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Growth differentiation factor-15 (GDF-15), soluble ST2 (sST2), and high-sensitivity troponin I (hsTnI) are emerging predictors of adverse clinical outcomes. We sought to examine whether circulating concentrations are related to the development of kidney disease in the community.
Plasma GDF-15, sST2, and hsTnI concentrations were measured in 2,614 Framingham Offspring cohort participants (mean age 57 years, 54% women) at the sixth examination cycle (1995–1998). Associations of biomarkers with incident chronic kidney disease (CKD, eGFR<60 ml/min/1.73m2, n=276), microalbuminuria (urinary albumin to creatinine ratio ≥ 25 mg/g in women and 17 mg/g in men, n=191), and rapid decline in renal function (decline in eGFR ≥ 3 ml/min/1.73m2 per year, n=237), were evaluated using multivariable logistic regression; P<0.006 was considered statistically significant in primary analyses.
Participants were followed over a mean of 9.5 years. Higher plasma GDF-15 was associated with incident CKD (multivariable-adjusted OR 1.9 per 1-unit increase in log-GDF-15, 95% CI 1.6–2.3, P<0.0001) and rapid decline in renal function (OR 1.6, 95% CI 1.3–1.8, P<0.0001). GDF-15, sST2, and hsTnI had suggestive associations with incident microalbuminuria but did not meet the pre-specified P-value threshold after multivariable adjustment. Adding plasma GDF-15 to clinical covariates improved risk prediction of incident CKD: the c-statistic increased from 0.826 to 0.845 (P=0.0007), and categorical net reclassification was 6.3% (95% CI 2.7–9.9%).
Higher circulating GDF-15 is associated with incident renal outcomes, and improves risk prediction of incident CKD. These findings may provide insights into mechanisms of renal injury.
Over 82 million Americans have cardiovascular disease and over 20 million Americans have underlying kidney disease (1). Additionally, worsening renal function may both be a risk factor for and a consequence of progressive cardiovascular disease (2, 3). Indeed, clinical studies suggest that impaired renal function is a risk factor for the development of cardiovascular disease and subsequent prognosis (2). Conversely, experimental evidence suggests that cardiac injury may in turn accelerate loss of renal function (3).
Although the underlying mechanisms leading to chronic kidney disease (CKD) are multifactorial, immune and inflammatory responses are potential shared biologic pathways that can contribute to both worsening of kidney function (4) and cardiovascular disease (5). Two novel cardiovascular biomarkers, growth differentiation factor-15 (GDF-15) and soluble ST2 (sST2), are upregulated in response to inflammation (6, 7) as well as cardiovascular stress (8, 9) and, accordingly, share some similarities to troponins in reflecting potential insult to the cardiovascular system. Indeed, all three biomarkers, GDF-15, sST2, and troponin have emerged as strong predictors of mortality in individuals with existing cardiovascular disease (10–12), as well as community-dwelling adults (13–15).
Despite a growing body of evidence substantiating the role of GDF-15, sST2, and troponin in prognosticating cardiovascular disease, less is known about the potential association with the development of kidney disease. Previous studies have shown higher GDF-15 levels to be associated with progressive deterioration of kidney function and adverse prognosis in patients with existing kidney disease (16, 17). Cross-sectionally, higher sST2 levels appear to be associated with worse kidney function in participants with cardiovascular disease (18). Elevated circulating troponins are commonly observed in patients with CKD, and are associated with progression to end-stage renal disease and death (19). Despite these observations, the role of these biomarkers in the development of incident renal disease in individuals without pre-existing kidney disease is unknown. The association of each of these biomarkers of cardiovascular stress with kidney function may provide insights into potential mechanisms of renal injury, and the complex interaction of cardiovascular and renal disease. Recognizing the limitations of serum creatinine in detecting early decreases in renal function, these biomarkers may also improve our ability to predict early CKD. Thus, the purpose of this study was to examine the association of GDF-15, sST2, and highly sensitive troponin I (hsTnI), with incident renal outcomes including CKD, microalbuminuria, and progressive decline in renal function.
