Search

Aims: To observe the effect of calcitriol on tubular epithelial cells apoptosis in diabetic nephropathy (DN) and to explore the possible mechanism of its renal protection., Methods: In vivo, DN rats established by streptozocin (STZ) were treated with or without calcitriol by gavage. Rats were killed at 18 weeks after treatment. In vitro, HK-2 cells were cultured in high glucose with or without 1,25-dihydroxyvitamin D3. In some experiments, P38MAPK activator anisomycin was applied to incubate HK-2 cells. Cell apoptosis was detected by TUNEL or Annexin V-FITC/PI staining with flow cytometry. Immunohistochemical staining was used to observe the expression of VDR in kidney. Protein expression of cleaved caspase-3, Bax, Bcl-2, VDR, pp38MAPK and p38MAPK was assessed by western blotting., Result: Calcitriol treatment ameliorated the severity of proteinuria and reduced renal tubular epithelial cells apoptosis in DN rats. In addition, calcitriol treatment significantly increased renal VDR expression and reduced the expression of p-p38MAPK in rats. In vitro, 1,25-dihydroxyvitamin D3 decreased the apoptotic rate of HK-2 cells induced by high glucose. In accord with the results from animal study, 1,25-dihydroxyvitamin D3 increased VDR expression, but decreased p-p38MAPK expression in HK-2 cells. Moreover, P38MAPK activator anisomycin blocked the anti-apoptotic effect of 1,25-dihydroxyvitamin D3 on HK-2 cells., Conclusions: Calcitriol attenuates renal tubular cells apoptosis via VDR activation which inhibits p38MAPK signaling in DN rats.

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.

This study aimed 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 activation. We observed that in renal tissue biopsies, the expression of CD68 and M1 was apparent in the glomeruli and interstitium, while accumulation of M2 and TREM-1 was primarily observed in the interstitium. The numbers of CD68, M1, and M2 macrophages infiltrating in the DN group were increased in a process-dependent manner compared with the control group, and the intensities of the infiltrates were proportional to the rate of subsequent decline in renal function. M1 macrophages were recruited into the kidney at an early stage (I+IIa) of DN. The M1-to-M2 macrophage ratio peaked at this time, whereas M2 macrophages predominated at later time points (III) when the percentage of M1/M2 macrophages was at its lowest level. In an in vitro study, we showed that under high glucose conditions, macrophages began to up-regulate their expression of TREM-1, M1, and marker iNOS and decreased the M2 marker MR. However, the above effects of high-glucose were abolished when TREM-1 expression was inhibited by TREM-1 siRNA. In conclusion, our study demonstrated that there was a positive correlation between the M1/M2 activation state and the progress of DN, and TREM-1 played an important role in high-glucose-induced macrophage phenotype transformation., Competing Interests: The authors have declared that no competing interests exist.

Aim: Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). This study aimed to investigate whether active vitamin D (VD) suppresses macrophage transition to the M1 phenotype via inhibiting the high glucose-induced STAT-1 phosphorylation to reduce TREM-1 expression., Methods: In vivo, pathological changes in kidney tissue were detected and the expression of CD68 TREM-1, STAT-1, M1 makers, and M2 makers were acquired in renal tissue of patients with DN and 18w DN rats. In vitro, RAW 264.7 cells were incubated in the presence of high glucose with or without VD . Silencing and overexpression of TREM-1 and silencing and activate of STAT-1 were explored to elucidate the underlying mechanism. The expression of TREM-1 and STAT-1 and the changes of macrophage phenotype were examined separately by western blot and immunofluorescence staining., Results: (a) Expression of TREM-1, p-STAT-1, and M1 markers (iNOS and TNF-α) were increased and positively correlated in kidneys from patients with DN. (b) In DN rats, the enlargement of glomerular surface area, expansion of glomerular mesangial matrix, the expression of CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) were significantly increased in comparison with the normal control group, whereas above changes were markedly decreased in the diabetic group treated with the VD group. (c) In vitro, VD significantly decreased high glucose-induced CD68, TREM-1, p-STAT-1, and M1 marker (iNOS) expression. However, above-mentioned effects of VD are abolished when TREM-1 is overexpressed or STAT-1 is activated. Reductions in STAT-1 expression decreased the TREM-1 expression., Conclusion: VD can inhibit macrophage transition to the M1 phenotype through the STAT-1/TREM-1 pathway., (© 2018 Wiley Periodicals, Inc.)

