IL-34 is associated with the presence and severity of renal dysfunction and coronary artery disease in patients with heart failure

Pro-inflammatory mediators are identified in patients with heart failure (HF), some of which may be used as biomarkers with diagnostic or prognostic value. As an additional ligand of Colony Stimulating Factor-1 Receptor (CSF-1R), interleukin-34 (IL-34) has been identified as a pro-inflammatory cytokine participating in chronic heart failure (CHF). However, the potential impact of IL-34 in CHF complications remains unknown. In order to determine the clinical significance of serum IL-34 in CHF patients, especially those with kidney dysfunction and coronary artery disease (CAD) comorbid conditions, serum IL-34 was measured in 510 consecutive patients with CHF in a cross-sectional study. The present study demonstrated that higher serum IL-34 levels were associated with poorer renal function and more severe anemia in patients with CHF. After adjusting for age, gender, conventional risk factors, and other significant covariates, IL-34 positively correlated with the presence and severity of renal dysfunction (as measured by eGFR and cystatin C) on multivariable linear and logistic regression analysis. IL-34 was also demonstrated to be an independent risk factor for CAD among HF patients. In conclusion, elevated serum IL-34 levels were demonstrated to be independently associated with renal insufficiency and CAD in patients with CHF, regardless of the systolic function.

IL-34 as an independent risk factor for renal dysfunction in CHF patients. In patients with HF, serum IL-34 levels were not significantly different between patients with and without hypertension (117.64 ± 7.86 pg/mL vs. 113.59 ± 11.03 pg/mL, p = 0.764), as well as those with and without diabetes mellitus (106.15 ± 8.90 pg/mL vs. 122.04 ± 8.67 pg/mL, p = 0.202), indicating that there was no relationship between IL-34 and the presence of hypertension or diabetes mellitus. However, among HF patients, serum IL-34 levels were significantly higher in those combined with kidney dysfunction than those without (145.87 ± 14.68 pg/mL vs. 104.24 ± 6.65 pg/mL, p = 0.001) (Fig. 1A). IL-34 levels were also significantly higher in patients with cystatin C ≥ 1.550 (median) compared to those with cystatin C < 1.550 (135.26 ± 10.38 pg/mL vs. 98.00 ± 7.75 pg/mL, p < 0.0001) among patients with HF (Fig. 1B). When all HF patients were divided into tertiles according to IL-34 levels, the upper tertile had a significantly higher proportion of CKD patients compared to the lowest tertile (39.4% vs. 21.2%, p = 0.001).
To further establish the correlation between IL-34 and CKD, we separately analyzed IL-34 as a log-transformed standardized continuous variable, as an ordinal variable with values expressed as tertiles, and as a categorical variable by using the lowest tertile as a reference value. Each of the models confirmed that IL-34 was an independent predictor for the presence of CKD among patients with HF, either adjusted for age and gender, or adjusted for the full model including age, gender, BMI, hypertension, diabetes mellitus, hsCRP, NT-proBNP, hemoglobin, albumin and NYHA functional class (Table 3).
On the other hand, subgroup analysis by using a logistic regression model revealed IL-34 to be a better predictor for worsening renal function in male patients with older age. The predictive value was also better in those concomitant coronary artery disease, hypertension, or diabetes mellitus. Moreover, IL-34 could predict the risk of kidney dysfunction between both patients with reduced and preserved ejection fraction. (Fig. 1C).

IL-34 is independently associated with the presence and severity of CAD in patients with HF.
In the present study, HF patients with CAD had higher IL-34 levels compared to those without CAD (115.68 ± 6.28 pg/mL vs 92.34 ± 6.64 pg/mL, p = 0.001) (Fig. 1D). Serum IL-34 levels were significantly associated with the CAG Gensini score (r = 0.176, p < 0.001) on simple linear correlation analysis, indicating that patients with higher serum IL-34 levels tended to have more severe coronary artery disease. Moreover, univariate and multivariable logistic regression analyses demonstrated that serum IL-34 remained a significant independent risk factor for CAD among HF patients, as well as when adjusted for age, gender, BMI, smoking, history of hypertension, diabetes mellitus, dyslipidemia, statin use, and laboratory tests including triglyceride, total cholesterol, low-density lipoprotein (LDL), glycosylated hemoglobin (HbA1c), hsCRP (Table 4).

