Increased circulating galectin-1 levels are associated with the progression of kidney function decline in patients undergoing coronary angiography

Galectin-1 modulates acute and chronic inflammation, and is associated with glucose homeostasis and chronic renal disease. Whether the serum galectin-1 level can predict short-term and long-term renal outcomes after contrast exposure in patients undergoing coronary angiography (CAG) remains uncertain. This study aimed to evaluate the relationship between the serum galectin-1 level and the incidence of contrast-induced nephropathy (CIN), and to investigate the predictive role of the circulating galectin-1 level for renal function decline in patients undergoing CAG. In total, 798 patients who had undergone CAG were enrolled. Baseline creatinine and serum galectin-1 levels were determined before CAG. CIN was defined as an increase in the serum creatinine level of 0.5 mg/dl or a 25% increase from baseline within 48 h after the procedure, and renal function decline was defined as > 30% reduction of the estimated glomerular filtration rate from baseline. All patients were followed for at least 1 year or until the occurrence of death after CAG. Overall, CIN occurred in 41 (5.1%) patients. During a median follow-up period of 1.4 ± 1.1 years, 80 (10.0%) cases showed subsequent renal function decline. After adjustment for demographic characteristics, kidney function, traditional risk factors, and medications, higher galectin-1 levels were found to be associated independently with a greater risk of renal function decline [tertile 2: hazard ratio (HR) 5.56, 95% confidence interval (CI) 1.79–17.22; tertile 3: HR 5.56, 95% CI 1.97–16.32], but not with CIN, regardless of the presence of diabetes. In conclusion, higher baseline serum galectin-1 levels were associated with a greater risk of renal function decline in patients undergoing CAG, but were not associated independently with CIN.


Results
Baseline patient characteristics. In total, 798 subjects who underwent elective CAG and/or percutaneous coronary intervention (PCI) were enrolled in this analysis (Fig. 1). The mean age of the study population was 67 ± 12 years, and 67.5% of patients were male. Table 1 summarizes the clinical and demographic characteristics of the patients, grouped by galectin-1 concentration. Patients with higher plasma galectin-1 concentrations were older and had higher incidences of hypertension, diabetes, CKD, heart failure, and multiple vessel disease. Subjects in the highest galectin-1 tertile had significantly increased levels of fasting glucose, proteinuria, and decreased hemoglobin levels, estimated glomerular filtration rates (eGFRs), and left ventricular ejection fractions (LVEFs).
Kaplan-Meier survival analysis was performed to investigate the potential impact of the baseline galectin-1 level on adverse event-free survival (Fig. 2). Patients in the highest galectin-1 group were at significantly greater risk of renal function decline than were those in the lowest galectin-1 group (p < 0.001). Patients with prior CIN were also at significantly greater risk of renal function decline than were those without prior CIN (p < 0.001).
Baseline characteristics according to the presence of diabetes. Of the 798 study subjects, 275 (34.5%) were diabetic. Table 2 shows the clinical and demographic characteristics of patients grouped by the presence of diabetes mellitus. Patients with diabetes were older and had higher incidences of hypertension, CKD, and peripheral arterial disease. In addition to anti-diabetic agents, diabetic patients also received more antiplatelet therapy, statins, and anti-hypertensive agents. Compared with nondiabetic patients, patients with diabetes had higher levels of serum galectin-1, more proteinuria, lower hemoglobin and total cholesterol levels, and reduced LVEFs. www.nature.com/scientificreports www.nature.com/scientificreports/ independent correlates of contrast-induced nephropathy and predictors of renal function decline. In univariate logistic regression analysis, a higher galectin-1 level, older age, history of diabetes, lower hemoglobin level, decreased baseline eGFR, and presence of proteinuria were associated significantly with a greater risk of CIN. To identify independent predictors of CIN, multivariable logistic regression analysis was performed. After adjustment for age, sex, and baseline eGFR, the highest serum galectin-1 level was associated significantly with CIN [odds ratio (OR), 2.64; 95% confidence interval (CI), 1.03-6.76; p = 0.04]. After adjustment for age, sex, and all factors that were significant in the univariate analysis, the serum galectin-1 level was not associated significantly with CIN, but associations persisted with age (OR, 1.04; 95% CI, 1.01-1.08; p = 0.026), and the hemoglobin level (OR, 0.67; 95% CI, 0.54-0.83; p < 0.001; Table 3).
In a univariate Cox regression analysis, older age, histories of hypertension and diabetes, proteinuria, higher serum galectin-1 level, lower hemoglobin level, reduced eGFR, and lower LVEF, as well as prior CIN, were associated significantly with a higher incidence of renal function decline. In a multivariable Cox regression analysis, the circulating galectin-1 level [tertile 2: hazard ratio (HR) 5 Table 1. Baseline characteristics of the study cohort by tertiles of the serum galectin-1 concentration. BMI, body mass index; ACEi/ARB, angiotensin-converting enzyme inhibitor/angiotensin receptor blocker; HbA1c, glycosylated hemoglobin A1c; eGFR, estimated glomerular filtration rate; LVEF, left ventricular ejection fraction; CAD, coronary artery disease; PCI, percutaneous coronary intervention; CIN, contrast-induced nephropathy.
Subgroup analysis. The study cohort was stratified according to the presence of diabetes, presence of proteinuria, and PCI status. Increasing galectin-1 concentrations were associated significantly with renal function decline, irrespective of underlying diseases, such as diabetes and proteinuria, or of PCI status (p interaction > 0.05; Table 5).

