Abstract Background and Aims Vascular calcification is an independent risk factor for all-cause mortality in patients with CKD. Macrophages play an important role in vascular calcification, which involve in the osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells (VSMCs). Recent research shows SGK1 is a highly attractive candidate for developing VSMCs calcification. Previous studies indicated that TGFβ1 induces vascular calcification by regulating osteo-/chondrogenic transdifferentiation of VSMCs. This study focus on the effects of macrophages-derived SGK1 on VSMCs transdifferentiation via regulating TGFβ1 expression. Method Before induced into M2 phenotype with IL-4, RAW 264.7 cells were treated with SGK1 inhibitor EMD638683 for 24 hours to reduce SGK1 expression. The above cells were grouped as: M0, M2, M2+EMD to explore the expression of SGK1 and TGFβ1 by using western blot, qRT-PCR and immunofluorescence staining, respectively. The culture medium (CM) of the above group macrophages was collected. Then mouse aortic smooth muscle cells (MAoSMCs ) were cultured in these supernatants with normal medium or osteogenic medium (OM) with or without TGFβ1 receptor inhibitor SB-431542 for 3 weeks to explore the transdifferentiation and calcification of MOVAS by using western blot, RT-qPCR, immunofluorescence staining, calcium quantification, Alizarin Red and Von Kossa Staining. Results As Alizarin Red and Von Kossa Staining shown, MAoSMCs contained more calcium deposits in the M2-CM group compared with either in the group of NC nor M0-CM. Consistent with the above results, We also found that M2-CM promoted MAoSMCs transdifferentiation, which was characterized by markedly increase of expression of osteo-/chondrogenic markers (Runx2, ALPL, FGF23) and decrease of the MAoSMCs marker (SM22α). Exploring the mechanism of the above phenomenon we found the expression of SGK1 and TGFβ1 were significantly increased in M2 group compared with M0 group. Interestingly, both SGK1 inhibitor EMD638683 which reduced TGFβ1 expression in M2 and TGFβ1 receptor inhibitor SB-431542 could partially blocked MAoSMCs osteo-/chondrogenic transdifferentiation and calcification. Furthermore, recombinant mouse TGFβ1 Protein increased calcium content in MAoSMCs by using calcium quantification, Alizarin Red and Von Kossa Staining and promoted MAoSMCs osteo-/chondrogenic transdifferentiation, which was characterized by markedly increase of expression of osteo-/chondrogenic markers (Runx2, ALPL, FGF23) and decrease of the MAoSMCs marker (SM22α). Conclusion Our findings shed light M2 macrophages promotes MAoSMCs osteo-/chondrogenic transdifferentiation and calcification by up-regulating TGFβ1 expression.

Objective: Macrophage infiltration in kidney is a major pathological feature of diabetic nephropathy (DN), which has been demonstrated associate with macrophages autophagy homeostasis. However, the relationships between autophagy and the infiltration response related of macrophages adhesion and migration are unknown. This study aims to investigate the impact of macrophages adhesion and migration by modulating autophagy. Methods: In vivo, rats were randomly distributed into control (NC) and DN groups. The pathological changes in renal tissue were assessed, and expression of CD68, LC3, P62 were analyzed. In vitro, RAW264.7 cells were divided into NC and high glucose (HG) groups. The capacity of macrophages adhesion migration and the expression of autophagy markers were observed with and without autophagy modulators (rapamycin, 3-methyladenine, chloroquine, and bafilomycin A1 for RAPA, 3-MA, CQ, BAFA). The macrophages autophagosome and the process of degradation and fusion of autophagosome-lysosome were observed by electron microscopy. Results: In vivo, renal injury is aggravated in diabetic rat compared with NC group. The autophagy level is inhibited in renal tissues of DN group with the increasing expression of CD68 and P62, while expression level of LC3 decreased (p < .05). In vitro, HG and 3-MA reduce the numbers of autophagosome of macrophages to inhibit autophagy level with decrease expression of LC3 and Beclin-1, but increase expression of P62, which promote the adhesion and migration capacity of macrophages (p < .05). Moreover, CQ and BAFA suppress autophagy level by inhibiting the process of autophagosome-lysosome degradation and fusion of macrophages, as well as the expression of LC3 and Beclin-1. We notice an increase expression of P62 by CQ and BAFA stimulation (p < .05). CQ and BAFA further facilitate the adhesion and migration capacity of macrophages. However, RAPA increases the numbers of macrophages autophagosome that inhibited by HG, resulting in a recovery of autophagy level with increase expression of LC3 and Beclin-1, whereas a reduction expression of P62, which lead to inhibition of adhesion and migration of macrophages induced by HG (p < .05) Conclusions: High glucose efficiently reduced the level of macrophage autophagy, following macrophages adhesion and migration enhanced when autophagy is suppressed. Activation of autophagosome improve the level of autophagy, but leading to a reduction of the macrophages adhesion and migration. While, inhibiting the process of degradation and fusion of autophagosome-lysosome suppress the level of autophagy and promote the macrophages adhesion and migration. These results indicate that high glucose may play an important role in macrophages adhesion and migration through modulating autophagy activities in diabetic nephropathy.