Microglia-Derived NLRP3 Activation Mediates the Pressor Effect of Prorenin in the Rostral Ventrolateral Medulla of Stress-Induced Hypertensive Rats

Abstract

Increased microglial activation and neuroinflammation within autonomic brain regions such as the rostral ventrolateral medulla (RVLM) have been implicated in stress-induced hypertension (SIH). Prorenin, a member of the brain renin-angiotensin system (RAS), can directly activate microglia. The present study aimed to investigate the effects of prorenin on microglial activation in the RVLM of SIH rats. Rats were subjected to intermittent electric foot-shocks plus noise, this stress was administered for 2 h twice daily for 15 consecutive days, and mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) were monitored. The results showed that MAP and RSNA were augmented, and this paralleled increased pro-inflammatory phenotype (M1) switching. Prorenin and its receptor (PRR) expression and the NLR family pyrin domain containing 3 (NLRP3) activation were increased in RVLM of SIH rats. In addition, PLX5622 (a microglial depletion agent), MCC950 (a NLRP3 inhibitor), and/or PRO20 (a (Pro)renin receptor antagonist) had antihypertensive effects in the rats. The NLRP3 expression in the RVLM was decreased in SIH rats treated with PLX5622. Mito-tracker staining showed translocation of NLRP3 from mitochondria to the cytoplasm in prorenin-stimulated microglia. Prorenin increased the ROS-triggering M1 phenotype-switching and NLRP3 activation, while MCC950 decreased the M1 polarization. In conclusion, upregulated prorenin in the RVLM may be involved in the pathogenesis of SIH, mediated by activation of the microglia-derived NLRP3 inflammasome. The link between prorenin and NLRP3 in microglia provides insights for the treatment of stress-related hypertension.

Keywords: Hypertension; Microglia; NLRP3; Prorenin; Stress.

Fig. 1

Expression of prorenin/PRR in the RVLM of rats. A qRT-PCR showing both prorenin and PRR are increased in the RVLM of SIH rats. B Representative western blots of prorenin and PRR in the RVLM of SIH rats. C, D Densitometric quantification showing prorenin and PRR are higher in the SIH group than in controls. Data are presented as the mean ± SEM. *P < 0.05 vs Ctrl, n = 6/group.

Fig. 2

Effects of intracisternal infusion of PRO20 on the MAP and RSNA in SIH rats. A Representative original traces demonstrating the effect of PRO20 infusion on RSNA, MAP, and HR (1, basal RSNA; 2, maximum RSNA; 3, noise level). The maximum occurred 1–2 min after the rat was euthanized. Note that the maximum RSNA did not significantly differ among the 3 groups, while the SIH/SIH + aCSF rats had a greater basal RSNA level than control and/or PRO20-treated SIH rats, and intracisternal infusion PRO20 attenuated the MAP and baseline RSNA level of SIH rats. B Statistical data for MAP. C Statistical data for baseline nerve activity. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs SIH. ABP, arterial blood pressure; MAP, mean arterial pressure; HR, heart rate; RSNA, renal sympathetic nerve activity; SIH, stress-induced hypertensive rats; aCSF, artificial cerebrospinal fluid.

Fig. 3

The ratio of M1/M2 polarization and release of pro-inflammatory/immune-regulatory factors in the RVLM of SIH rats. A Immunofluorescence detection of M1 and/or M2 phenotype microglia in the RVLM showed robust switching to M1 phenotype in SIH rats. B Relative ratio of M2 versus M1 phenotype microglia in the RVLM expressed as a percentage. C, D qRT-PCR showing the release of pro-inflammatory/immune-regulatory factors was increased in the RVLM of SIH rats. E, F The PRR antagonist, PRO20, decreased the M1 polarization in SIH rats. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs SIH; scale bars, 200 μm in (A) and (E).

Fig. 4

Expression of NLRP3 is downregulated by PRO20, a PRR antagonist. A Effect of PRO20 on the mRNA expression of NLRP3/ASC/caspase-1 using qRT-PCR analyses. B, C Representative immunoblots and quantitative analysis showing that NLRP3, ASC, caspase-1, and pro-IL-1β expression in the RVLM of SIH rats was upregulated, while PRO20 treatment downregulated their expression. D Representative images showing that NLRP3 (red) co-localized with the microglial marker (OX42). E, F Relative fluorescence intensity calculation of NLRP3-immnoreactivity with or without OX42-immnoreactivitiy in different groups. G–I Representative images and quantitative analysis showing co-localization of PGP 9.5 (neuronal marker, green) and NLRP3 (red) in the RVLM. J, K Representative images of double immunofluorescent staining for GFAP (astrocyte marker, green) and NLRP3 (red) and densitometric quantification of immunoreactivity in the RVLM. Notably, immunofluorescence staining analysis showed that NLRP3 mostly localized with microglia and neurons (arrows). Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs SIH; scale bars, 200 μm in (D), (G), and (J).

