Neuropeptide Y protects kidney from acute kidney injury by inactivating M1 macrophages via the Y1R-NF-κB-Mincle-dependent mechanism

Abstract

Neuropeptide Y (NPY) is produced by the nerve system and may contribute to the progression of CKD. The present study found the new protective role for NPY in AKI in both patients and animal models. Interestingly, NPY was constitutively expressed in blood and resident kidney macrophages by co-expressing NPY and CD68+ markers, which was lost in patients and mice with AKI-induced by cisplatin. Unexpectedly, NPY was renoprotective in AKI as mice lacking NPY developed worse renal necroinflammation and renal dysfunction in cisplatin and ischemic-induced AKI. Importantly, NPY was also a therapeutic agent for AKI because treatment with exogenous NPY dose-dependently inhibited cisplatin-induced AKI. Mechanistically, NPY protected kidney from AKI by inactivating M1 macrophages via the Y1R-NF-κB-Mincle-dependent mechanism as deleting or silencing NPY decreased Y1R but increased NF-κB-Mincle-mediated M1macrophage activation and renal necroinflammation, which were reversed by addition of NPY or by silencing Mincle but promoted by blocking Y1R with BIBP 3226. Thus, NPY is renoprotective and may be a novel therapeutic agent for AKI. NPY may act via Y1R to protect kidney from AKI by blocking NF-κB-Mincle-mediated M1 macrophage activation and renal necroinflammation.

Keywords: AKI; Inflammation; Macrophage; Mincle; Neuropeptide Y; Y1R.

Figure 1

NPY is produced by macrophages but rapidly decreased in patients and mice with AKI. a. Serum levels of NPY by ELISA. Each dot represents one health individual or AKI patient. b. NPY-expressing macrophages (NPY+EMER1+) in the blood by flow cytometry. Each dot represents one health or AKI patient, and each bar is the mean ± SEM for groups of health people (n=6) or AKI patients (n=15). c. Serum levels of NPY from normal or AKI mouse. d. Serum levels of creatinine. e. Expression of renal NPY by western blotting. f. Tubular necrosis (*) by H&E staining, and NPY expression and F4/80+ macrophage infiltration in day 3 AKI kidneys by immunohistochemistry. g-i. Quantitative analysis of tubular necrosis, NPY expression, and F4/80+ macrophage infiltration in day 3 AKI kidneys. J. Tow-color immunofluorescence reveals NPY (green)-expressing F4/80+ macrophages (red) in the normal kidney are lost in the AKI kidney on day 3. k. Two-color flow cytometry detects a marked decrease in NPY-expressing macrophages in the AKI kidney on day 3. Each dot represents one mouse, and each bar is the mean ± SEM for groups of 5-8 mice (c-k). ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Ctrl group. Scale bar, 100 μm.

Figure 2

Mice lacking NPY develop much more severe AKI induced by cisplatin. a. H&E staining shows that mice lacking NPY are largely promoted tubular necrosis (*). b. Quantitative analysis of tubular necrosis. c, d. Serum creatinine and blood urea nitrogen (BUN). e. Renal iNOS+F4/80+ M1 macrophages detected by two-color flow cytometry. f. Renal mRNA expression of proinflammatory cytokines (IL-1β, IL-6, TNF-α and IL-10) by real-time PCR. g. Western blot analysis of renal iNOS expression. Each dot represents one mouse, and each bar is the mean ± SEM for groups of 6 mice. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 as indicated. Scale bar, 100 μm.

Figure 3

Mice lacking NPY develop much more severe AKI induced by IRI. a. H&E staining shows that mice lacking NPY are largely promoted IRI-induced tubular necrosis (*). b. Quantitative analysis of tubular necrosis. c, d. Serum creatinine and blood urea nitrogen (BUN). e f. Western blot analysis of renal Y1R and p-p65, iNOS, Mincle expression. g. Renal mRNA expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) by real-time PCR. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 as indicated. Scale bar, 100 μm.

Figure 4

Treatment with NPY protects against cisplatin-induced AKI in mice. a, d. H&E staining shows that treatment with exogenous NPY dose-dependently inhibits cisplatin-induced tubular necrosis (*). b, e. Immunohistochemistry detects that treatment with exogenous NPY dose-dependently inhibits F4/80+ macrophage infiltration in the AKI kidney. c. serum levels of creatinine. f. Two-color flow cytometry detects that treatment with NPY (50 μg/kg/day) inhibits iNOS+F4/80+ M1 macrophages infiltrating the AKI kidney. g. Real-time PCR shows that treatment with exogenous NPY dose-dependently upregulated renal Y1R but inhibits expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α). Each dot represents one mouse, and data represent the mean ± SEM for groups of 6 mice. NPY25, NPY50 and NPY100 indicate that the dose of NPY treatment is 25, 50 and 100 μg/kg/day, respectively. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. AKI without NPY treatment. Scale bar, 100 μm.