The Framingham Heart Study offspring cohort is a longitudinal community-based cohort that was recruited in 1971, and includes the children (and spouses’ children) of the original cohort participants. Participants have undergone serial examinations, including routine questionnaires, a physical examination, anthropometry, and blood testing (20). Plasma GDF-15, sST2, and hsTnI concentrations were measured at the sixth examination (1995–1998). Of the 3,532 attendees at the sixth examination, 2,786 also attended the eighth examination (2005–2008). Sample characteristics of those who did and did not attend the follow-up examination are shown in Supplemental Table 1. A total of 132 participants were excluded due to missing creatinine, 14 due to missing biomarker data, and 26 due to missing covariates, leaving 2,614 participants for this analysis. For each outcome, we further excluded participants with prevalent disease at baseline (164 with prevalent CKD for the incident CKD analyses, 698 with prevalent microalbuminuria, or missing covariates that were additionally adjusted for in the microalbuminuria analyses). The study was approved by the institutional review board of Boston University Medical Center, and all participants provided written informed consent.
Serum creatinine was measured using the modified Jaffe method, and calibrated as previously described (21). Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration formula (22). CKD was defined as stage 3 CKD or lower (eGFR < 60 ml/min/1.73m2). A rapid decline in renal function was defined as a decline in eGFR ≥ 3 ml/min/1.73m2 per year (23).
At the sixth and eighth examinations, spot morning urine samples were collected and initially frozen at −20°C and then transferred to −80°C. Urine albumin concentration was measured using immune-turbimetry (Tina-quant Albumin assay; Roche Diagnostics, Indianapolis, IN), and urinary creatinine was measured using the modified Jaffe method, with an intra-assay coefficient of variation of 1.7–3.8%. Urinary albumin to creatinine ratio was used (UACR) as a measure of urinary albumin excretion (24). Microalbuminuria was defined as a UACR ≥ 25 mg/g in women and 17 mg/g in men (25). Dipstick proteinuria was assessed on spot urine samples (Ames Labstix, Elkhart, Ind).
Blood samples were collected after an overnight fast and immediately centrifuged and stored at −80°C until assayed. Plasma GDF-15 concentrations were measured with a pre-commercial, automated electrochemiluminescent immunoassay on a Cobas e 411 analyzer (Roche Diagnostics). The assay has a limit of detection below 10 ng/L, a linear measuring range up to 20,000 ng/L, and an inter-assay coefficient of variation of 2.3% and 1.8% at GDF-15 concentrations of 1,100 ng/L and 17,200 ng/L, respectively (Roche Diagnostics, data on file). sST2 levels were measured using a high-sensitivity sandwich immunoassay (Presage™ ST2; Critical Diagnostics, San Diego, CA, USA) (26), with a lower detection limit of 2 ng/mL and total coefficient of variation of 4.0%. hsTnI concentrations were measured with an ultra-sensitive immunoassay for cardiac troponin I, utilizing a novel, single-molecule counting technology (Erenna hsTnI, Singulex), with a limit of detection of 0.2 pg/mL, a detection range of 0.5 to 70 pg/mL, and coefficient of variation of 10% (27). Plasma homocysteine, aldosterone, B-type natriuretic peptide (BNP), and cystatin C were measured as previously described (Supplemental Data Methods).
Participants underwent a comprehensive clinical assessment at the baseline examination. Hypertension was defined as a systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or current antihypertensive drug treatment. Diabetes mellitus was defined as a fasting glucose ≥ 126 mg/dL or the use of insulin or oral hypoglycemic medications. Current smoking was defined as smoking at least one cigarette per day over the past year. Total and high-density lipoprotein cholesterol levels were measured after an overnight fast. Cardiovascular events were adjudicated by a 3-physician panel after systematic review of outpatient and hospital medical records.
