Neutrophil-to-lymphocyte ratio is a predictor of renal dysfunction in diabetic patients

Background: Neutrophil-to-lymphocyte ratio (NLR) has been widely evaluated as a biomarker in various medical and surgical prognoses, but its usefulness in diabetic kidney disease is not yet known. Methods: This prospective observational study included outpatients, comprised of 190 men aged 73 ± 11 (mean ± standard deviation) years and 175 women aged 77 ± 10 years at baseline, from a rural hospital. We examined the relationship between baseline NLR calculated by analyzing the differential leukocyte counts in the complete blood count and the 1.5-year estimated glomerular ltration rate (eGFR) decline rate (i.e. 1.5-year eGFR − baseline eGFR) *100/baseline eGFR. Rapid eGFR decline rate was dened as a value <(cid:0)15%. Results: Multiple linear regression analysis using rapid eGFR decline rates as objective variables, adjusted for confounding factors as explanatory variables, showed that tertiles of NLR (β=0.120, p=0.018) as well as hemoglobinA1c, presence of antidiabetic medication, and urinary albumin excretion were signicantly and independently associated with a rapid eGFR decline rate. The multivariate-adjusted odds ratios (95% condence interval) of the 2 nd and 3 rd tertiles of baseline NLR for rapid eGFR decline rate were 3.94 (1.34-11.6) and 3.93 (1.26-12.3), respectively. Multivariate-adjusted mean eGFR (95% condence interval) values after 1.5 years categorized by tertile of baseline NLR were: 1 st , 64.2 (62.1-66.3); 2 nd , 60.5 (58.5-62.6); and 3 rd , 59.1 (57.0-61.2). Conclusions: These results suggest that baseline NLR might be a useful biomarker for renal function decline in diabetic outpatients.

albuminuria and decreased eGFR among diabetic outpatients [19]. However, the usefulness of NLR as a predictor of DKD has not been studied and its importance in nephrology has not yet been established.
Thus, the aim of this study was to examine the relationship between NLR, potential risk factors (such as age, gender, history of CVD, hypertension, hyperglycemia, lipids, serum uric acid [SUA]), and renal function by examining prospective data from diabetic outpatients.

Subjects
Participants were continuously recruited from diabetic outpatients that visited the medical department of Seiyo Municipal Nomura Hospital from April to June 2017 [19]. Those with an estimated glomerular ltration rate (eGFR) of <30 ml/min/1.73 m 2 were excluded. Patients who had severe tissue damage, acute massive hemorrhage, acute poisoning, cancer, acute coronary artery disease, heart failure, active infection, or blood diseases affecting neutrophils and lymphocytes were also excluded. Patients with medications that may affect neutrophils and lymphocytes were also excluded. The study complies with the Declaration of Helsinki and was approved by the Ehime University Ethics Committee (IRB Approval Number: 1709006). Written informed consent was obtained from each subject.

