The Association of Serum Anti-Lysosomal-Associated Membrane Protein-2 Antibody with Vasculitis Combined with Hypertension

The aim of this study was to explore the association of serum anti-lysosomal-associated membrane protein-2 (anti-LAMP-2) antibody with vasculitis combined with hypertension (VAS-HTN). A total of 51 VAS-HTN patients, 46 essential hypertension (EH) patients, and 46 healthy controls (HC) were included in the study. Serum anti-LAMP-2 antibody levels are increased in VAS-HTN patients as compared with EH and HC (all P < 0.05). Serum anti-LAMP-2 antibody levels were significantly higher in active stage patients than those in non-active stage patients and HC (all P < 0.05). The correlation analysis showed a significant positive correlation between serum anti-LAMP-2 antibody levels and the Birmingham Vasculitis Activity Score (BVAS) and hypersensitive C-reactive protein (Hs-CRP) (all P < 0.05). Among the subsets of VAS-HTN, the levels of serum anti-LAMP-2 antibody were remarkably higher in all VAS-HTN subsets compared with HC (all P < 0.05). More interestingly, the levels of serum anti-LAMP-2 antibody were remarkably increased in polyarteritis nodosa (PAN) patients compared with ANCA-associated vasculitis and Takayasu arteritis patients (all P < 0.05). In addition, there was a significant positive correlation between serum anti-LAMP-2 antibody levels and BAVS and Hs-CRP in PAN patients (all P < 0.05). Multivariate logistic regression analysis showed that the anti-LAMP-2 antibody was independently associated with VAS-HTN. The levels of serum anti-LAMP-2 antibody were remarkably increased in VAS-HTN patients compared to EH and HC and might reflect the disease activity. The anti-LAMP-2 antibody may be a potential biomarker for diagnosis and estimating the disease activity in VAS-HTN.


Introduction
Hypertension is a global public health problem. It is currently the main contributor to the global burden of disease [1]. Numerous studies have confirmed that an increasing number of patients with hypertension is attributed to various diseases (e.g., kidney disease, primary aldosteronism, and vasculitis) [2][3][4][5]. Among them, the incidence of vasculitis is very insidious and harmful [6]. Because of the particularity of etiology, conventional antihypertensive therapy is not feasible. Furthermore, the target organ (renal, lung, heart, brain, etc.) damage caused by vasculitis is very similar and more serious to that caused by hypertension [7,8]. Consequently, differential diagnosis of patients with vasculitis combined with hypertension (VAS-HTN) is extremely important in the hypertensive population.
Vasculitis is a disabling disease featuring inflammatory injury and destruction that affects all sizes of vessels in the body [9]. Recently, the incidence of vasculitis has gradually increased, which has brought a heavy economic burden to society [10,11]. Hypertension is a common clinical feature of vasculitis [12,13]. In clinical, hypertension specialists will also encounter patients with vasculitis. However, vasculitis presents with a wide range of clinical presentations and lacks specific laboratory indicators. e identification and evaluation of disease in vasculitis is a tremendous challenge. erefore, finding novel biomarkers is very important for VAS-HTN patients to be identified from hypertensive populations and to create conditions for future treatment.
Lysosomal-associated membrane protein-2 (LAMP-2) is a transmembrane protein that participates in molecular chaperone-mediated autophagy and the presentation of intracellular antigens [14][15][16][17]. Kain et al.'s study found that LAMP-2 was expressed on the surface of neutrophils and was considered the anti-neutrophil cytoplasmic antibody (ANCA) antigen [18]. Recent studies suggest that anti-LAMP-2 antibody has been involved in the pathogenesis of vasculitis [19][20][21]. Some research showed that the levels of serum anti-LAMP-2 antibody were observed to increase in small vessel vasculitis [22][23][24]. Nevertheless, other research demonstrated that the levels of serum anti-LAMP-2 antibody were similar in ANCA-associated vasculitis (AAV) patients compared to healthy controls (HC) [22,23,25]. erefore, whether the anti-LAMP-2 antibody is associated with vasculitis remains controversial. Furthermore, serum anti-LAMP-2 antibody levels have not been evaluated in VAS-HTN, particularly in Takayasu arteritis (TA) and polyarteritis nodosa (PAN). e goal of this study was to examine whether there is a relationship between serum anti-LAMP-2 antibody levels and VAS-HTN.