Discussion
This observational study demonstrates that in patients with chronic heart failure, increased serum levels of IL-34 were independently associated with the presence of kidney dysfunction and coronary artery disease, as well as their severity.
Renal dysfunction is common in HF patients and plays a critical role in the progression, and prognosis of heart failure as well as coronary heart disease 13,14 . A landmark analysis that included 1906 patients with HF demonstrated eGFR as the most powerful predictor of mortality exceeding the significance of left ventricular ejection fraction and NYHA functional class, establishing its role as a biomarker for renal dysfunction especially predicting poor outcome in patients with heart failure 15 . Another biomarker, cystatin C, also appears to be sensitive in the early detection of kidney dysfunction in HF patients 16,17 . In the present study, serum levels of IL-34 were found to be independently associated with renal biomarkers including eGFR and cystatin C among patients with HF, suggesting that IL-34 may play an important role in the process of heart failure mainly owing to its effect on renal function.
However, it is unclear whether IL-34 plays a maladaptive or a compensatory role in the pathogenesis of renal impairment, or only reflects the underlying renal dysfunction in patients with heart failure. On the basis of several experimental studies, it is reasonable to conclude that IL-34 has a participatory role in the pathophysiology of renal impairment, in addition to reflecting the severity of heart failure. First, IL-34, generated by renal tubule epithelial cells (TECs), is up-regulated in kidney and serum during ischemia in a murine model of kidney ischemic reperfusion injury. Its overexpression is known to promote renal dysfunction in both the acute and chronic phases by the following two mechanisms: enhancing intra-renal macrophage proliferation and elevating bone marrow myeloid cell proliferation. These responses result in an increase    in the circulating monocytes attracted to the kidney by chemokines 11 . These results indicate that IL-34 contributes to kidney dysfunction during ischemia. The reduced cardiac output in heart failure is principally responsible for low renal blood flow and increased renal venous pressure, and is one of the major determinants of renal impairment 18 . Renal destruction secondary to low renal blood flow is similar to that with ischemic injury, which in part explains the reason why serum IL-34 is up-regulated in heart failure-induced kidney dysfunction. However, this mechanism needs to be further explored. Second, the role of IL-34 as an inflammatory mediator has been suggested by its ability to induce proinflammatory cytokines and chemokines such as IL-6, IL-8 and CCL2 in human whole blood 7 . Increased IL-34 levels are seen along with the increased expression of tumor necrosis factor alpha (TNF-α ), IL-1β and IL-17, which are involved in regulating monocyte-and/or macrophage-induced inflammation associated with tubular destruction and lupus nephritis 10,19 . Thus, IL-34 may contribute to kidney function deterioration by its pro-inflammatory effects.
On the other hand, our present study demonstrated that higher serum IL-34 levels were associated with more severe coronary artery disease, which is in accordance with the results of a previous study with limited sample size that including only those subjects who had CAD 20 . Furthermore, another study illustrated that serum IL-34 may be superior to CRP as a potential inflammatory biomarker predicting the risk of vascular diabetic complications 21 . However, all these studies excluded patients with cardiac dysfunction or renal dysfunction, while our present study included those patients, thus expanding the scope of application of IL-34 as a predictive biomarker for CAD.
As discussed above, IL-34 may contribute to ischemic myocardial injury and atherosclerosis mainly owing to its function as a regulator of pro-inflammatory cytokines and innate immunity 11,22 . Moreover, pro-inflammatory cytokines can in turn induce increased expression and secretion of IL-34 23 , suggesting the presence of crosstalk between IL-34 and other cytokines. These findings support the role of an inflammatory process contributing to CAD. Segaliny et al. demonstrated that IL-34 could result in an in vitro increase in monocyte/CD34 + cell adhesion to activated human umbilical vein endothelial cell (HUVEC)-monolayers under physiological shear-stress conditions 24 . Hence it is also possible that IL-34 plays a part in CAD, partly by regulating mononuclear phagocyte adhesion to the endothelium, angiogenesis and macrophage recruitment.
Our present study suggested that IL-34 affect the severity of heart failure mainly through its involvement in the process of renal dysfunction during heart failure, and may influence the prognosis of heart failure, although it has not been shown to directly impair the process of cardiac remodeling, fibrosis or myocardial contraction. Second, an experimental study demonstrated that macrophages stimulated by IL-34 expressed galectin-3 25 . It is well known that galectin-3 is an important marker of cardiac remodeling as well as cardiac diastolic dysfunction 26    and higher concentrations of galectin-3 are associated with increased risk for incident HF and mortality in the community 27,28 . Thus, IL-34 may have a role to play in the pathogenesis of heart failure mediated by galectin-3. However, the direct impact of IL-34 on heart failure and the underlying mechanisms needs to be further studied. The present study however has several limitations. First, the study was performed in a single center as a cross-sectional study. A larger prospective cohort is required to accurately assess the predictive value of serum IL-34 in the development of worsening renal function (WRF), which is common in HF, as well as to analyze its prognostic value in patients with heart failure 29 . In addition, both in vivo and in vitro studies are required to understand the precise mechanisms underlying the observations made in our present study, and to verify their clinical significance. Second, in order to confirm the effect of IL-34 on tubular damage, it is necessary to analyze serum IL-34 levels together with some tubular damage markers such as Neutrophil Gelatinase Associated Lipocalin (NGAL) and Kidney Injury Molecule 1 (KIM-1) in future studies.