Evaluation of confounding factors
All enrolled patients were interviewed and clinically examined at presentation. Their medical history, present conditions, smoking status, alcohol consumption, and medications (e.g. antihypertensives, antidyslipidemics, and antidiabetics) were obtained by interview using a structured questionnaire. Body mass index (BMI) is calculated as a person's weight in kilograms divided by height in meters squared. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded using an automatic oscillometer at the right upper arm of patients while in a sedentary position. Smoking status was de ned as the number of cigarette packs per day multiplied by the number of years smoked (pack-year), and the participants were classi ed as never smokers, past smokers, light smokers (<20 pack-year) or heavy smokers (≥ 20 pack-year). Alcohol consumption was measured using the Japanese liquor unit, in which a unit corresponds to 22.9 g of ethanol, and the participants were classi ed as never drinkers, occasional drinkers (<1 unit/day), daily light drinkers (< 2 units/day), or daily heavy drinkers (≥ 2 units/day). Total cholesterol (T-C), high-density lipoprotein cholesterol (HDL-C), hemoglobin A1c (HbA1c), creatinine (Cr), SUA, and WBC count were measured during fasting. NLR was the ratio of neutrophil to lymphocyte. Non-HDL cholesterol (Non-HDL-C) was calculated by subtracting HDL-C from T-C. Urinary spot samples were collected after rest in the morning for analysis of urinary albumin and creatinine. Urinary albumin excretion (UAE) was expressed by the albumin: creatinine ratio (mg/g), and the participants were classi ed into the A1 stage (<30 mg/g Cr), A2 stage (30 to 300 mg/g Cr), or A3 stage (≥ 300 mg/g Cr). The eGFR was calculated using the CKD Epidemiology Collection (CKD-EPI) equations modi ed with a Japanese coe cient: Male, Cr ≤ 0.9 mg/dl, 141 × (Cr/0.9) -0.411 × 0.993 age × 0.813; Cr > 0.9 mg/dl, 141 × (Cr/0.9) -1.209 × 0.993 age × 0.813; Female, Cr ≤ 0.7 mg/dl, 144 × (Cr/0.7) -0.329 × 0.993 age × 0.813; Cr > 0.7 mg/dl, 144 × (Cr/0.7) -1.209 × 0.993 age × 0.813 [20]. Moreover, past ischemic stroke, ischemic heart disease, and obstructive arteriosclerosis were de ned as CVD.

Outcomes
The eGFR decline rate was calculated from the difference between the eGFR value after 1.5 years and that of the baseline year, divided by baseline eGFR (i.e. [1.5 year eGFR−baseline eGFR]*100/baseline eGFR). A rapid eGFR decline rate was de ned as a 1.5-year eGFR decline rate of < 15%.

Statistical analysis
All continuous variables with normal distribution were presented as the mean ± standard deviation (SD), and nonnormal variables (e.g. TG, HbA1c, and NLR) were reported as the median (interquartile range). In all analyses, parameters with non-normal distributions were used after log-transformation. Subjects were divided into three groups based on tertile of baseline NLR (1 st , 0.65-1.56; 2 nd , 1.57-2.33; 3 rd , 2.34-15.3). Differences in means and prevalence between the three groups were examined by analysis of variance (ANOVA) for continuous data and a χ 2 test for categorical data. Pearson's correlations were calculated in order to characterize the associations between baseline characteristics and rapid eGFR decline rate. Multiple logistic regression analysis was used to evaluate the contribution of each confounding factor, including NLR, to the rapid eGFR decline rate. Analysis of covariance (ANCOVA) was performed with the use of a general linear model approach to determine the association between baseline NLR and multivariate-adjusted eGFR after 1.5 years. In these analyses, eGFR values after 1.5 years were considered the dependent variables and the three ranges of NLR were considered the independent variables.
Statistical analysis was performed using IBM SPSS Statistics software, version 21 (Statistical Package for Social Science Japan, Inc., Tokyo, Japan). A value of p < 0.05 was considered signi cant.

Results
Baseline characteristics of subjects categorized by tertile of baseline NLR Characteristics of the subjects categorized by tertile of baseline NLR are illustrated in Table 1. This prospective observational study included outpatients, comprised of 190 men aged 73 ± 11 years and 175 women aged 77 ± 10 years at baseline. Age was higher in correlation with increased tertile of NLR, but BMI, DBP, and eGFR were lower.
There were no inter-group differences based on gender, period of diabetes, smoking status, daily alcohol consumption, CVD, SBP, presence of antihypertensive medication, HDL-C, Non-HDL-C, presence of antilipidemic medication, HbA1c, presence of antidiabetic medication, SUA, SUA-lowering medication, or UAE stage. The change in mean eGFR during the 1.5 years of the 1 st NLR tertile was more gradual compared with the 2 nd and 3 rd NLR tertiles. As shown in Table 2, the 1 st NLR tertile had fewer patients (4.1%) with a primary outcome (i.e. 1.5year eGFR decline rate < 15%) compared with the 2 nd and 3 rd NLR tertiles, which had more patients with primary outcomes (19.5% and 17.4%, respectively) with a signi cant p-value of 0.006.
Relationships between baseline confounding factorsand early eGFR decline rate Table 3 shows the simple relationship between baseline confounding factors and rapid eGFR decline rate.
Pearson's correlation coe cient showed that tertile of NLR, age, SBP, presence of antihypertensive medication, HDL-C and UAE stage were signi cantly correlated with a rapid eGFR decline rate. Multiple linear regression analysis using rapid eGFR decline rate as an objective variable, adjusted for baseline confounding factors as explanatory variables, showed that tertile of NLR, age, non-HDL-C, and UAE stage was signi cantly and independently associated with a rapid eGFR decline rate.
Non-adjusted and adjusted odds ratio (95% CI) of the tertiles of baseline NLR for the early 1.5-year eGFR decline rate As shown in Table 4, the non-adjusted and multivariate-adjusted odds ratios (ORs) (95% con dence interval [CI]) of the 1 st , 2 nd , and 3 rd tertiles of baseline NLR for rapid eGFR decline rate were1, 5 (Table 5).