Study Subjects.
All enrolled VAS-HTN patients were seen at the People's Hospital of Xinjiang Uygur Autonomous Region from January 2013 to December 2017, and the diagnosis was made based on clinical presentation, laboratory tests, imaging examinations, or/and pathological biopsies. Finally, 51 VAS-HTN patients (including 24 PAN, 20 AAV, and 7TA) were included in this study. e diagnostic flow chart of VAS-HTN screening is shown in Figure 1. All vasculitis patients fulfilled the 1990 ACR and/or 2012 revised CHCC classification criteria [26][27][28][29][30].
e criteria for the diagnosis of vasculitis are shown in Figure 2. Also, 46 patients with age-and sex-matched essential hypertension (EH) and 46 HC subjects were include in the study.

Data Collection and Measurements.
All clinical information data is derived from the electronic medical records of patients during their hospitalization. Details of the data collection and measurements can be found in previously published papers [31,32] and online supplemental material.

Definitions of Disease
Activity. Disease activity was assessed according to the third version of the Birmingham Vasculitis Activity Score (BVAS) [33]. e BVAS � 0 was considered inactive in VAS-HTN patients, and the BVAS ≥ 1 was considered active in VAS-HTN patients.

Definitions of Renal Injury.
Renal injury was defined as any proteinuria and/or hematuria and/or elevated serum Scr. Proteinuria was defined as a urine routine of more than 1+ and/or urine containing more than 0.15 g of protein was collected for 24 hours. Hematuria was defined as more than 5 red blood cells per high magnification view in the urine sediment. Elevated serum Scr was defined as a female Scr > 84 μmol/L or a male Scr > 104 μmol/L [31].

Serum Sample Collection and Anti-LAMP-2 Antibody
Measurement. Blood samples from all participants were drawn into procoagulation tubes, and the serum was collected after centrifugation at 3000 g at 4°C for 15 minutes as soon as possible. e serum was stored in a refrigerator at −80°C for standby. e levels of serum anti-LAMP-2 antibodies were measured using a commercial ELISA kit according to the manufacturer's instructions (Uscn Life Sciences, Wuhan, China).

Statistical
Analysis. SPSS software version 20.0 and GraphPad Prism software 5.0 were employed for statistical analysis and graphing. Mean ± standard deviation, or median and interquartile range, was used to present continuous variables, as appropriate. Categorical variables are expressed as totals and percentages. e t-test/one-way ANOVA or rank-sum test is used for comparisons across groups, as appropriate. Spearman or Pearson rank correlations were used to assess correlations. Binary logistic regression was used to determine independent factors for the diagnosis of VAS-HTN. Receiver operating characteristic (ROC) curve analysis was used to determine the optimal cutoff value for anti-LAMP-2 antibodies. P value <0.05 was regarded as significant.

Clinical and Laboratory
Features. e mean age of the patients with VAS-HTN in this study was 40.22 ± 9.62 years, and women accounted for 43.1% of all patients. Among the VAS-HTN patients, 24 were diagnosed with PAN, 20 with AAV, and 7 with TA. 16 patients were in the inactive phase and 35 patients were in the active phase according to BVAS assessment. e basic characteristics of 51 VAS-HTN patients, 46 EH patients, and 46 HC patients are presented in Tables 1 and 2.  (Figure 3(d)).

Correlations between Serum Anti-LAMP-2 Antibody
Levels and Clinical and Laboratory Parameters. We evaluated whether serum anti-LAMP-2 antibody levels are in correlation with clinical and laboratory parameters in VAS-HTN patients.. e correlation analysis showed that there was a positive correlation between serum anti-LAMP-2    Table 3, multivariate logistic regression analysis showed that anti-LAMP-2 antibody (OR: 1.028, P � 0.004) and Scr (OR: 1.079, P � 0.003) were all independently associated with VAS-HTN.

ROC Curve Analysis.
e diagnostic performance of serum anti-LAMP-2 antibody levels for detecting VAS-HTN patients using the ROC curves. e best cutoff values for serum anti-LAMP-2 antibody levels were determined using the highest value of the Youden index. e results showed that the best cutoff value, sensitivity, and specificity were at 76.99 ng/ml, 84.3%, and 83.7%. When assessing the active stage with serum anti-LAMP-2 antibody levels in VAS-HTN patients, a cutoff value of 117.58 ng/ml with a sensitivity of 65.7% and a specificity of 68.8% was observed according to ROC curve analysis. Further evaluation of patients with the VAS-HTN subgroup revealed that the optimal anti-LAMP-2 antibody cutoff value for identifying PAN and VAS-HTN was 132.38 ng/ml, with a sensitivity of 62.5% and specificity of 85.2%. All estimated values are shown in Table 4.