Figure 5

Cisplatin-induced loss of Y1R while enhancing NF-κB-Mincle signaling and M1 macrophage activation in the AKI kidney is largely enhanced in NPY KO mice but inhibited by treatment with exogenous NPY. a. Western blots reveal that cisplatin-induced AKI in NPY WT mice is associated with inhibition of Y1R but upregulation of NF-κB/p65-Mincle signaling, which is further enhanced in NPY KO mice. b, c. Two-color flow cytometry for Mincle+F4/80+ M1 macrophages infiltrating the AKI kidney of NPY WT or KO mice. d. Real-time PCR for renal Mincle mRNA expression in the AKI kidney of NPY WT or KO mice. e. Western blots show that treatment with NPY (50 μg/kg/day) for 3 days largely upregulates Y1R but blocks NF-κB/p65-Mincle signaling. f, g. Therapeutic effect of NPY on renal Mincle+F4/80+ M1 macrophages by two-color flow cytometry. h. Therapeutic effect of NPY on renal Mincle mRNA expression by real-time PCR. Each dot represents one mouse, and each bar is the mean ± SEM for groups of 5-6 mice. ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. AKI without treatment.

Figure 6

BMDMs lacking NPY results in a loss of Y1R but largely enhances LPS-induced activation of NF-κB/p65-Mincle signaling and M1 macrophage activation in vitro. a b. Western blots detect that BMDMs lacking NPY result in a loss of Y1R while largely promoting LPS (100ng/ml) -induced NF-κB/p65-Mincle signaling and activation of M1 macrophages (Mincle+ iNOS+), which are reversed by addition of NPY (0, 100, 200 mg/ml) in a dose-dependent manner. c, d. Real-time PCR shows that addition of NPY (0, 100, 200 mg/ml) to BMDMs dose-dependently inhibits LPS-induced loss of Y1R and upregulation of Mincle, IL-1β, IL-6, TNFα, and MCP-1 mRNA, which are largely reversed by silencing NPY. Each dot represents one independent experiment, and each bar is the mean ± SEM for 3-4 independent experiments. ^*P<0.05, ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. LPS-stimulated group.

Figure 7

Treatment with a Y1R antagonist BIBP 3226 abolishes the protective effect of NPY on cisplatin-induced AKI by inhibiting NPY-Y1R but largely enhancing NF-κB/p65-Mincle-Syk signaling in mice. a. H&E staining detects that treatment with a Y1R antagonist BIBP3226 (0, 100, 300 mg/kg/day) dose-dependently blocks the renoprotective effect of NPY (50 μg/kg/day) on cisplatin-induced tubular necrosis (*). b. Quantitative analysis of tubular necrosis and serum levels of creatinine. c, d. Two-color flow cytometry detects that treatment with a Y1R antagonist BIBP3226 (100 mg/kg/day) reverses the suppressive effect of NPY (50 μg/kg/day) on M1 macrophage (Mincle+iNOS+F4/80+) activation in cisplatin-induced AKI kidneys. e. Wester blots detect that treatment with BIBP 3226 (0, 100, 300 mg/kg/day) blocks NPY-Y1R but inhibits NF-κB/p65-Mincle-Syk signaling in the AKI kidney in a dose-dependent manner. Each dot represents one mouse, and data represent the mean ± SEM for groups of 5-6 mice. BI100 and BI300 indicate that the administration dose of BIBP3226 is 100 and 300 mg/kg/day, respectively. ^*P<0.05, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 as indicated. Scale bar, 50 μm.

Figure 8

Blockade of Y1R with BIBP 3226 blunts the inhibitory effect of NPY on LPS-induced NF-κB/p65-Mincle-Syk signaling and M1 macrophage activation by DMBMs in vitro. a. Western blots reveal that addition of BIBP 3226 (300 mg/ml) to BMDMs blocks NPY (200 mg/ml)-induced Y1R but enhances LPS (100 ng/ml)-induced activation of NF-κB/p65-Mincle-Syk signaling and M1 macrophages with high levels of Mincle and iNOS. b. Real-time PCR for expression of Y1R, Mincle, and IL-β by BMDMs treated with or without NPY and BIBP 3226. c, d. Two-color flow cytometry detects Mincle and iNOS-expressing macrophages (Mincle+iNOS+F4/80+) after treatment with or without NPY and BIBP 3226. Each dot represents one independent experiment, and each bar is the mean ± SEM for 3 independent experiments. BIBP, BIBP3226. ^**P<0.01, ^***P<0.001 vs. Ctrl group; ^#P<0.05, ^##P<0.01, ^###P<0.001 as indicated.