Background
Idiopathic pulmonary fibrosis (IPF), a chronic, progressive, fibrotic interstitial lung disease (ILD) with a poor prognosis, is largely unaffected by currently available medical treatments [
1]. IPF is associated with the histopathologic and/or radiologic pattern of a usual interstitial pneumonia (UIP). It is characterized by progressive worsening of dyspnea and lung function. The incidence and mortality of IPF are increasing [
1,
2], and the median survival time is 2 to 3 years from the time of diagnosis [
3]. Identification of peripheral blood biomarkers may facilitate the diagnosis, estimation of prognosis, and selection and evaluation of a treatment as well as the development of new therapeutic intervention. A number of candidate blood biomarkers for IPF including cytokines, chemokines, enzymes, collagen relevant products and products of type II epithelial cells, have been studied for their diagnostic and predictive values. Serum levels of mucin-like glycoprotein Krebs von den Lungen 6 antigen (KL-6) [
4], surfactant protein (SP)-A and SP-D [
5,
6], matrix metalloproteinase (MMP)1 and MMP7 [
7], brain natriuretic peptide [
8], and, most recently CC-chemokine ligand 18 [
9] are elevated in patients with IPF. KL-6, SP-A, and SP-D in blood are considered to derive from proliferating epithelial cells and/or disruption of the epithelial barrier.
Napsin A, an aspartic proteinase, is expressed in type II pneumocytes and in alveolar macrophages presumably secondary to phagocytosis [
10,
11]. It is abundant and active in the alveolar space, correlating with the levels of SP-B, proSP-B, and SP-C [
11]. Therefore, it is possible that circulating napsin A may increase upon type II pneumocyte hyperplasia and/or epithelial barrier breakdown, such as IPF and acute lung injury. In addition, immunohistochemistry for napsin A marks most cases of lung adenocarcinomas and is negative in most squamous cell carcinomas and adenocarcinomas of other organs [
12,
13]. Its local expression is reported to be useful both for classifying primary lung tumors as adenocarcinoma and for identifying lung origin in the setting of a metastatic adenocarcinoma [
12,
13].
We hypothesized that serum napsin A levels would be increased in patients with IPF and would correlate with severity of disease [
14,
15]. To test this hypothesis, we quantitated levels of circulating napsin A in patients with IPF, primary pulmonary adenocarcinomas, and controls, after that we analyzed the correlations between the serum levels of napsin A and those of KL-6, SP-A, SP-D respectively, and lung function as measured by percent-predicted forced vital capacity (FVC) in IPF patients. Furthermore, napsin A is also expressed in the proximal convoluted tubules of the kidney [
10], and we measured serum napsin A levels in patients with kidney disease to determine whether renal dysfunction might affect serum Napsin A levels.
Discussion
In the present study we demonstrated that circulating levels of napsin A are increased in patients with IPF, as compared with healthy controls, and correlate with those of KL-6, SP-A, SP-D, and the severity of disease. In addition, the serum napsin A levels were not elevated in patients with pulmonary adenocarcinoma without ILD or in kidney disease. These findings suggest that serum napsin A may be a candidate biomarker for IPF.
Our findings demonstrating elevated serum levels of KL-6, SP-A, and SP-D in IPF are consistent with those reported previously [
4‐
6], as well as the cut-off levels in this study for KL-6, SP-A and SP-D were similar to those in previous reports [
17‐
19]. Compared to these serum markers, napsin A showed the largest AUC for distinguishing IPF from controls. A comparison of KL-6, SP-A, and SP-D for the diagnostic values in patients with ILD including IPF previously demonstrated that KL-6 was superior to other markers [
6], and the findings of present study for IPF regarding the order of the AUC values obtained from ROC curves is the same as that study [
6], in which KL-6 preceded SP-A and SP-D. In our findings, serum napsin A levels showed greater diagnostic accuracy for distinguishing IPF from controls.
