iRhom2 loss alleviates renal injury in long-term PM2.5-exposed mice by suppression of inflammation and oxidative stress

Abstract

Particulate matter (PM2.5) is a risk factor for organ injury and disease progression, such as lung, brain and liver. However, its effects on renal injury and the underlying molecular mechanism have not been understood. The inactive rhomboid protein 2 (iRhom2), also known as rhomboid family member 2 (Rhbdf2), is a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells, and has been explored in the pathogenesis of chronic renal diseases. In the present study, we found that compared to the wild type (iRhom2^+/+) mice, iRhom2 knockout (iRhom2^-/-) protected PM_2.5-exposed mice from developing severe renal injury, accompanied with improved renal pathological changes and functions. iRhom2^-/- mice exhibited reduced inflammatory response, as evidenced by the reduction of interleukin 1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and IL-18 in kidney samples, which might be, at least partly, through inactivating TNF-α converting enzyme/TNF-α receptors (TACE/TNFRs) and inhibitor of α/nuclear factor κ B (IκBα/NF-κB) signaling pathways. In addition, oxidative stress was also restrained by iRhom2^-/- in kidney of PM_2.5-exposed mice by enhancing heme oxygenase/nuclear factor erythroid 2-related factor 2 (HO-1/Nrf-2) expressions, and reducing phosphorylated c-Jun N-terminal kinase (JNK). In vitro, blockage of HO-1 or Nrf-2 rescued the inflammatory response and oxidative stress that were reduced by iRhom2 knockdown in PM_2.5-incubated RAW264.7 cells. Similar results were observed in JNK activator-treated cells. Taken together, our findings indicated that iRhom2 played an essential role in regulating PM_2.5-induced chronic renal damage, thus revealing a potential target for preventing chronic kidney diseases development.

Keywords: Inflammation; JNK; Oxidative stress; PM2.5; Renal injury; iRhom2.

Graphical abstract

Fig. 1

The expression profiles of iRhom2 in PM_2.5-incubated different cell lines. Different cell lines, including RAW264.7, BMDM, HEK-293, MPC5 and HK-2, were treated with PM_2.5 at the described concentrations (0, 2.5, 5, 25, 50 and 100 μg/ml) for 24 h, followed by RT-qPCR analysis of (A) iRhom2 and (B) TACE. Different cell lines of RAW264.7, BMDM, HEK-293, MPC5 and HK-2 were treated with 100 μg/ml PM_2.5 for the exhibited time (0, 4, 8, 12, 24 and 48 h), followed by RT-qPCR analysis of (C) iRhom2 and (D) TACE. Data are represented as mean ± SEM (n = 8). ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 versus the Con group.

Fig. 2

iRhom2 suppression negatively regulates inflammatory response in RAW264.7 cells. (A) Luciferase reporter analysis with mouse RAW264.7 cells that were co-transfected with the indicated reporter plasmids plus empty vector (EV) or iRhom2 and then left untreated or treated with 100 μg/ml PM_2.5 for 24 h. (B) RT-qPCR analysis of IL-1β, IL-6, TNF-α and IL-18 in mouse RAW264.7 cells that were co-transfected with the indicated reporter plasmids plus EV or iRhom2 and then treated with or not 100 μg/ml PM_2.5 for 24 h. ^***p < 0.001 versus the EV/PM_2.5/- group; ⁺p < 0.05 and ⁺⁺p < 0.01 versus the EV/PM_2.5/+ group. Mouse RAW264.7 cells that were co-transfected with the indicated reporter plasmids plus EV or iRhom2 and then left untreated or treated with 100 μg/ml PM_2.5 for 24 h. (C) RT-qPCR analysis of the indicated inflammation-related genes in mouse RAW264.7 cells transfected with negative control (NC) siRNA or iRhom2 siRNA for 24 h before exposure to 100 μg/ml PM_2.5 for 24 h. ^##p < 0.01 and ^###p < 0.001. (D) WB analysis of iRhom2, p-IκBα and p-NF-κB in mouse RAW264.7 cells transfected with NC or iRhom2 siRNAs for 24 h before incubation with 100 μg/ml PM_2.5 for 24 h. ^***p < 0.001 versus the NC group; ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 versus the PM_2.5 group. Data are represented as mean ± SEM (n = 8).