Discussion
Recently, it has been reported that anti-LAMP-2 antibody was associated with small-vessel vasculitis. Kain et al. research findings suggest that LAMP-2 autoantibodies can be detected in most AAV patients, and possibly related to the disease activity [34]. However, Roth et al. concluded that LAMP-2 antibodies may be present at very low titers in a minority of AAV patients [25]. e reason for the difference between the two results may be due to the different case selection and detection methods [35]. erefore, the role of anti-LAMP-2 antibodies in vasculitis is still far from clear. As far as we know, no published research has reported the relationship between serum anti-LAMP-2 antibody levels and VAS-HTN in hypertensive populations.
In this study, all the selected VAS-HTN patients complained of hypertension and visited our center. Vasculitis International Journal of Hypertension included seven categories according to the 2012 CHCC classification criteria [26]. Our study has three types of vasculitis, which were included in this study. is also shows that these three types of vasculitis are more likely to lead to hypertension. is is consistent with some reports [12,13]. We investigated further the serum anti-LAMP-2 antibody levels in VAS-HTN patients, EH, and HC based on ELISA.
Our results showed that the levels of serum anti-LAMP-2 antibody were significantly higher in VAS-HTN patients compared with EH and HC. Furthermore, we found that the active stage of VAS-HTN patients was closely related to the elevated anti-LAMP-2 antibody levels. Multivariate logistic regression analysis showed that the anti-LAMP-2 antibody was an independent factor associated with VAS-HTN. On account of these results, we believe that the anti-LAMP-2 antibody could be an independent factor of VAS-HTN and might reflect the disease activity. In addition, the levels of serum anti-LAMP-2 antibody in patients with the inactive Peschel et al. discovered autoantibodies to hLAMP-2 that conjugate to natural glomeruli, suggesting an important role in the pathogenesis of ANCA-negative oligoimmune focal necrotizing glomerulonephritic lesions [36]. erefore, we investigated whether serum anti-LAMP-2 antibody related to renal injury in VAS-HTN. However, whether it is directly engaged in the renal damage process is unclear. As a next step, we need to expand the volume of samples to further prove this result.
In the present study, we found that all VAS-HTN subsets presented increased markedly serum anti-LAMP-2 antibody levels than in EH and HC. To our surprise, the levels of serum anti-LAMP-2 antibody were increased significantly in PAN patients compared with AAV and TA patients. Moreover, our results showed that serum anti-LAMP-2 antibody levels were significantly positively correlated with BAVS and Hs-CRP. Our results indicate that anti-LAMP-2 antibody could play a role in the pathogenesis of PAN and can reflect the disease activity. Further studies examining anti-LAMP-2 antibody levels during the course of disease in PAN are needed. e quality of the anti-LAMP-2 antibody as a biomarker was evaluated on account of ROC analysis. For the diagnosis of VAS-HTN, the optimal cutoff values of serum anti-LAMP-2 antibody were determined to be 76.99 ng/ml. We further assess the active stage with serum anti-LAMP-2 antibody levels in VAS-HTN patients, with a cutoff value of 117.58 ng/ml with a sensitivity of 65.7% and a specificity of 68.8%. Furthermore, the analysis of patients with VAS-HTN subsets revealed that the best anti-LAMP-2 antibody cutoff value for differentiating PAN from VAS-HTN is 132.38 ng/ ml with 62.5% sensitivity and 85.2% specificity. It suggests that anti-LAMP-2 antibody might serve as a potential biomarker for diagnosing VAS-HTN and to assist in the assessment of the disease activity. In addition, we believe that the detection of serum anti-LAMP-2 antibody may be a feasible method to distinguish PAN from AAV and TA.
In the present study, our data seem robust enough to conclude that the anti-LAMP-2 antibody is a suitable biomarker for the clinical diagnosis and disease assessment for VAS-HTN. It is helpful for the differential diagnosis of SV in the hypertensive population, the rational formulation of the treatment plan, the effective evaluation of treatment effect, the prediction of recurrence, and the better clinical management of VAS-HTN patients in the future. However, our study has some limitations that need to be considered. First, the present study failed to investigate the dynamics of serum anti-LAMP-2 antibody levels. Further longitudinal studies are needed to observe the dynamic changes. Second, the low number of patients requires further expanding the sample size. Finally, our study did not evaluate the comparison of diagnostic performance between anti-LAMP-2 and other vasculitis diagnosis markers, such as anti-endothelial cell  International Journal of Hypertension antibody (AECA). Further studies are needed to verify the diagnostic performance of anti-LAMP-2 antibodies in the future.

Conclusion
e study suggests that anti-LAMP-2 antibodies were higher in patients with VAS-HTN compared to EH and HC, and might reflect the disease activity, particularly in patients with PAN. e anti-LAMP-2 antibody may be a potential biomarker for diagnosis and estimating the disease activity in VAS-HTN.

Data Availability
Data supporting the results of this research are accessible from the first author.

Disclosure
Xintian Cai and Bin Zhu are the co-first authors for this paper.

Conflicts of Interest
e authors declare that they have no conflicts of interest.