Discussion
In this single-center observational study, the serum galectin-1 concentration was associated with eGFR decline during a mean follow-up period of 1.4 ± 1.1 years in 798 patients with stable angina undergoing elective CAG, irrespective of the presence of diabetes. To our knowledge, this longitudinal study is the first to explore the relationship between galectin-1 and subsequent renal function deterioration in patients undergoing CAG. These results demonstrate the involvement of galectin-1 in chronic renal function impairment, and suggest that the circulating galectin-1 level is an independent predictor of chronic renal function decline.
Much research attention has focused on galectin-1 and galectin-3, given their apparent major roles in cancer biology 23 . Recent reviews and meta-analyses have demonstrated that galectin-1 is associated with tumor formation, progression, metastasis, angiogenesis, and prognosis in several kinds of cancer 23,24 . Intracellularly, the galectin-1 protein provides a carbohydrate-independent scaffold for intracellular signaling pathways; extracellularly, it governs β-galactoside binding. Multivalent interactions between galectin-1 and glycoproteins in the extracellular matrix contribute to cancer metastasis. A few in vitro and animal studies have mentioned the potential link between galectin-1 and kidney diseases [20][21][22][25][26][27][28] , but no previous clinical report has described an association between the galectin-1 level and CKD.
The mechanisms linking galectin-1 elevation to CKD progression are not fully clear. Several groups have postulated the existence of mechanisms relating galectin-1 to CKD 20 , especially in the context of diabetes 21,22 . Increased galectin-1 expression has been reported in diabetes 19 , diabetic retinopathy 29 and diabetic nephropathy 21,22 . Galectin-1 has been shown to regulate podocin production and damage, and diabetic nephropathy progression, in podocytes 22 . In addition, recent evidence has revealed that galectin-1 is a fibrosis protein that is highly expressed in the kidneys of mice with types 1 and 2 diabetes, and activated in proximal tubular epithelial cells under high-glucose conditions 22 . The phosphorylation and activation of Akt also may play crucial roles in the modulation of activating enhancer binding protein 4 (AP4) to up-regulate the galectin-1 protein under hyperglycemic conditions in diabetic mice 22 . AP4 was identified as a protein binding to the galectin-1 promoter that regulates various functions under high glucose stimulation 22 .
Less is known about the association between galectin-1 and CKD in non-diabetic contexts. Emerging evidence suggests that the progression of cardiac and renal functions is often interconnected 30,31 . Cardiac and renal diseases have several common pathways, including those of enhanced systemic inflammation and stress-mediated neurohormonal responses, the development of anemia and bone and mineral disorders, and acid-base and fluid imbalances 31 . Chronic myocardial dysfunction is also a well-known mediator of progressive renal function decline. The pathophysiology of this so-called type 2 cardiorenal syndrome remains under investigation 30 . Interestingly, a recent review suggested that galectin-1 is an emergent mediator of cardiovascular inflammation 32 . In this study, we also found that galectin-1 was associated with the LVEF and CAD in univariate analysis. We speculate that the parallel process affecting both heart and kidney functions also plays an essential role in the non-diabetic www.nature.com/scientificreports www.nature.com/scientificreports/ association between galectin-1 and CKD. Our findings, in agreement with previous reports, illustrate the association of galectin-1 with renal function decline irrespective of diabetes. Galectin-1 thus may play a critical role in renal function decline in diabetic and non-diabetic patients.
Interestingly, we found no significant association between galectin-1 and CIN in patients undergoing CAG or PCI procedures. One possible explanation is that the mechanisms of CIN differ from those of chronic renal decline. Contrast media may alter nitric oxide, endothelin, and adenosine levels, inducing renal vasoconstriction and leading to renal medulla ischemia and acute tubular necrosis 33,34 . Contrast media may also have cytotoxic effects via the upregulation of reactive oxygen species 33,35 or direct induction of osmotic tubular nephrosis 36 . Another possible reason is the lower incidence of CIN than renal function decline in our study sample. Furthermore, galectin-1 and diabetes were correlated in this study and in previous reports 17,18 . Before adjustment for diabetes, tertile 3 of the galectin-1 level correlated with CIN. After adjustment for all related risk factors, including diabetes, this association was insignificant.
This study has some limitations that should be considered. First, the study population was relatively small, and consisted of Asian patients treated at a single hospital. Further studies larger numbers of different participants are required to confirm our findings. Second, patients enrolled in our study were elders (mean age, 67 ± 12 years). Caution should be taken when applying our findings to younger populations. Finally, the eGFR was used as the   www.nature.com/scientificreports www.nature.com/scientificreports/ sole renal function endpoint, without consideration of other clinical endpoints affecting renal outcomes, including newly diagnosed diabetes mellitus and the progression of proteinuria. Nevertheless, our study demonstrated that the serum galectin-1 level is a novel risk marker for renal outcomes in patients with suspected CAD undergoing CAG or PCI procedures.

conclusions
Although not a predictor of CIN, the circulating serum galectin-1 level is an independent prognostic marker for subsequent renal function decline in patients undergoing CAG, irrespective of diabetes. These findings provide novel evidence of galectin-1's involvement in the pathogenesis of renal dysfunction in patients with suspected CAD. Further research exploring the underlying mechanism is needed.

