Levels of serum VEGF-C/D and CXCL-12 are related to the severity of proteinuria in diabetic nephropathy

Objective To investigate the relationship between serum levels of lymphangiogenic factors and type 2 diabetic nephropathy and to evaluate the role of lymphangiogenesis in diabetic nephropathy. The patients were divided into a normal albuminuria group (UmAlb < 30 mg/24 h, n = 30), microalbuminuria group (UmAlb 30–300 mg/24 h, n = 30) and massive albuminuria group (UmAlb > 300 mg/24 h, n = 30), and 30 healthy individuals were enrolled as the healthy control group (n = 30). Levels of serum vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor-D (VEGF-D) and chemokine ligand 12 (CXCL-12) were quantied by enzyme-linked immunosorbent assay kits. in diabetic nephropathy, which has high value for the early diagnosis and evaluation of disease severity in diabetic nephropathy. These ndings suggest that serum VEGF-C,VEGF-D and CXCL-12 levels may be a useful surrogate marker of clinical outcome in DN. eGFR: estimated glomerular ltration rate; FBS: fasting blood sugar; TG: triglyceride; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; UmAlb: urinary microalbumin.

Levels of serum VEGF-C/D and CXCL-12 are related to the severity of proteinuria in diabetic nephropathy

Introduction
Diabetes mellitus (DM) is a chronic metabolic disease that has seriously affected people's quality of life and health. According to a 2010 survey, the prevalence of prediabetes reached 50.1%, which means that approximately 500 million Chinese adults may have DM, and the prevalence of DM has increased dramatically 1 . DM vascular disease is one of the common complications and is also the main cause of death among DM patients. The most common vascular diseases are cardiovascular disease, cerebrovascular disease, and microvascular disease of the kidney, retina and skin. Diabetic nephropathy (DN), a common microvascular complications of DM, has become a major risk factor for end-stage renal disease and a major reason for renal replacement therapy [2] . DN is characterized by microalbuminuria, progressive renal impairment, hypertension, edema, and severe renal failure in the late stage.
At present, the main markers examined for the clinical diagnosis of DN are glomerular ltration rate, urinary microalbumin excretion rate, creatinine level, urea nitrogen level and serum cystatin level.
Although these markers can be used to effectively evaluate the function of the kidney, they indicate only late changes of kidney function and cannot re ect early structural changes in the kidney 2,3 . Therefore, we need to nd new markers that can be used for the early diagnosis of DN.
With the discovery of speci c markers of lymphatic endothelial cells, we are aware of the role of lymphangiogenesis in various diseases. Recent studies in mice with DN induced by streptozotocin and a high-fat diet showed that compared with the control group, the experimental group showed a signi cant increase in the number of lymphatic vessels in the cortex and medulla of the kidney, suggesting that DN may induce lymphangiogenesis of the kidney or at least lymphatic dilatation of the kidney 4 . Vascular endothelial growth factors (VEGF) belong to the platelet-derived growth factor family and play an important role in regulating angiogenesis and lymphangiogenesis. VEGF-C/D is a major member of the VEGF family, which binds to and activates the receptor of vascular endothelial growth factor receptor 3 (VEGFR-3), promotes the proliferation of lymphatic endothelial cells and lymphangiogenesis, and regulates angiogenesis and lymphatic endothelial cell regeneration 5,6 . CXCL-12, also known as stromal cell-derived factor-1 (SDF-1), is a small protein called a cytokine that belongs to the chemokine protein family. CXCL-12 binds to its receptor CXCR4,a novel axis that regulates lymphangiogenesis, which has a strong chemotactic effect on lymphocytes and plays an important role in the development process 7,8 .
In this study, we measured serum VEGF-C,VEGF-D and CXCL-12 levels in DN patients at different clinical stages to explore the relationship between lymphangiogenesis and DN, further clarify the role of lymphangiogenesis in DN, and infer whether VEGF-C, VEGF-D and CXCL-12 can be used as markers in the early diagnosis of DN.

Study population
We adopted a prospective observational study method, and ninety patients with type 2 DM (T2DM) who were hospitalized in our hospital from January 2018 to June 2019 were selected as the study subjects. According to the Mogenson stage, the patients were divided into a normal albuminuria group (UmAlb < 30 mg/24 h, n = 30), microalbuminuria group (UmAlb 30-300 mg/24 h, n = 30) and massive albuminuria group(UmAlb > 300 mg/24 h, n = 30), and 30 healthy individuals were enrolled in this study as the healthy control group (n = 30).