The association of each biomarker (GDF-15, sST2, and hsTnI) with each renal outcome (incident CKD, incident microalbuminuria, rapid decline in renal function) was modeled using multivariable logistic regression. Biomarkers were natural log-transformed due to right-skewed distributions. The primary models for incident CKD and rapid decline were adjusted for age, sex, baseline eGFR, hypertension, diabetes, and baseline proteinuria by dipstick; microalbuminuria analyses were adjusted for age, sex, hypertension, diabetes, BMI, smoking, baseline log-UACR, and HDL cholesterol. Based on previous analyses of predictors of incident kidney disease (28, 29), models were further adjusted for homocysteine, and aldosterone in secondary analyses; additionally microalbuminuria analyses were adjusted for BNP. To account for the number of biomarkers (n=3) and kidney traits tested (n=3), primary analysis results were considered significant at a corrected P-value threshold of 0.05/9 = 0.006.
In secondary analyses, we excluded participants with baseline diabetes mellitus, hypertension, or prevalent coronary heart disease or heart failure from GDF-15 analyses. We also adjusted for cystatin C (measured at exam 7) in secondary analyses.
To assess the incremental benefit of each biomarker (if found significantly associated with renal function), the c statistic was compared in models with traditional risk factors with and without the addition of the biomarker. We estimated the Integrated Discrimination Improvement (IDI), and the category-free Net Reclassification Improvement (NRI) metric (30). Category-based NRI metrics were calculated using the same empirical groupings of risk as previously reported: low- (0 to <3%), intermediate- (3 to 6%), and high-risk (>6% 8-year risk of CKD or microalbuminuria) (28). Analyses were conducted with SAS version 9.2 for Windows.
The study sample included 2,614 participants of the Framingham Offspring cohort with a mean age of 57 years and slightly more than half were women. Thirty-seven percent had hypertension, 7% had diabetes, and 3% had a history of coronary heart disease at baseline. Median and first/third quartile concentrations for each biomarker are displayed in Table 1.
Over 9.5 years of follow-up, 276 participants (140 women) developed incident CKD, defined as an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73m2. Each 1-unit increase in log-GDF-15 was associated with a nearly two-fold increased odds of incident CKD (multivariable-adjusted odds ratio (OR) per unit increase log-GDF-15 1.91, 95% CI 1.61–2.26, P<0.0001, Table 2). Neither sST2 nor hsTnI was associated with incident CKD at the pre-specified threshold for statistical significance after accounting for clinical covariates (P=0.05 and P=0.03, respectively).
Multivariable-adjusted ORs of incident CKD by quartiles of GDF-15, sST2, and hsTnI are displayed in Figure 1A, and show a significant increase in CKD risk across GDF-15 (P for trend<0.0001). Compared with the lowest quartile, the upper GDF-15 quartile was associated with a 5.65-fold increased odds (95% CI 2.97–10.75, P<0.0001) of incident CKD. The number of incident CKD events by biomarker quartiles is shown in Supplemental Table 2.
A total of 191 participants (78 women) developed incident microalbuminuria after 9.5 years of follow-up, defined as a urinary albumin to creatinine ratio ≥25 mg/g in women and 17 mg/g in men. All three biomarkers had suggestive associations with incident microalbuminuria, but did not meet statistical significance after multivariable-adjustment, when considering the corrected P-value threshold (P=0.02 for GDF-15, P=0.007 for both sST2 and hsTnI, Table 2).
The risk of incident microalbuminuria by quartiles of GDF-15, sST2, and hsTnI is displayed in Figure 1B, and shows trends toward increasing risk of microalbuminuria across sST2 and hsTnI quartiles (P for trend=0.005 for both). The number of incident microalbuminuria events by biomarker quartiles is shown in Supplemental Table 2.
Over 9.5 years of follow-up, 237 participants (133 women) experienced a rapid decline in kidney function, defined as a decline in eGFR ≥3 ml/min/1.73m2 per year. GDF-15 was associated with rapid decline (OR per unit increase log-GDF 1.55, 95% CI 1.31–1.83, P<0.0001, Table 2). Neither sST2 nor hsTnI were associated with a rapid decline in eGFR after multivariable adjustment.