Study design and patient population. A team of cardiologists (under the direction of Prof. Po-Hsun
Huang) collected data for this study consecutively from June 2011 to August 2018. The sample size was not pre-calculated. In total, 855 subjects with stable coronary CAD admitted to Taipei Veterans General Hospital for elective CAG and/or PCI were screened. Each patient's serum creatinine concentration was measured before CAG, and the eGFR was calculated using the CKD Epidemiology Collaboration equation 37 . Patients with stage 5 CKD, defined  Table 3. Multivariable logistic regression analysis of the association between the serum galectin-1 concentration and the incidence of contrast-induced nephropathy. OR, odds ratio; CI, confidence interval; HTN, hypertension; Hb, hemoglobin; eGFR, estimated glomerular filtration rate; LVEF, left ventricular ejection fraction. *Adjusted for age, sex, and baseline eGFR; † adjusted for age, sex, and variables with p values < 0.05 in the univariable analysis.  www.nature.com/scientificreports www.nature.com/scientificreports/ as creatinine clearance < 15 ml/min/1.73 m 2 (n = 15), and those undergoing hemodialysis or peritoneal dialysis (n = 42) were excluded from the analysis. Thus, a total of 798 patients was enrolled in the study and included in the final analysis (Fig. 1). The patients were grouped into tertiles according to the serum galectin-1 concentration.
Each patient's chart was reviewed in detail to collect data on medications, smoking status, and risk factors for CIN, such as age, existing renal dysfunction, type 2 diabetes mellitus, and volume depletion. Patients' blood pressure was measured using electronic sphygmomanometers at least four times a day during hospitalization. Hypertension was defined as systolic blood pressure ≥ 140 mmHg, diastolic blood pressure ≥ 90 mmHg, or the use of antihypertensive medications. Type 2 diabetes mellitus was defined as fasting plasma glucose level ≥ 126 mg/dl or the use of hypoglycemic agents. The body mass index was calculated by dividing the weight of the patient (in kilograms) by the square of the height (in meters). A nonionic low-osmolality contrast medium (iopromide) was used for all patients' examinations. It was administered intra-arterially, mainly through transradial catheters. Metformin and nephrotoxic medications, such as non-steroidal anti-inflammatory drugs, were discontinued 48 h before contrast medium administration. Before and after contrast medium exposure, physiological (0.9%) saline was given intravenously at a rate of 1 ml/kg/h for 12 h. In patients with left ventricular dysfunction (ejection fraction < 40%) or apparent heart failure, the hydration rate was reduced to 0.5 ml/kg/h. This research was conducted according to the principles expressed in the Declaration of Helsinki. This study was approved by the research ethics committee of Taipei Veterans General Hospital, and all participants provided written informed consent.

Laboratory investigations and cardiac catheterization.
Blood samples were collected after an ≥ 8-h fast. The blood cell count; serum glucose, creatinine, and uric acid levels; and lipid profiles were determined using a Hitachi 7600 autoanalyzer (Hitachi Ltd., Tokyo, Japan). The serum creatinine concentration was assessed at the time of admission and daily for the following 3 days after contrast medium exposure. Urine dipstick analysis was performed using commercial test strips, and proteinuria was defined as a urine protein concentration ≥ 30 mg/100 ml. Plasma concentrations of galectin-1 were determined using the commercially available Human Galectin-1 Quantikine ELISA Kit DGAL10 (R&D Systems, Inc., Minneapolis, MN, USA); the sensitivity was 0.129 ng/ml and the assay range was 0.3-20 ng/ml. The intra-and interassay coefficients were 5.7-8.8% and 7.5-9.5%, respectively. Two experienced interventional cardiologists interpreted the coronary angiograms. Coronary lesions causing > 50% diameter narrowing were considered to represent significant stenosis. The LVEF was estimated by left ventriculography. The contrast consumption of each patient was also recorded.
Definition of study endpoints and renal function decline. All patients were evaluated for the occurrence of CIN, which was defined as an increase in the serum creatinine concentration of ≥0.5 mg/dl or a 25% increase from baseline within 48 h after CAG 38 . Patients were advised to visit outpatient clinics regularly after  Table 5. Stratified analysis of the effect of the serum galectin-1 concentration on renal function decline in patients grouped by diabetes, proteinuria, and percutaneous coronary intervention status. HR, hazard ratio; CI, confidence interval; PCI: percutaneous coronary intervention. *Adjusted for age, sex, hypertension, diabetes, hemoglobin, baseline estimated glomerular filtration rate, proteinuria, left ventricular ejection fraction, and prior contrast-induced nephropathy.