Tubular epithelial cell-to-macrophage communication forms a negative feedback loop via extracellular vesicle transfer to promote renal inflammation and apoptosis in diabetic nephropathy

Abstract

Background: Macrophage infiltration around lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). However, how these two types of cells communicate remains obscure. We previously demonstrated that LRG1 was elevated in the process of kidney injury. Here, we demonstrated that macrophage-derived, LRG1-enriched extracellular vesicles (EVs) exacerbated DN. Methods: We induced an experimental T2DM mouse model with a HFD diet for four months. Renal primary epithelial cells and macrophage-derived EVs were isolated from T2D mice by differential ultracentrifugation. To investigate whether lipotoxic TEC-derived EV (EV_e) activate macrophages, mouse bone marrow-derived macrophages (BMDMs) were incubated with EV_e. To investigate whether activated macrophage-derived EVs (EV_m) induce lipotoxic TEC apoptosis, EV_m were cocultured with primary renal tubular epithelial cells. Subsequently, we evaluated the effect of LRG1 in EV_e by investigating the apoptosis mechanism. Results: We demonstrated that incubation of primary TECs of DN or HK-2 mTECs with lysophosphatidyl choline (LPC) increased the release of EV_e. Interestingly, TEC-derived EV_e activated an inflammatory phenotype in macrophages and induced the release of macrophage-derived EV_m. Furthermore, EV_m could induce apoptosis in TECs injured by LPC. Importantly, we found that leucine-rich α-2-glycoprotein 1 (LRG1)-enriched EV_e activated macrophages via a TGFβR1-dependent process and that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-enriched EV_m induced apoptosis in injured TECs via a death receptor 5 (DR5)-dependent process. Conclusion: Our findings indicated a novel cell communication mechanism between tubular epithelial cells and macrophages in DN, which could be a potential therapeutic target.

Keywords: death receptor 5; diabetic nephropathy; extracellular vesicles; interleukin; leucine-rich α-2-glycoprotein 1; tumor necrosis factor α; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1

Lipotoxicity induces the release of tubular epithelial cell (TEC)-derived EV_e in vivo and in vitro. (A) Blood glucose assay. (B) Kidney tissues stained with periodic acid-Schiff (PAS). (C) Urinary albumin (ALB) assay. (D) Urinary creatinine (Cr) assay. (E) Expression of KIM-1 was detected by western blot. (F) Kidney tissues stained with Oil Red O (ORO). (G) Primary TEC-derived EV_e of DN representative image with nanoparticle tracking analysis (NTA). (H) Transmission electron photomicrographs of primary TEC-derived EV_e of DN. (I) The expression of TSG101, ARF6, CD63 and calnexin in primary TEC-derived EV_e from DN patients was detected by western blot. Similar results were obtained in 3 independent experiments with 10 mice per group.

Figure 2

Lipotoxic TEC-derived EV_e activates macrophages. (A) Immunohistochemical staining of F4/80 in kidney tissues. Scale bar, 50 μM; magnification, 20 ×. (B) Mouse bone marrow-derived macrophages (BMDMs) were incubated with purified EV_e (10¹⁰/mL) isolated from DN mouse-derived TECs. (C) The levels of CXCL12, TNF-α, and IL-1β in BMDMs treated with primed TEC-derived EV_e from DN mice were analyzed by quantitative real-time PCR. (D) The levels of CXCL12, TNF-α, and IL-1β in BMDMs treated with lipotoxic mTEC-derived EV_e were analyzed by quantitative real-time PCR. (E) The levels of CXCL12, TNF-α, and IL-1β in THP-1 cells treated with lipotoxic HK-2-derived EV_e were analyzed by quantitative real-time PCR. (F) The levels of CXCL12, TNF-α, and IL-1β in THP-1 cells treated with lipotoxic HK-2-derived EV_e and boiled lipotoxic HK-2-derived EV_e analyzed by quantitative real-time PCR. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 3