Multivariable-adjusted ORs of rapid decline in renal function by quartiles of GDF-15, sST2, and hsTnI are displayed in Figure 1C. Increasing GDF-15 quartiles were associated with a higher risk of rapid decline in renal function (P for trend<0.0001). Specifically, the upper GDF-15 quartile was associated with a 2.51-fold increased odds (95% CI 1.54–4.09, P=0.0002) of rapid decline compared with the lowest quartile. The number of rapid decline events are shown by biomarker quartile in Supplemental Table 2.
We next evaluated the incremental predictive value of GDF-15 when added to a base model of established clinical CKD risk factors. The addition of GDF-15 to a clinical model predicting incident CKD resulted in a statistically significant increase in the c-statistic (from 0.826 to 0.845, P=0.0007, Table 3), and also led to modest but significant improvements in reclassification (categorical NRI 6.3%, 95% CI 2.7–9.9%, Supplemental Table 3; category-free NRI 43.5%, 95% CI 31.1–55.9%).
After accounting for the three biomarkers simultaneously, in addition to previously established biomarkers associated with renal outcomes (aldosterone and homocysteine for incident CKD and rapid decline in renal function; aldosterone, homocysteine, and BNP for microalbuminuria) (28), GDF-15 remained a significant predictor of incident CKD (OR per 1-unit increase in log-GDF15 1.72, 95% CI 1.44–2.07, P<0.0001) and rapid decline in renal function (OR per unit increase log-GDF-15 1.34, 95% CI 1.11–1.62, P=0.002). After adjusting for the previously established biomarkers, GDF-15, sST2, and hsTnI were not significant predictors of incident microalbuminuria (P=0.13, 0.04, and 0.03, respectively).
After additional adjustment for cystatin C, GDF-15 remained an independent predictor of incident CKD (multivariable-adjusted OR per unit increase log-GDF-15 1.45, 95% CI 1.21–1.75, P<0.0001), whereas the association with rapid decline in renal function was attenuated but remained significant (OR per unit increase log-GDF-15 1.23, 95% CI 1.01–1.49, P=0.04).
In sensitivity analyses excluding participants with baseline diabetes, hypertension, and coronary heart disease/heart failure, GDF-15 remained a significant predictor of incident CKD and rapid decline in renal function (Supplemental Table 4).
Our findings demonstrate that GDF-15 is associated with incident CKD and rapid decline in renal function in participants from the Framingham Heart Study. The addition of GDF-15 to clinical covariates also resulted in improvement in the prediction of incident CKD, including the c-statistic and the categorical NRI. This suggests that GDF-15 may be useful in predicting the development of CKD, years prior to the clinical onset of disease.
GDF-15 is a stress-responsive member of the transforming growth factor-β cytokine superfamily (31). In the kidney, GDF-15 is expressed weakly along the entire nephron, and markedly upregulated in the outer medullary collecting duct in response to metabolic acidosis and potassium depletion, where it appears to trigger compensatory proliferation of acid-secreting collecting duct cells (32). Despite potential cardio- and renal-protective effects shown in experimental studies, higher GDF-15 levels have been associated with adverse prognosis in patients with cardiovascular disease (10), as well as in individuals in the community (13, 15). Higher GDF-15 levels are also associated with increased mortality in hemodialysis patients, and in patients with diabetic nephropathy (16, 17). In the latter group, elevated GDF-15 concentrations were associated with a greater decline in estimated glomerular filtration rate, and higher progression to end-stage renal disease (17). Our study extends these findings to a population-based sample, where we demonstrate a strong association with incident kidney disease. In addition, our findings establish the association of GDF-15 and incident kidney disease in a population with early CKD, where associations are less likely to be confounded by other metabolic derangements and reduced filtration, which accompany more advanced renal disease.
Whether GDF-15 actively contributes to the development of CKD due to its association with vascular dysfunction or via direct kidney effects remains to be investigated. In an experimental model of diabetic renal injury, elevated urinary GDF-15 was associated with a proximal tubule injury marker (33). Renal GDF-15 expression also appears to be upregulated in response to metabolic acidosis (34) and kidney injury (35). Outside of the kidney, GDF-15 is expressed in various tissue types including cardiomyocytes (8) in response to oxidative and/or metabolic stress and inflammation. In the community, elevated levels of GDF-15 are associated with endothelial dysfunction and other measures of subclinical cardiovascular disease (36). Further studies are needed to clarify whether GDF-15 is a marker or a causal mediator of cardiovascular and renal disease.