Inclusion criteria
The inclusion criteria were (1) age between 18 and 90 years old and(2) type 2 diabetes mellitus, meeting the World Health Organization (WHO) diagnostic and typing criteria for type 2 diabetes in 1999.

Exclusion criteria
The exclusion criteria were as follows: (1)

Basic data collection
Questionnaires were developed to collect basic information on patients, such as sex, age, blood pressure (SBP and DBP), fasting blood glucose (FBG) level, triglyceride (TG) level, total cholesterol (TC) level, low density lipoprotein cholesterol (LDL-C) level, high-density lipoprotein cholesterol (HDL-C) level, creatinine (Cr) level, glycosylated hemoglobin (HbA1c) level, urinary microalbumin (UmAlb) level, estimated glomerular ltration rate (eGFR), etc. All the above indicators were based on the results of the rst examination after admission.

Serum VEGF-C, VEGF-D and CXCL-12 level measurement
While the patients were in a fasted state, we collected 5 ml of venous blood on the second day after admission with a vacuum anticoagulant tube and centrifuged blood samples for 10 minutes (3000 r/min). The upper serum portion was separated into Eppendorf tubes and stored in a freezer at -80°C.

Statistical analysis
All data were analyzed by SPSS 17.0 software. First, the normality test was carried out on measurement data. Measurement data with a normal distribution were expressed as the mean ± standard deviation. Variance analysis was used for comparison among groups, homogeneity test of variance was carried out by the Levene method, the LSD-t test was used for comparison between groups, and the Tamhane T2 test was used for unequal variance. The count data were analyzed by the chi-square test. Pearson correlation analysis was used to analyze the correlation among the observed indicators. Receiver operating curves (ROCs) were drawn to analyze the value of serum levels of vascular endothelial growth factor-C, vascular endothelial growth factor-D and CXCL-12 in evaluating the severity of DN. P < 0.05 was statistically signi cant.

Basic data comparison
Ninety patients with DN were enrolled in this study, including 60 males and 30 females, aged 37-87 years with an average age of 60.22 ± 11.04 years. There were 30 patients in each study group; there were 25 males and 5 females in the healthy control group, aged 23-84 years, with an average age of 60.97 ± 16.52 years. There was no signi cant difference in sex or age among the groups (P > 0.05), which showed that the basic data of each group were balanced and comparable, as shown in Table 1. One-way ANOVA indicated that the relative expression levels of serum VEGF-C, VEGF-D and CXCL-12 among the massive albuminuria group, microalbuminuria group, normal albuminuria group and control group were signi cantly different (P < 0.01). The LSD-t test was used to compare two groups. Compared with those in the control group, the expression levels of serum VEGF-C, VEGF-D and CXCL-12 in the massive albuminuria group, microalbuminuria group, and normal albuminuria group increased gradually with the progression of DN, in the following order: massive albuminuria group > microalbuminuria group > normal albuminuria group > control group; the difference was signi cant (P < 0.05), as shown in Table 2.

Diagnostic value of serum levels of VEGF-C, VEGF-D and CXCL-12 in DN
ROC curve analysis showed that the areas under the ROC curve (AUCs) in the normal albuminuria group, microalbuminuria group, and massive albuminuria group were 0.668, 0.799 and 0.850. When the cutoff value were 152.40, 196.05 and 214.60 pg/ml, the sensitivity was 93.3%, 96.7% and 100.0%, and the speci city was 40.0%, 50.0% and 56.7%, respectively. The serum VEGF-D and CXCL-12 levels used to assess the area under the ROC curve in the massive albuminuria group, microalbuminuria group, and normal albuminuria group were higher than the VEGF-C level, which indicated that serum levels of VEGF-C, VEGF-D and CXCL-12 had a certain value for the early diagnosis and assessment of disease of severity of DN, as shown in Figs. 1, 2, and 3; Tables 4, 5, and 6.

