Serum and urine ANGPTL8 expression levels are associated with hyperlipidemia and proteinuria in primary nephrotic syndrome

This cross-sectional study was carried out between November 2017 and November 2019 in Gansu Provincial People’s Hospital. The study was approved by the Medical Ethics Committee of Gansu University of Traditional Chinese Medicine (No. syll20160037), and all methods were carried out in accordance with relevant guidelines and regulations. All participants provided oral and written informed consent prior to participation. A total of 193 subjects were recruited, including 133 patients with PNS and 60 healthy controls (HCs). Subjects underwent a detailed medical history record review, medical examination, comprehensive urine analysis, routine blood testing, endocrine profiling, biochemical analysis and ultrasound examination of the urinary system. We performed autoantibody tests, an HBV assay, and immune globulins assay, to identify secondary nephropathy. Renal biopsy was carried out in 78 patients based on their disease characteristics.

The PNS patient inclusion criteria were as follows: heavy proteinuria [24-h urine total protein (24 hUTP) > 3.5 g or urine protein/creatinine ratio > 3.0 mg/mg or 24 hUTP > 50 mg/kg] and hypoalbuminemia [serum albumin (ALB) ≦25 g/L], and various degrees of edema and hyperlipidemia [21].

The HCs had no concomitant health problems, and fasting blood lipid levels [22] [cholesterol (CHOL) < 200 mg/dl, triglycerides (TG) < 150 mg/dl, and low-density lipoprotein cholesterol (LDL-C) < 130 mg/dl] and urinary proteins (urinary microalbumin ≤150 mg/dl or negative urine qualitative test) were within the normal range.

The exclusion criteria were as follows: PNS patients without the required clinical and laboratory data, those with secondary nephrotic syndrome, previous history of other acute or chronic kidney disease, patients with abnormal ultrasound examination of the urinary system (e.g., deformities, cysts, hydrops, stones), identified acute or chronic illness (diabetes mellitus, thyroid dysfunction, polycystic ovary syndrome, obesity, fatty liver, familial hypercholesterolemia), and other systemic diseases, such as hematological diseases, cardiovascular diseases, connective tissue diseases, tumors, and obvious infections.

The medical records of the following parameters were collected for the study: age, sex, body mass index (BMI), calculated as weight divided by height squared (kg/m2), blood pressure, disease state including initial treatment, pathology results of renal biopsy provided by Guangzhou KingMed Diagnostics (an independent ISO15189-certified clinical laboratory) including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), and mesangial proliferative glomerulonephritis (MsPGN). Also collected were the levels of serum CHOL, TG, high-density lipoprotein cholesterol (HDL-C), LDL-C, ALB, creatinine (CREA), urea (Ur) [measured by automated biochemical analyzer (ARCHITECT c1600, Abbott, USA)], 24 hUTP, urine creatinine (UCr) [measured by automated chemiluminescence immunoanalyzer (UniCel DxI 800, Beckman-Coulter, USA)], and urine protein [measured by automated urine analyzer (FUS-2000, DIRUI, China)]. All assays were performed according to routine procedures specified by the clinical laboratory center (ISO15189 certified) of Gansu Provincial People’s Hospital. The severity of proteinuria was divided into three categories according to the 24 hUTP level: mild (≤ 1 g/d), moderate (1 ~ 3.5 g/d), and severe (≥ 3.5 g/d) proteinuria. The estimated glomerular filtration rate (eGFR) was calculated from the serum creatinine level using the Modification of Diet in Renal Disease (MDRD) study equation [23].

Blood samples were collected from the antecubital vein of subjects between 07:30 and 09:00 a.m. after an overnight fast (at least 10 h). The collected blood was coagulated for 30 min at 4 °C and then centrifuged for 20 min at 3000 rpm, 4 °C. A clean, midstream first-morning urine sample was also obtained from all subjects. The assay samples were immediately stored at − 20 °C prior to ELISA, and repeated freeze–thaw cycles were avoided. The serum and urine ANGPTL8 concentrations were measured with ELISA kits (Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China, Catalog No. E1766H1), and the procedures were conducted in compliance with the manufacturer’s protocol.

The distribution pattern of continuous variables was tested for normality using the Kolmogorov–Smirnov (n ≥ 100) or Shapiro–Wilk test (n < 100). For normally distributed variables, the data are presented as the mean ± standard deviation (S.D.). Differences were assessed by independent samples t-test, two-tailed Student’s t-test, or one-way ANOVA. Pearson correlation coefficients were estimated to determine the correlation. For non-normally distributed variables, the data are presented as the median [interquartile range]. Differences were assessed by the Mann-Whitney U test, the Kruskal-Wallis test, or Kruskal-Wallis one-way ANOVA, and correlations were analyzed by the Spearman correlation method. Categorical variables were reported as frequencies or percentages. Differences were assessed by the Pearson χ2 test or the continuity modified χ2 test. For grade variables, differences were assessed by the Wilcoxon signed-rank test. A two-sided P value < 0.05 was considered statistically significant. All statistical analyses were carried out with SPSS software (version 26.0).

Table 1 summarizes the clinical characteristics of the included patients. A total of 133 patients were enrolled in the PNS group; 94 males (70.68%) and 39 females (29.32%), and the male to female ratio was 2.41. Overall, 33 patients were less than 18 years of age, while there were 100 adult PNS patients. Regarding disease status, there were 48 initial cases, 51 partial remission cases and 34 relapse cases. Sixty subjects were enrolled in the control group; 40 males (66.67%) and 20 females (33.33%), with a male to female ratio of 2.41. The PNS patients had typical manifestations, including massive proteinuria [24 h proteinuria of 5.989 (2.297, 9.140)], hypoalbuminemia, and hyperlipidemia (Table 1). There were no differences in sex, age or blood pressure between the patient group and the HC group (P > 0.05).

Figures 1 and 2 show that PNS patients had significantly higher serum and urine ANGPTL8/UCr concentrations than the control group. However, no significant difference was found in the expression of serum ANGPTL8 and urine ANGPTL8/UCr in PNS patients with or without glucocorticoid treatment (Table 2).

The PNS patients were further grouped into three proteinuria groups according to the 24 hUTP level. As shown in Table 3, serum ANGPTL8 levels were not significantly different among the three proteinuria groups (P > 0.05), while urine ANGPTL8/UCr levels showed significant differences among the three proteinuria groups (P < 0.05).

As shown in Table 4, in PNS patients, the serum ANGPTL8 levels were significantly positively associated with CHOL (r = 0.209, P < 0.05) and TG levels (r = 0.412, P < 0.001), while no significant association was found between serum ANGPTL8 levels and 24-h UTP.

The correlation analysis of urine ANGPTL8 and lipid metabolism indicators showed that urine ANGPTL8/UCr levels were positively correlated with HDL-C (r = 0.201, P < 0.05) and negatively correlated with CREA (r = − 0.323, P < 0.001), eGFR (r = − 0.352, P = 0.000) and 24 h UTP (r = − 0.268, P < 0.05) (Table 4).

We investigated the serum and urine ANGPTL8 levels in 78 PNS patients with renal biopsy pathology results, which included 21 MCD, 8 MsPGN, 38 MN and 11 FSGS patients. No significant differences in serum ANGPTL8 levels were found across the different PNS types. However, the urine ANGPTL8/UCr level was significantly different between the MN and MsPGN groups (P < 0.05, Table 5).