The mechanism by which the circulating levels of napsin A are elevated in IPF is not known. It is probably due to a combination of a loss of integrity of the epithelial barrier caused by lung injury and an increased mass of type II cells due to hyperplasia as SP-A and SP-D [
20]. The molecular weights of SP-A and SP-D are 26–38 kDa and 43 kDa, respectively [
20‐
22]; that of napsin A is approximately 38 kDa [
12], while that of KL-6 is estimated to be greater than 200 kD [
20,
23]. Serum KL-6 possibly requires cleavage by a proteinase to liberate its extracellular domain in order to leak into the bloodstream [
20,
24]. These differences may account for differences in the detected levels of these markers in IPF.
Serum napsin A levels were correlated with serum KL-6, SP-A, and SP-D in patients with IPF. Moreover, we found that napsin A levels were more strongly correlated with SP-A, and SP-D levels than with KL-6 levels, and this is supported by previous findings that napsin A is protease that relates to maturation of SP-B and SP-C [
11]. Consequently, napsin A is also a useful type II pneumocytes marker, as it the case with existing biomarker for IPF: KL-6, SP-A, and SP-D. The serum markers for IPF are similar to those for type II pneumocytes; it is possible that these biomarkers reflect type II pneumocyte activity.
A concern regarding serum biomarkers is that elevated levels of some markers can be found in IPF as well as in malignancies, while these diseases may coincide. Serum levels of KL-6 or VEGF were reported to be increased in patients with IPF but also in lung cancer patients [
25,
26]. The production of SP-A and SP-D by lung adenocarcinoma cells obtained from malignant pleural effusions has also been previously reported [
27]. In lung tumors, the sensitivity and specificity of napsin A immunostaining are high for identifying adenocarcinomas [
12,
13,
28‐
30]. We compared the serum levels of napsin A, KL-6, SP-A, and SP-D in patients with IPF and primary pulmonary adenocarcinomas. The ROC curves demonstrated that napsin A, KL-6 and SP-D were superior to SP-A as serum markers distinguishing IPF from adenocarcinomas. The limitation in falsely positive cases with lung cancer may be able to be corrected by using in combination.
In addition to type II pneumocytes, napsin A is expressed in the epithelium of the proximal and convoluted tubules of the kidney [
31]. In this study, none of the subjects with IPF, lung cancer, or controls exhibited any signs of renal dysfunction or renal cell carcinoma. Serum napsin A levels of patients with kidney disease indicated no elevation compared with those of control subjects. Therefore, it is unlikely that our data were influenced by kidney disease.
There were some limitations in this study. The study was retrospective and included only limited numbers of patients. The role of napsin A in the pathogenesis of lung disease is unknown, and it is possible that several other diseases including other types of ILD and pneumonia can cause an increase in serum napsin A levels. Therefore, a large cohort study will be required to confirm our results. We will also need to clarify the relationship of these markers to the histological patterns of ILD.
Conclusions
We have shown that napsin A is found in increased quantities in the circulation of patients with IPF, in whom the levels correlate with those of KL-6, SP-A, SP-D, and lung function. Napsin A is superior to KL-6, SP-A and SP-D for distinguishing IPF from controls. Although these findings do not allow us to determine whether napsin A is useful for predicting the outcome in IPF yet, they support the hypothesis that napsin A is a candidate biomarker for diagnosing the presence of disease in an individual.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Challenging Exploratory Research from JSPS, and a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and by the National Institute of Biomedical Innovation, Japan. We thank Ayako Kitanosono and Mariko Araki for their assistance in data collection.
Competing interests
All authors except for Masahiro Maeda have no potential conflicts of interest exist with any companies/organizations. Masahiro Maeda is an employee of Immuno-Biological Laboratories.
Authors’ contributions
TS: contributed to the planning, data collection, data analysis, and writing of the manuscript. TH: contributed to data collection, data analysis, and writing of the manuscript. HU: contributed to data analysis, and writing of the manuscript. MY: contributed to data collection, data analysis, and writing of the manuscript. GT: contributed to data collection, data analysis, and writing of the manuscript. TN: contributed to data collection, data analysis, and writing of the manuscript. MM: contributed to data analysis, and writing of the manuscript. TH: contributed to data analysis, and writing of the manuscript. HT: contributed to data analysis, and writing of the manuscript. HI: contributed to the planning, data collection, data analysis, and writing of the manuscript. All authors read and approved the final manuscript.