Conclusions
In conclusion, the present study confirmed that circulating IL-34 levels were significantly increased in HF patients with kidney dysfunction, and in HF patients with CAD. IL-34 levels significantly correlated with eGFR, cystatin C, and CAG Gensini score. These findings also indicate that baseline serum IL-34 levels may serve as independent predictors for kidney dysfunction and CAD in patients with chronic heart failure regardless of the level of systolic function. The role of IL-34 in assessing the prognosis of heart failure needs to be assessed in future cohort studies.

Methods
We first studied serum IL-34 in a cohort of chronic heart failure patients to investigate its association with other renal haemodynamic measurements such as eGFR and cystatin C.
Participants. This was a cross-sectional study that consecutively enrolled a total of 510 patients with chronic heart failure. The group consisted of patients who satisfied the following criteria: stable chronic heart failure with ≥ 3 months of diuretics use, hospitalization for heart failure at least once within a year before enrollment, and NT-proBNP levels of over 300 pg/mL for patients in sinus rhythm, and 900 pg/mL for those with atrial fibrillation at baseline. Included patients had heart failure either with preserved ejection fraction (HFpEF) or with reduced ejection fraction (HFrEF), however, all HF patients should have cardiac structural changes at baseline proved by echocardiography, including either left atrial enlargement, left ventricle enlargement or left ventricle hypertrophy. Patients with acute coronary syndrome during the four weeks preceding assessment or with significant concomitant diseases such as cancer, infections or autoimmune diseases were excluded. Patients who had renal replacement therapy or heart transplantation were also excluded. All included subjects were of age 18 years or above.
Data recorded at enrollment included results of routine laboratory tests, and details regarding concomitant diseases and cardiac medications. For each patient, echocardiography was performed at the same day when taking blood samples or within a week. The study was approved by the ethics review committee of our institution and a prior signed informed consent was obtained from each patient. All experiments and methods were performed in accordance with relevant guidelines and regulations.

Measurements.
Fasting venous blood samples were obtained to measure the levels of serum IL-34, NT-proBNP, hsCRP (high sensitivity C reactive protein), creatinine, and cystatin C, along with other routine biochemical indices. Once collected, the samples were immediately immersed in ice and centrifuged within 30 minutes at 2000 rpm for 20 min to obtain platelet-poor serum. All samples were stored at − 80 °C before analysis.

Coronary artery disease
The diagnosis of CAD included involvement of at least one vessel, which was defined as > 50% narrowing of the luminal diameter on coronary angiography, with or without angina pectoris. Coronary angiography (CAG) score was calculated by using the CAG Gensini score 30 .

IL-34
Serum IL-34 was measured by using a ELISA (Human IL-34 Quantikine ELISA Kit, D3400, R&D Systems, USA) according to the manufacturer guidelines.
Statistical analyses. Continuous variables were expressed as mean and standard error (SE) if normally distributed. Values that were not normally distributed were natural log transformed before analysis. Categorical data were summarized as proportions and frequencies. Continuous variables were compared using independent t-test and one-way ANOVA test. Categorical variables were compared using Chi-square test or Fisher's exact test as appropriate. The relationships between variables were tested by simple linear regression analyses. Both univariate and multivariable linear as well as logistic regression models were conducted to determine the influence of baseline levels of IL-34 on decline in eGFR and elevation in cystatin C, and on the presence of CKD, respectively. The predictive value of IL-34 was adjusted for age, gender, BMI, history of hypertension, history of diabetes mellitus, hsCRP, NT-proBNP, hemoglobin, albumin and NYHA functional class. Logistic regression analysis was also performed to assess the independent predictive value of IL-34 for CAD among heart failure patients. Odds ratios (OR) were presented as unadjusted, adjusted for age and gender, and further adjusted for age, gender, BMI,