This study intends to investigate the effect of active vitamin D (VD) on the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) in the renal tissues of diabetic nephropathy (DN) rats and to explore the impact of TREM-1 on macrophage adhesion and migration. We find that the expressions of TREM-1 and CD68 protein are higher in DN rats compared with rats in the normal control group and that these changes are decreased in the DN + VD group. In vitro, the capacity for macrophage adhesion and migration and the expression of TREM-1 are increased under high-glucose conditions, but VD inhibits this progress. TREM-1 siRNA decreases high-glucose-induced macrophage adhesion and migration, whereas over-expression of TREM-1 inhibits its action. However, VD cannot suppress high glucose-induced TREM-1 expression and macrophage adhesion and migration when TREM-1 is over-expressed. These results demonstrate that VD can suppress macrophage adhesion and migration by reducing the expression of TREM-1., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy (DN). Macrophages mainly exhibit M1 phenotype, which contributes to the inflammation and fibrosis in DN. Studies indicate that autophage plays an important role in M1/M2 activation. However, the effect of mitophage on M1/M2 macrophage phenotype transformation in DN is unknown. This study investigates the role of mitophage on macrophage polarization in DN. In vivo experiments show that macrophages are exhibited to M1 phenotype and display a lower level of mitophagy in the kidney of streptozocin (STZ)-induced diabetic rats. Additionally, inducible nitric oxide synthase (iNOS) expression is positive correlated with the P62 expression, while negative correlated with LC3. Electronic microscope analysis shows mitochondria swelling, crista decrease and lysosome reduction in DN rats compared with NC rats. In vitro, RAW264.7 macrophages switch to M1 phenotype under high glucose conditions. Mitophagy is downregulated in such high glucose induced M1 macrophages. Furthermore, macrophages tend to switch to the M1 phenotype, expressing higher iNOS and TNF-α when impair mitophagy by 3-MA. Rapamycin, an activator of mitophagy, signifcantly blocks high-glucose induced M1 makers (iNOS and TNF-α) expression, meanwhile enhances M2 makers (MR and Arg-1) expression. These results demonstrate that mitophage participates in the regulation of M1/M2 macrophage phenotype in diabetic nephropathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Macrophages, especially their activation state, are closely related to the progression of diabetic nephropathy. Classically activated macrophages (M1) are proinflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. 1,25-Dihydroxyvitamin D3 has renoprotective roles that extend beyond the regulation of mineral metabolism, and PPARγ, a nuclear receptor, is essential for macrophage polarization. The present study investigates the effect of 1,25-dihydroxyvitamin D3 on macrophage activation state and its underlying mechanism in RAW264.7 cells. We find that, under high glucose conditions, RAW264.7 macrophages tend to switch to the M1 phenotype, expressing higher iNOS and proinflammatory cytokines, including TNFα and IL-12. While 1,25-dihydroxyvitamin D3 significantly inhibited M1 activation, it enhanced M2 macrophage activation; namely, it upregulated the expression of MR, Arg-1, and the anti-inflammatory cytokine IL-10 but downregulated the M1 markers. However, the above effects of 1,25-dihydroxyvitamin D3 were abolished when the expression of VDR and PPARγ was inhibited by VDR siRNA and a PPARγ antagonist. In addition, PPARγ was also decreased upon treatment with VDR siRNA. The above results demonstrate that active vitamin D promoted M1 phenotype switching to M2 via the VDR-PPARγ pathway.

Podocyte injury plays a critical role in the development and progression of diabetic nephropathy (DN). Over expression of TRPC6 on the podocytes has been revealed to cause podocyte injury in non-diabetic states. Besides, the emerging evidence from clinic revealed that vitamin D could reduce albuminuria and improve renal function, which was associated with podocyte protection. Our study aimed to investigate whether calcitriol ameliorating podocyte impairment is associated with regulation of the expression of TRPC6 in STZ-induced rats. Sprague-Dawley rats were randomly divided into three groups: normal control, DN, and DN treated with calcitriol (DN + VD); VD rats were treated with 0.1 μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozocin. The rats were sacrificed after 18 weeks treatment. DN rats exhibited increased proteinuria accompanied by elevated TRPC6 expression. Treatment with calcitriol not only reduced proteinuria, but also normalized TRPC6 expression. Meanwhile, in DN rats, the expression of podocyte specific markers including nephrin and podocin was significantly decreased, accompanied by increased desmin, a marker of podocyte injury. Treatment with calcitriol reversed above changes. In addition, vitamin D receptor (VDR) was significantly decreased, whereas this reduction was attenuated by the calcitriol treatment. Moreover, TRPC6 was positively correlated with both 24 h urinary protein and desmin. In contrast, TRPC6 was negatively correlated with both VDR and nephrin expression in podocytes. Calcitriol can ameliorate podocyte injury, which is contributed by the inhibition of enhanced TRPC6 expression in the early stages of DN rats.

