Macrophage Phenotype And The Effect Of TREM-1 In Its Regulation In Human Diabetic Nephropathy

Objectives:Heterogeneity and plasticity are hallmarks of macrophages. Classically activated macrophage (Ml) is characterized with tissue inflammation and damage, while alternatively activated macrophage (M2) displays anti-inflammatory and tissue repair. Macrophage accumulation is the feature of pathological changes in the renal of diabetic nephropathy (DN), but the possible role of macrophage phenotype in the progression of DN has not been elucidated. Triggering receptor expressed on myeloid cells (TREM-1), a cell surface molecule expressed on macrophages, implicates in the amplification of inflammatory responses. Recent studies demonstrate that TREM-1 is crucial for modulating macrophage polarization, and plays a pivotal role in the development of the obstructive nephropathy. However, whether TREM-1 has the similar effect in DN has not yet been characterized. This study tried to examine the macrophage phenotype and its relationship to renal function and histological changes in human DN and the effect of TREM-1 on high-glucose induced macrophage phenotype switch.Method:We studied retrospectively 46 patients with DN who were confirmed by diagnosis of the renal biopsy between 2011 and 2015. Biopsies were divided into ?, ?a, ?b, ?, ? classes according to the pathologic classification of DN. The other four normal renal tissue specimens taken from patients with renal trauma or renal tumor were as control group. Serum creatinine and proteinuria were collected before renal biopsy. Kidney tissues were used for PAS staining to assess histological changes. Immunohistochenical staining was used to detect numbers of CD68+, MR+ macrophage and TREM-1. Immunofluorescene staining was used to detect the number of (iNOS+CD68)+ macrophage. TREM-1 siRNA pre-treatment with RAW264.7 cells were done before culturing macrophages in high-glucose media (25mM) for 24 hours. TREM-1, M1 marker iNOS and M2 marker MR were detected by RT-PCR and Western blot.Results:(1) CD68, M1 macrophages infiltration were apparent in the glomeruli and interstitium, while accumulation of M2 macrophages were mainly observed in the interstitium. Numbers of CD68, Ml and M2 macrophages infiltration in DN group were increased in a process dependent manner compared with control group (P<0.05). (2) There were positive correlations between the glomeruli CD68, M1 and serum creatinine and proteinuria. Likewise, interstitium CD68, Ml and M2 expression correlated strongly with serum creatinine and proteinuria. (3) To further investigate M1/M2 macrophage activation phenotype during the course of DN, we found that there was increased expression of M1 at an early stage (I+IIa) of DN (P<0.05). The ratio of M1 to M2 macrophage peaked at this time. In contrast, M2 macrophages predominated at later time points(III) (P<0.05), and the percentage of M1/M2 macrophage was at the lowest level. (4) In DN group, TREM1-positive cells were apparent in the interstitium, and the expression levels significantly correlated with the course of DN (P<0.05). In vitro study, after macrophages have being treated with 25mmol/L glucose for 24h, RT-PCR and Western blot analysis showed Ml marker iNOS and TREM-1 were up-regulated (P<0.05), whereas M2 markers MR was significantly decreased (P<0.05). (3) However, the above effects of high-glucose were inhibited when TREM-1 was blocked by TREM-1 siRNA (iNOS mRNA:high-glucose versus TREM-1 siRNA:5.048±0.645 vs 2.260±0.062, P<0.05; MRmRNA:high-glucose versus TREM-1 siRNA: 1.042±0.036 vs 2.214±0.083, P<0.05).Conclusion:1. In biopsy renal tissue of human DN, the number of macrophages was significantly increased and the intensity of the infiltration correlated strongly with the classes of DN2. There was a positive correlation between the M1/M2 activation state and the progress of DN3. TREM-1 played an important role in high-glucose induced macrophage phenotype switch.