Our findings suggest that GDF-15 may be useful in the prediction of incident CKD. The addition of GDF-15 to the clinical model resulted in a modest increment in the c-statistic and increases in NRI that were comparable to improvements seen with a multi-marker panel consisting of aldosterone, BNP, and homocysteine in the same population (28). These improvements in CKD risk prediction stand in contrast to results observed from the use of biomarkers in cardiovascular disease prediction in the general population, where biomarkers have been variably successful in improving risk prediction (37). Biomarkers may be more informative in CKD risk prediction, because the diagnosis of CKD is made on the basis of biochemical data. Future studies are needed to validate our findings in additional large cohorts, and to evaluate the clinical utility of using GDF-15 in CKD prevention efforts will be needed.
We also observed nominal associations of GDF-15, sST2, hsTnI and incident microalbuminuria, although these associations did not persist after accounting for multiple hypothesis testing, or adjustment for previously studied biomarkers. ST2 is a member of the interleukin-1 receptor family whose production in cardiomyocytes is upregulated in response to mechanical stress (38). Like GDF-15, ST2 and its ligand IL-33 are expressed in endothelial cells in response to inflammatory cytokines (6), and the ST2/IL-33 pathway appears to be a contributor to endothelial dysfunction and early atherosclerosis (39). Vascular dysfunction is known to precede the development of microalbuminuria (40), and may be one of the plausible mechanisms by which elevated GDF-15 and sST2 may be relevant to kidney injury. Elevated circulating troponins are observed commonly in patients with CKD, and are associated with increased risk of kidney disease progression (19). In experimental studies, myocardial damage is directly associated with the development of proteinuria and focal glomerulosclerosis (3), suggesting that the association of circulating troponin and kidney damage is related to cardiac injury, rather than diminished clearance (19). Further studies will be needed to firmly establish any association of sST2, hsTnI and kidney outcomes.
There are several limitations to our study that deserve mention. Kidney function was estimated based on a single creatinine and urinary albumin measurement, rather than persistent changes over time, which may have resulted in misclassification of renal outcomes. Our results may have also been influenced by survivor bias, as only individuals who attended both initial and follow-up examinations for assessment of renal function were included. Thus, sicker individuals at greater risk for developing kidney disease may not have been captured. However, both of these potential limitations would have likely biased our results toward the null. Our sample was restricted to a predominantly white of European ancestry, and generalization of our findings to other ethnicities is unclear.
In summary, we found that GDF-15 was associated with new onset kidney disease and rapid decline in renal function in the community. The addition of GDF-15 to established risk factors resulted in improvements in CKD risk prediction. This suggests that GDF-15 may help to identify individuals at high risk for developing CKD and, in turn, may aid in targeting preventive efforts in the future.
This work was partially supported by the National Heart, Lung and Blood Institute’s Framingham Heart Study (Contract No. N01-HC-25195). Dr. Ho was supported by an American Heart Association Fellow-to-Faculty Transition award. Dr. Wollert was supported by the German Ministry of Education and Research (BMBF, BioChancePlus). Dr. Cheng is supported by an award from the Ellison Foundation. Dr. Januzzi is partially supported by the DeSanctis Clinical Scholar Endowment. Measurement of sST2 was performed by Critical Diagnostics, Inc. GDF-15 assays were provided by Roche Diagnostics, Inc. The urinary albumin excretion assays were supported by a grant from the American Diabetes Association and by donation of reagents by Roche Diagnostics, Inc. Measurement of hsTnI was performed by Singulex, Inc.
Statement of competing financial interests: Dr. Januzzi has received research grant funding from Roche Diagnostics and Critical Diagnostics, and has served on the advisory board for Critical Diagnostics. Drs. Wollert and Kempf are named as co-inventors on a patent for the use of GDF-15 for cardiovascular applications, and have a contract with Roche Diagnostics for the development of a GDF-15 assay.