Associations of serum amyloid A and 25‐hydroxyvitamin D with diabetic nephropathy: A cross‐sectional study

Abstract Background The present study investigated the relationships between serum amyloid A (SAA), 25‐hydroxyvitamin D (25(OH)VD) and diabetic nephropathy (DN) to provide evidence for the prevention and management of DN. Methods A total of 182 patients with type 2 diabetes mellitus (T2DM) were enrolled in this study. The levels of SAA, 25(OH)VD, and other conventional indicators were measured and analyzed. Receiver operating characteristic curve analysis was applied for the combined measurement of SAA and 25(OH)VD, and risk factors for DN were evaluated using binary logistic regression analysis. Results The levels of SAA in T2DM patients were significantly higher than those in healthy subjects, and the level significantly increased with the progression of DN (p < 0.05). In contrast, the level of 25(OH)VD in T2DM patients was significantly lower than that in healthy subjects, and the level significantly decreased with the progression of DN (p < 0.05). The combined measurement of SAA and 25(OH)VD distinguished DN patients from T2DM patients better than the measurement of SAA or 25(OH)VD alone. SAA was an independent risk factor for DN, and 25(OH)VD was an independent protective factor for DN. Conclusion SAA and 25(OH)VD might be used as potential markers to identify patients at increased risk of developing DN.

Serum amyloid A (SAA) is an acute-phase response protein. 5 When inflammation occurs, the expression of SAA in the liver, fat, and kidney increases significantly. 6,7 SAA in the kidney is secreted by glomerular cells, including podocytes and mesangial cells. Its main role is to promote the expression of inflammatory cytokines in fibroblasts, macrophages and podocytes. 6,7 Previous studies have reported that SAA is closely related to the occurrence and progression of DN. 8,9 Serum 25-hydroxyvitamin D (25(OH)VD) is a metabolite of vitamin D in the body, is relatively stable and is considered to be the best indicator of vitamin D levels in the body. 10 Previous studies have reported a close relationship between vitamin D and T2DM, and there is evidence that vitamin D deficiency leads to the occurrence of diabetes. 11

| Subjects
This cross-sectional study was conducted at the Department of Endocrinology and Laboratory Medicine, the Second People's Hospital of Lianyungang, from August 2020 to February 2021.
A total of 240 patients with T2DM participated in this study, but only 182 patients were included in the end (58 patients were excluded based on the exclusion criteria). The selection process of subjects with T2DM are shown in Figure 1. In addition, based on the urinary albumin-to-creatinine ratio (UACR), patients with T2DM were divided into three groups 17

| Anthropometric and laboratory measurement
General information on sex, age, smoking status, course of disease and medical records was obtained from the questionnaire. Height and weight were measured, and body mass index (BMI) was calculated as weight (kg) divided by the square of height (m 2 ). Blood pressure was measured as the mean of two results measured in a sitting position, and the interval between the two measurements was at least 15 min.
All subjects fasted for 8-10 h, and venous blood samples were collected from 6 AM to 8 AM. EDTA K 2 anticoagulated venous blood (2 ml) was used for the detection of glycated hemoglobin A1c  Beckman Coulter). The levels of SAA and urinary albumin were detected using immunoturbidimetry (AU5800 biochemical analyzer, Beckman Coulter). The 25(OH)VD level was measured using the liquid chromatography tandem mass spectrometry method (API 3200 mass spectrometer, Sciex). The minimum detection level for SAA was 0.1 mg/L, and its intra-and inter-assay coefficients of variation were 5% and 10%, respectively. The minimum detection level for 25(OH)VD was 1.0 ng/ml, and its intra-and inter-assay coefficients of variation were 4% and 6%, respectively.
Before specimen detection, the detection systems were calibrated and maintained. The internal quality control met the analytical performance, and external quality assessments were qualified.

| Statistical analyses
Statistical analyses were performed using IBM SPSS Version 19 (IBM Corp.). The Kolmogorov-Smirnov test was used to analyze the normality of quantitative variables, and normally distributed data were expressed as the means and standard deviations. Unpaired t tests and chi-square tests were used for comparisons between groups with quantitative and categorical variables, respectively. One-way analysis of variance was used to compare the means among multiple groups, and the LSD test was used for pairwise comparisons. The combined detection of SAA and 25(OH)VD was analyzed by using receiver operating characteristic (ROC) curves, and risk factors for DN were evaluated using binary logistic regression analysis. Two-tailed p-values less than 0.05 were considered statistically significant.

| Characteristics of patients with T2DM
This study included a total of 182 patients with T2DM and 180 healthy subjects (matched for sex and age). The age, sex, BMI, blood pressure (SBP and DBP), smoking status, duration of T2DM, Urea, Crea, and other laboratory indicators of T2DM and control subjects are listed in Table 1. There were no differences in the means of BMI, Urea, Crea, TC, TG, HDL-C, or LDL-C in either group (p > 0.05). The levels of blood pressure (SBP and DBP), FBG, HbA1c, UACR, and SAA were significantly higher in the T2DM group than in the control group (p < 0.05). In addition, the levels of eGFR and 25(OH)VD were significantly lower in the T2DM group than in the control group (p < 0.05).

| Levels of SAA and 25(OH)VD among T2DM patients grouped by UACR
According to the levels of UACR, patients with T2DM were divided into three groups (NA group, MA group and CP group). Table 2 and

| Combined measurement of SAA and 25(OH) VD in patients with DN
According to the American Diabetes Association guidelines, 18 the MA and CP groups were defined as having DN (UACR ≥30 mg/g).
The ROC curve analysis indicated that the combined measurement of SAA and 25(OH)VD distinguishes between patients with T2DM and patients with DN better than the detection of SAA or 25(OH)VD alone (p < 0.05, Figure 3).

| Independent risk factors for DN
Group allocation (NA, MA, and CP groups) was the dependent variable, and age, sex, BMI, SBP, DBP, smoking status, Urea, Crea, TC, TG, HDL-C, LDL-C, FBG, HbA1c, eGFR, SAA and 25(OH) VD were the independent variables. Binary logistic regression analysis showed that for each 1 mg/L increase in SAA, the risk of T2DM progressing to DN increased significantly (OR = 1.133, p = 0.000). The study also indicated that for each 1 ng/ml decrease in 25(OH)VD, the risk of T2DM progressing to DN increased significantly (OR = 0.838, p = 0.001). Therefore, our study demonstrated that the SAA level was an independent risk factor for DN, but the level of 25(OH)VD was an independent protective factor for DN (Table 3).  In the present study, we also found that combined measure- are required to clarify the causal relationships between SAA, 25(OH) VD and DN and the regulatory mechanisms in the occurrence and development of DN.

| DISCUSS ION
In conclusion, our study shows that patients with T2DM had higher SAA and lower 25(OH)VD and that the measurement of SAA and 25(OH)VD might be used to identify patients at increased risk of developing DN. However, many more prospective large-scale trials are necessary to elucidate the regulatory mechanisms between SAA, 25(OH)VD and DN.

CO N FLI C T S O F I NTE R E S T
The authors declare no potential conflicts of interest with respect to the study, authorship, and/or publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are available upon request from the corresponding author (Fumeng Yang).