Discussion
The lymphatic system is an important auxiliary system of tissue uid re ux, which plays an important role in maintaining the dynamic balance of tissue uid, lipid absorption, immune monitoring and other physiological processes. In many pathological conditions (such as in ammatory reactions, tissue wound repair, organ transplantation, and tumors), lymphatic vessel formation can be reactivated 9,10 . At present, research on lymphangiogenesis both domestically and internationally is mainly concentrated in the eld of cancer, and the mechanism of lymphangiogenesis in DN is not clear. It is well known that in ammation is considered to be an important factor in lymphangiogenesis, and the involvement of in ammatory factors (such as interleukin-1 and tumor necrosis factor alpha) and macrophages can increase the production of vascular endothelial factors, thus promoting lymphangiogenesis 11,12 . In recent reports on human kidney diseases, we found that lymphangiogenesis is affected by the duration of in ammation and brosis progression, rather than acute in ammation. As a type of chronic interstitial in ammation, DN is characterized by mesangial dilatation, podocyte loss, glomerular proliferation, glomerular basement membrane thickening and tubular epithelial cell dysfunction, which leads to glomerular capillary occlusion; therefore, lymphatic vessel formation is considered a kind of renal edema and hypertension compensation response 4,13,14 .
DN is a serious complication of DM and initially manifests as microalbuminuria. Persistent diabetesrelated metabolic and hemodynamic disorders can lead to in ammatory changes in the kidney, and promote the process from injury to repair of the kidney, leading to renal brosis, especially after stages III and IV, and the degree of renal brosis is more signi cant 15,16 . Many studies have shown that renal brosis is accompanied by lymphangiogenesis. One study induced renal brosis by constructing a unilateral ureteral obstruction model in mice and found that the degree of renal lymphangiogenesis was positively correlated with renal brosis 17 . To understand the relationship between lymphangiogenesis and renal brosis, another study used unilateral ureteral obstruction in rats to analyze the relationship between in ammation, brosis, lymphangiogenesis and growth factor expression; it was found that transforming growth factor-β 1 (TGF-β1) and VEGF-C were present in renal tubular epithelial cells and monocytes, where their levels gradually increased and reached a peak at 14 days after ureteral obstruction 18 . Sakamoto I et al, through pathological examination of 124 renal biopsy specimens, found an increase in lymphatic vessel number in patients with tubulointerstitial disease compared with the control group and that this was related to the degree of tissue damage; moreover, the correlation with the brotic area was stronger than that with the in amed area. In addition, compared with other renal diseases, lymphangiogenesis in DN patients was more signi cant 19 . Therefore, lymphangiogenesis is a common feature of tubulointerstitial brosis.
The advantages and disadvantages of lymphangiogenesis in DN are still controversial. Lymphatic vessels, an important regulator of uid balance, immune cell transport and immune recognition, play an important role in many diseases. DN is the main cause of end-stage renal disease worldwide. Hyperglycemia-induced oxidative stress and in ammation play an important role in the occurrence and development of DN. Lymphangiogenesis is an important part of the in ammatory process of tissues and organs. Many studies have shown that the dilated lymphatic system is necessary to resolve in ammation, and lymphatic vessels play an important role in uid clearance and immune cell transport, thus achieving the effect of reducing or alleviating in ammation 20,21 . However, recent studies have shown that lymphangiogenesis is not necessarily bene cial to the progression of DN. Functional lymphatics play an important role in the process of body uid balance and immune monitoring, but disordered expansion of lymphatics can lead to the failure of immune cell clearance, which leads to chronic in ammation. Recent studies have found that in DN mice, selective inhibition of VEGFR-3 to inhibit lymphatic proliferation could reduce the serum cholesterol level, free fatty acids and proteinuria, thus decreasing in ammation and oxidative stress in the kidney and alleviating renal brosis 22 . Another study also questioned the involvement of lymphatic function DN. With the development of chronic in ammation in DN, the expression of various in ammatory factors was not balanced, resulting in the excessive growth of lymphatic vessels, which eventually led to structural incompleteness and dysfunction. Studies have shown that by reducing renal lipid toxicity, renal lymphatic dysfunction can be alleviated in DN, thereby alleviating the degree of renal in ammation and brosis 23 .

Conclusion
The results of this study showed that the serum levels of VEGF-C, VEGF-D and CXCL-12 were closely related to DN. With the development of DN, the expression levels of serum VEGF-C, VEGF-D and CXCL-12 increased gradually, which is also consistent with the ndings of many studies, proving that lymphangiogenesis accompanies the development of DN and is positively correlated with the severity of the disease. ROC curve analysis showed that the serum levels of VEGF-C, VEGF-D and CXCL-12 had certain value in the early diagnosis of DN, and could at least be used to predict the progression of the disease. At present, there is still controversy about whether lymphangiogenesis is good or bad for DN, but many studies have shown that inhibiting lymphangiogenesis can alleviate in ammation and brosis of DN to a certain extent, which provides us with new ideas and perspectives for the treatment in DN, but further studies and clinical trials are needed to prove this.  The ROC curves of serum VEGF-D in different groups for diagnosis of DN.