LRG1-enriched EV_e-mediated macrophage activation is TGFβR1-dependent. (A) Representative transmission electron photomicrographs immunogold-labeled with an anti-LRG1 antibody of primary TEC-derived EV_e of DN. Scale bar: 100 nm. (B) Representative transmission electron photomicrographs immunogold-labeled with an anti-LRG1 antibody of lipotoxic HK-2-derived EV_e. Scale bar: 100 nm. (C) Expression of LRG1 in HK-2-derived EV_e and mTEC-derived EV_e treated with LPC was detected by western blot. (D) Effects of various concentrations of rhLRG1 on TNF-α mRNA levels in THP-1 cells analyzed by quantitative real-time PCR. (E) Effects of rhLRG1 (10 ng/mL) on CXCL12, TNF-α and IL-1β mRNA levels in THP-1 cells analyzed by quantitative real-time PCR. (F) THP-1 cells were differentiated using PMA treatment for 48 h. Thereafter, THP-1 cells were pretreated with cytochalasin D (1 μM) followed by 2 hours of incubation with EV_e (10¹⁰/mL) isolated from LPC-treated HK-2 cells. Prior to this incubation, EV_e were labeled using the fluorescent dye PKH67. EV_e uptake by macrophages was visualized using confocal microscopy and quantified. (G) Differentiated macrophage-like THP-1 cells were pretreated with cytochalasin D (1 μM) for 1 hour followed by 2 hours of incubation with lipotoxic HK-2-derived EV_e (10¹⁰/mL) for 2 hours. Effect of cytochalasin D on CXCL12, TNF-α and IL-1β mRNA levels in THP-1 cells analyzed by quantitative real-time PCR. (H) The efficiency of TGFβR1 knockdown in THP-1 cells analyzed by quantitative real-time PCR. (I) CXCL12, TNF-α and IL-1β mRNA levels in THP-1 cells treated with siRNA-TGFβR1 analyzed by quantitative real-time PCR. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 4

Lipotoxic TEC-derived, LRG1-enriched EV_e induce the release of macrophage-derived EV_m. (A) Representative images of BMDM-derived EV_m treated with primary TEC-derived EV_e from DN with nanoparticle tracking analysis (NTA). (B) Transmission electron photomicrographs of BMDM-derived EV_m treated with primary TEC-derived EV_e of DN. (C) Expression of TSG101, ARF6 and CD63 in BMDM-derived EV_m treated with primary TEC-derived EV_e from DN patients was detected by western blot. (D) Representative images of THP-1-derived EV_m treated with rhLRG1 with nanoparticle tracking analysis (NTA). (E) Transmission electron photomicrographs of THP-1-derived EV_m treated with rhLRG1. (F) The expression of TSG101, ARF6 and CD63 in THP-1-derived EV_m treated with rhLRG1 was detected by western blot. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 5

Activated macrophage-derived EV_m induce lipotoxic TEC apoptosis. (A) Representative images of TUNEL staining in various groups. Scale bar, 50 μm. (B) Tubular epithelial cells of DN were incubated with purified EV_m (10¹⁰/mL) isolated from DN mice-derived macrophages. (C) Flow cytometry analysis of primary TECs in each group. (D) Flow cytometry analysis of HK-2 in each group. (E) Flow cytometry analysis of mTEC in each group. (F) The activity of caspase-3 in HK-2 cells induced by THP-1-derived EV_m. (G) The activity of caspase-3 in mTECs induced by BMDM-derived EV_m. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 6

Activated macrophage-derived EV_m induce tubular epithelial cell apoptosis in a TRAIL-DR5-dependent manner. (A) HK-2 cells were pretreated with cytochalasin D (1 μM) followed by 2 hours of incubation with THP-1-derived EV_m (10¹⁰/mL). Prior to this incubation, EV_m were labeled using the fluorescent dye PKH67. EV_m uptake by HK-2 cells was visualized using confocal microscopy and quantified. (B) Flow cytometry analysis of HK-2 in each group. (C) The expression of DR5 in primary TECs was detected by western blot. (D) The expression of DR5 in HK-2 cells treated with LPC was detected by western blot. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 7

Activated macrophage-derived EV_m induces tubular epithelial cell apoptosis in a TRAIL-DR5-dependent manner. (A) Effect of primary TEC-derived EV_e on TRAIL mRNA levels in BMDMs analyzed by quantitative real-time PCR. (B) Effect of mTEC-derived EVs on TRAIL mRNA levels in BMDMs analyzed by quantitative real-time PCR. (C) The levels of CXCL12, TNF-α, IL-1β, TRAIL and calnexin in macrophage-derived EV_m of DN mice analyzed by western blot. (D) Representative transmission electron photomicrographs immunogold-labeled with an anti-TRAIL antibody of macrophage-derived, TRAIL-enriched EV_m of DN mice. Scale bar: 100 nm. (E) The expression of TRAIL in BMDM-derived EV_m treated with primary TEC-derived EV_e from DN mice was detected by western blot. (F) The expression of TRAIL in BMDM-derived EV_m treated with mTEC-derived EV_e from DN mice was detected by western blot. (G) Effect of rhLRG1 (10 ng/mL) on TRAIL mRNA levels in BMDMs analyzed by quantitative real-time PCR. (H) Effect of rhLRG1 (10 ng/mL) on TRAIL protein levels in BMDMs analyzed by western blot. (I) Flow cytometry analysis of HK-2 in each group. Similar results were obtained in 3 independent experiments or in triplicate culture assays.

Figure 8

Tubular epithelial cell-to-macrophage forms a feedback loop via extracellular vesicle transfer promotes renal inflammation and apoptosis in diabetic nephropathy.