Therefore in the present study we first

Therefore, in the present study, we first examined the ATRAP Biocytin in human leukocytes of healthy subjects. We next analyzed possible relevant clinical factors affecting ATRAP expression in leukocytes of patients with NCDs. Furthermore, we examined the possible effect of ATRAP downregulation on the inflammatory profile of leukocytes in animals after low-dose lipopolysaccharide (LPS) injection, a model of low-grade inflammation in patients with NCDs [27,28], using bone marrow ATRAP-deficient chimeric mice.
Materials and methods
Results
Discussion Recent developments in the field of quantitative analysis of gene expression by ddPCR may provide the opportunity to reduce the quantitative variability currently seen using RT-qPCR analyses and obtain absolute values instead of relative levels, particularly in human samples of small quantity [31, 32]. Quantifying absolute levels is particularly useful in the clinical setting, allowing for the establishment of cut-offs for predicting the clinical endpoints and the observation of the trajectory of clinical markers without any reference samples for calibration. In the present study, precisely analyzing the gene expression in human leukocytes was crucial. A major advantage of this study was that we measured the absolute levels of genes using the ddPCR method. We measured leukocyte ATRAP mRNA level, but not the protein level. With respect to the in vivo regulation of ATRAP expression, we previously reported that a high-salt diet reduced the renal mRNA and protein levels of ATRAP, and accelerated the progression of hypertensive kidney injury in Dahl salt-sensitive rats [38]. We also showed that chronic angiotensin II infusion reduced the mRNA and protein levels of ATRAP in the heart and kidneys of mice, and accelerated the progression of hypertension and cardiac hypertrophy [22, 39]. In UUO-treated mice, renal mRNA and protein levels of ATRAP were decreased as renal fibrosis progressed [40]. Another group also reported that ATRAP mRNA and protein levels decreased in the injured femoral artery of mice [41]. On the other hand, it was reported that bortezomib, a proteasome inhibitor, could affect the ATRAP protein level in a post-translational manner [42]. These results indicate that ATRAP mRNA level closely reflects ATRAP protein level except under specific conditions, such as proteasome inhibitor treatment. To our knowledge, this is the first study to examine the expression and distribution of ATRAP in leukocytes and its clinical relevance in patients with NCDs. Here, we demonstrated for the first time that ATRAP was abundantly expressed in human leukocytes, predominantly in monocytes and granulocytes, from healthy subjects. We also showed that leukocyte ATRAP expression positively correlated with inflammatory parameters, such as the granulocyte and monocyte count, hsCRP and proinflammatory cytokine and chemokine levels in leukocytes of patients with NCDs. Furthermore, low-dose LPS enhanced leukocyte ATRAP and IL-1β gene expression in wild-type mice. In addition, after injection of low-dose LPS, the level of leukocyte IL-1β in bone marrow ATRAP-deficient chimeric mice was significantly upregulated in comparison to control wild-type chimeric mice, a model of low-grade inflammation in patients with NCDs [27]. These results suggest that ATRAP expression in leukocytes is closely linked to systemic inflammation and the leukocyte inflammatory status and that there is likely a compensatory upregulation of leukocyte ATRAP expression to improve the inflammatory profile in response to pathological stimuli in patients with NCDs. RAS in local tissues is reportedly associated with the inflammatory status [8, 43]. The serum hsCRP level is a well-established inflammatory marker that predicts the incidence and progression of NCDs, including hypertension [44], cardiovascular diseases [45, 46] and CKD [47]. However, previous studies on the relationship between leukocyte expression of RAS components and hsCRP have reported inconsistent findings [[48], [49], [50]]. A study reported that expression of RAS components in dendritic cells from patients with or without coronary artery disease correlated significantly with hsCRP levels [48]. Furthermore, peak AT1R and ACE levels in T-lymphocytes after angiotensin II stimulation were associated with hsCRP in normotensive and hypertensive patients [49]. In contrast, leukocyte AT1R expression did not correlate with hsCRP in healthy subjects and CKD patients in other study [50]. Although these inconsistent results might be due to differences in patient populations, medication status and methods for measuring RAS components in leukocytes, no studies have yet identified any RAS component that consistently reflects the circulating hsCRP levels and systemic inflammatory profile. Based on these previous findings and the results of the present study, we propose that leukocyte ATRAP is an emerging marker capable of reflecting the systemic and leukocyte inflammatory profile.