Objectives: The present study aimed to investigate the relationship between PI3K/p-Akt signaling pathway and podocyte impairment in DN rats as well as the protective effect of calcitriol., Methods: SD rats were randomly divided into four groups: normal control (NC), normal treated with calcitriol (NC+VD), diabetic nephropathy (DN) and DN treated with calcitriol (DN+VD); all VD rats were treated with 0.1 μg/kg/d calcitriol by gavage. DN model rats were established by intraperitoneal injections of streptozotocin (STZ). Rats were sacrificed after 18 weeks of treatments., Results: In the present study, increased albuminuria was observed as early as 3 weeks of diabetes and continued to increase more than six-fold throughout the length of the study (18 weeks). Expectedly, animals receiving the treatment with calcitriol was protected from this increase, lower about one third. Meanwhile, the expression of podocyte specific markers, including nephrin and podocin, together with PI3K/p-Akt was significantly decreased in DN rats, whereas calcitriol reversed these above changes accompanied by elevated the expression levels of VDR. Additionally, a positive correlation was observed between the expression levels of nephrin and VDR (r = 0.776, P < 0.05). Likewise, the expression of nephrin was positively correlated with both PI3K-p85 and p-Akt (r = 0.736, P < 0.05; r = 0.855, P < 0.05, respectively)., Conclusion: PI3K/p-Akt signaling pathway participates in calcitriol ameliorating podocyte injury in DN rats. The manipulation of calcitriol might act as a promising therapeutic intervention for diabetic nephropathy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aim: Imbalanced M1/M2 macrophage phenotype activation is a key point in diabetic kidney disease (DKD). Macrophages mainly exhibit the M1 phenotype, which contributes to inflammation and fibrosis in DKD. Studies have indicated that autophagy plays an important role in M1/M2 activation. However, the mechanism by which autophagy regulates the macrophage M1/M2 phenotype in DKD is unknown. Thus, the aim of the present study was to explore whether high glucose-induced macrophages switch to the M1 phenotype via the downregulation of STAT-3-mediated autophagy. Methods: DKD model rats were established in vivo via the intraperitoneal injection of streptozocin (STZ). The rats were sacrificed at 18 weeks for histological and molecular analysis. RAW264.7 cells were cultured in vitro with 30 mM glucose in the presence or absence of a STAT-3 activator (colivelin) and an autophagy activator (rapamycin). Moreover, M1 and M2 macrophage activation models were established as a control group. Immunofluorescence and Western blot analyses were used to detect the expression of autophagy-related proteins (LC3 and Beclin-1), M1 markers (iNOS and CD11c) and M2 markers (MR and CD206). Results: In DKD, macrophages exhibit an M1 phenotype. Under high-glucose conditions, RAW264.7 macrophages switched to the M1 phenotype. Autophagy was downregulated in high glucose–induced M1 macrophages. Both the STAT-3 activator and the autophagy activator promoted the transition of glucose-induced M1 macrophages to M2 macrophages. Moreover, STAT-3 activation increased the expression of autophagy markers (LC3 and Beclin-1). However, the autophagy activator had no effect on STAT-3 phosphorylation. Conclusion: High glucose promotes macrophage switching to the M1 phenotype via the downregulation of STAT-3-mediated autophagy. [ABSTRACT FROM AUTHOR]

Macrophage filopodia, which are dynamic nanotube-like protrusions, have mainly been studied in the context of pathogen clearance. The mechanisms by which they facilitate intercellular communication and mediate tissue inflammation remain poorly understood. Here, we show that macrophage filopodia produce a unique membrane structure called "filopodial tip vesicle" (FTV) that originate from the tip of macrophages filopodia. Filopodia tip-derived particles contain numerous internal-vesicles and function as cargo storage depots via nanotubular transport. Functional studies indicate that the shedding of FTV from filopodia tip allows the delivery of many molecular signalling molecules to fibroblasts. We observed that FTV derived from M1 macrophages and high glucose (HG)-stimulated macrophages (HG/M1-ftv) exhibit an enrichment of the chemokine IL11, which is critical for fibroblast transdifferentiation. HG/M1-ftv induce renal interstitial fibrosis in diabetic mice, while FTV inhibition or targeting FTV IL11- alleviates renal interstitial fibrosis, suggesting that the HG/M1-ftvIL11 pathway may be a novel mechanism underlying renal fibrosis in diabetic nephropathy. Collectively, FTV release could represent a novel function by which filopodia contribute to cell biological processes, and FTV is potentially associated with macrophage filopodia-related fibrotic diseases. 8XMabVtTRQ4kBojsCRSFmY Video Abstract [ABSTRACT FROM AUTHOR]