Fig. 4

Expression of NLRP3 is downregulated by PRO20, a PRR antagonist. A Effect of PRO20 on the mRNA expression of NLRP3/ASC/caspase-1 using qRT-PCR analyses. B, C Representative immunoblots and quantitative analysis showing that NLRP3, ASC, caspase-1, and pro-IL-1β expression in the RVLM of SIH rats was upregulated, while PRO20 treatment downregulated their expression. D Representative images showing that NLRP3 (red) co-localized with the microglial marker (OX42). E, F Relative fluorescence intensity calculation of NLRP3-immnoreactivity with or without OX42-immnoreactivitiy in different groups. G–I Representative images and quantitative analysis showing co-localization of PGP 9.5 (neuronal marker, green) and NLRP3 (red) in the RVLM. J, K Representative images of double immunofluorescent staining for GFAP (astrocyte marker, green) and NLRP3 (red) and densitometric quantification of immunoreactivity in the RVLM. Notably, immunofluorescence staining analysis showed that NLRP3 mostly localized with microglia and neurons (arrows). Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs SIH; scale bars, 200 μm in (D), (G), and (J).

Fig. 5

The NLRP3 inhibitor, MCC950, has an anti-hypertensive effect on SIH, which might be associated with microglia-derived NLRP3. A, B Representative immunoblots and densitometric analysis showing that NLRP3 expression is decreased in both MCC950 (NLRP3 inhibitor) and PLX5622 (CSF1R inhibitor)-treated SIH rats. C, D The efficiency of microglia elimination by PLX5622 evaluated by decreased microglia marker of Iba-1 using immunofluorescent staining. E SBP measurements showing that both MCC950 and PLX5622 had a depressor effect in SIH rats. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs SIH.

Fig. 6

NLRP3 mediates M1 polarization in prorenin-treated microglia. A Analysis of the expression profile of CD11b by flow cytometry confirming that the purity of isolated primary microglia was > 94%. B Representative fluorescence-activated cell sorting plots showing the M1 (CD86+) and M2 (CD206+) phenotypes by flow cytometry in Ctrl, prorenin, and prorenin + MCC950-treated microglia. C Statistical data showing the proportions of M1/M2 phenotypes in Ctrl, prorenin, and prorenin + MCC950-treated microglia. D qRT-PCR analysis showing that the pro-inflammatory factors TNF-α and IL-β are increased in prorenin-treated microglia, while co-treatment with prorenin and MCC950, an NLRP3 inhibitor, decreases the release of TNF-α and IL-β. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs prorenin.

Fig. 7

ROS mediate the prorenin-induced activation of the NLRP3 inflammasome. A Representative images of cytoplasmic ROS production in control, prorenin, and prorenin with NAC groups using DCFH-DA-associated fluorescence assays. B ROS quantification indicated by fluorescent intensity in the different groups. C Immunoblots of the components of the NLRP3 inflammasome (NLRP3, ASC, and caspase-1), pro-IL-1β, and its mature product IL-1β in prorenin with or without the ROS scavenger NAC. D Statistics for experiments as in C. E Caspase-1 activity. Data represent the mean ± SEM; n = 6, *P < 0. 05 vs Ctrl; ^#P < 0. 05 vs prorenin, one-way ANOVA with unpaired t test; scale bar, 15 μm.

Fig. 8

The increased NLRP3 inflammasome activation and NLRP3 translocation from cytoplasm to mitochondria are attenuated by co-treatment with prorenin and NAC. A Representative images of primary microglia co-stained with NLRP3 (red) and ACS (green). B Densitometric quantification of immunofluorescent staining for NLRP3 in different groups. C Co-localization of NLRP3 and ASC in control, prorenin, and prorenin plus NAC groups. D Representative images of primary microglia stained with NLRP3 (red) and mito-tracker (green). E Percentage co-localization of NLRP3 imunoreactivity with mitochondria. Data represent the mean ± SEM; n = 6; *P < 0.05 vs Ctrl; ^#P < 0.05 vs prorenin, one-way ANOVA with unpaired t test; scale bars, 15 μm in A and D.

Fig. 9

Schematic diagram illustrating the putative mechanisms of the pressor effect of prorenin via ROS-triggered microglia-derived NLRP3-IL-1β activation in the RVLM. PRR, (pro)renin receptor; ROS, reactive oxygen species; NAC, N-acetylcysteine; NLRP3, NLR family pyrin domain containing 3; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; IL-1β, interleukin-1β; BP, blood pressure; RSNA, renal sympathetic nerve activity.