(2):241-246.

Figure 1
Figure 1 The relationship between Cystatin C with all-cause, CVD and cancer mortality by curve fitting Adjusted for age, BMI, race, education level, marriage status, drinking, smoking, annual family income and eGFR.Fig1A The curve fitting of Cysc and all-cause mortality in metabolic syndrome participants Fig1B The curve fitting of Cysc and CVD mortality in metabolic syndrome participants Fig1C The curve fitting of Cysc and cancer mortality in metabolic syndrome participants

Figure 3
Fig2A The Kaplan-Meier survival curves of CysC and all-cause mortality in metabolic syndrome participants Fig2B The Kaplan-Meier survival curves of CysC and CVD mortality in metabolic syndrome participants Fig2C The Kaplan-Meier survival curves of CysC and cancer mortality in metabolic syndrome participants Downloaded from https://academic.oup.com/jcem/advance-article/doi/10.1210/clinem/dgae225/7643125 by guest on 13 April 2024

Table The HRs of CysC with all-cause, CVD and cancer mortality in the participants with metabolic syndrome
Adjusted for age, sex, and body mass index.Model 3: Adjusted for age, sex, race, marital status, body mass index, education, alcohol user, smoking status, annual family income and eGFR.Model 4: Adjusted for age, sex, race, marital status, body mass index, education, alcohol user, smoking status, annual family income, eGFR, Asthma, congestive heart failure, coronary heart disease, chronic kidney disease, chronic obstructive pulmonary disease (COPD), diabetes mellitus, hypertension, hyperlipidemia, stroke and cancer.