Discussion
In this prospective study of 365 diabetic outpatients, we determined the rate of decline in renal function as assessed by eGFR. Baseline NLR was assessed for its signi cance in the development of DKD, independently of potential confounding factors. The important nding of this study was that baseline NLR levels were found to be signi cantly associated with 1.5-year eGFR decline rate and future eGFR after 1.5 years among diabetic outpatients, even after adjusting for possible confounders such as gender, age, BMI, lifestyle, history of CVD, medication, blood pressure, lipids, glucose control, SUA, and UAE stage. Since NLR values are used extensively in the medical eld, NLR may be used as a cost-effective predictor of in ammation. Finally, we demonstrated that threshold values for NLR determine early eGFR decline rate.
NLR has been reported as an in ammatory-based marker that is widely available, easy to obtain, and inexpensive to measure, providing values that can aid in the risk strati cation of patients with various diseases [14] [18] [  However, some limitations could not be avoided. First, estimation of GFR using CKD tends to be less accurate in subjects with normal kidney function and CKD compared to inulin clearance. It is, however, more accurate than Cr or eGFR when the Modi cation of Diet in Renal Disease (MDRD) formula is used [20]. Second, baseline confounders and eGFR are based on a single assessment of blood, which can cause misclassi cation bias. Third, the failure to perform a renal biopsy cannot eliminate the potential impact of underlying diseases and medications on hypertension, diabetes, and dyslipidemia on NLR and renal dysfunction. However, renal diseases other than DKD and diseases that may affect NLR were eliminated as much as possible. Nevertheless, the demographics and referral source may limit generalization.

Conclusions
This study suggested that baseline NLR might be a surrogate marker of early eGFR decline rate in diabetic outpatients. The underlying mechanisms behind this relationship are completely unknown, and these factors seem to be independent of baseline confounding factors, such as age, gender, smoking status, alcohol consumption, history of CVD, hypertension, lipids, HbA1c, and SUA. Further studies are needed to evaluate whether baseline NLR is a useful marker to predict the incidence of early renal function decline.

Declarations
Authors' contributions TA and RK participated in the design of the study, performed the statistical analysis and drafted the manuscript. TA, RK, DN, and AK contributed to the acquisition and interpretation of data. TA, RK, DN, and TK contributed to the conception and design of the statistical analysis. All authors read and approved the manuscript.

Availability of data and materials
The data from this study can be acquired from the corresponding author upon reasonable request.

Consent for publication
Not applicable.

Ethics approval and consent to participate
This study conformed to the guidelines of the Declaration of Helsinki, and the study procedures were reviewed and approved by the medical research ethics committee of Ehime University Graduate School of Medicine. Each patient agreed to participate and signed the informed consent form.   neutrophil to lymphocyte ratio; HDL, high-density lipoprotein; UAE, urinary albumin excretion. r, Pearson's correlatio ient. β, standard coefficient. R 2 , multiple coefficient of determination. Data for hemoglobin A1c, NLR, and UAE were d and log-transformed for analysis. § Adjusted for all confounding factors in Table 1 by multiple linear regression is (Model 1: forced entry method; Model 2: stepwise method). The numbers in bold indicate significance.