Fig. 3

iRhom2 deficiency alleviates PM_2.5-induced renal dysfunction. (A) H&E, and Masson Trichrome staining of renal tissue sections from the indicated groups of mice. Quantification of (B) renal score and (C) collagen contents based on histological staining. (D) WB analysis of NPSH2. (E) Determination of serum BUN, proteinuria and KIM1. Data are represented as mean ± SEM (n = 8). ^**p < 0.01 and ^***p < 0.001 versus the iRhom2^+/+/Con group. ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 versus the iRhom2^+/+/ PM_2.5 group.

Fig. 4

iRhom2 deficiency alleviates PM_2.5-induced inflammation. (A) Measurements of TACE using IHC and RT-qPCR analysis. (B) Calculation of serum TNF-α, TNFR2 and TNFR1 using ELISA analysis. (C) RT-qPCR analysis of IL-1β, IL-6, and IL-18 in kidney. (D) IHC analysis of renal p-NF-κB. (E) WB analysis of p-IκBα and p-NF-κB in kidney. (F) WB analysis of iRhom2 mRNA levels in kidney of iRhom2^+/+ mice. Data are represented as mean ± SEM (n = 8). ^**p < 0.01 and ^***p < 0.001 versus the iRhom2^+/+/Con group. ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 versus the iRhom2^+/+/ PM_2.5 group.

Fig. 5

iRhom2 knockdown reduces oxidative stress and JNK activation in PM_2.5-induced RAW264.7 cells in vitro. Mouse RAW264.7 cells were transfected with NC or iRhom2 siRNAs for 24 h before incubation with 100 μg/ml PM_2.5 for 24 h. (A) Oxidative stress in cells were calculated through assessing cellular total ROS using DCF analysis, H₂O₂, MDA, XO, (B) SOD, and TAC levels. (C,D) WB analysis of HO-1, Nrf-2, Keap-1 and XO-1, as well as (E) phosphorylated JNK. Data are represented as mean ± SEM (n = 6). ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 versus the NC group; ⁺p < 0.05 and ⁺⁺p < 0.01 versus the PM_2.5 group.

Fig. 6

iRhom2 inhibition reduces oxidative stress and JNK activation in PM_2.5-induced renal injury in vivo. (A) Determination of oxidative tress-associated indexes, including total ROS, H₂O₂, MDA, and XO, as well as (B) anti-oxidant of SOD in renal of mice from the indicated groups. (C) WB analysis of kidney HO-1, Nrf-2, Keap-1, XO-1, and (D) phosphorylated JNK. (E) IHC analysis of phosphorylated JNK in renal tissue sections. Data are represented as mean ± SEM (n = 8). ^**p < 0.01 and ^***p < 0.001 versus the iRhom2^+/+/Con group. ⁺p < 0.05, ⁺⁺p < 0.01 and ⁺⁺⁺p < 0.001 versus the iRhom2^+/+/ PM_2.5 group.

Fig. 7

PM_2.5-induced renal injury via iRhom2-regulated oxidative stress and inflammation. (A) WB analysis of HO-1 in mouse RAW264.7 cells after 5 μM SnPP pre-treatment for 3 h. (B) WB analysis of Nrf-2 in mouse RAW264.7 cells after transfected with NC or Nrf-2 siRNA for 24 h. (C) WB analysis of phosphorylated JNK in mouse RAW264.7 cells after 5 μM ANI pre-treatment for 3 h. Mouse RAW264.7 cells were pre-treated with SnPP or ANS for 3 h, or with Nrf-2 siRNA for 24 h, and then were subjected to si-iRhom2 transfection for 24 h. Finally, all cells were incubated with or without 100 μg/ml PM_2.5 for 24 h. (D) RT-qPCR analysis of TACE, TNFR2, and TNFR1. (E) RT-qPCR analysis of inflammatory cytokines. (F) Assessments of DCF fluorescent intensity, H₂O₂ and SOD levels in cells. Data are represented as mean ± SEM (n = 6). ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001.

Fig. 8

Model showing the role of iRhom2 as a positive regulator of PM_2.5-induced renal injury. iRhom2 played an essential role in regulating the progression of renal injury in PM_2.5-exposed mice, most likely through activating TACE/TNFRs and IκBα/NF-κB signaling pathways to promote inflammation. In addition, iRhom2 inactivated HO-1/Nrf-2 pathway, whereas activated JNK expression to enhance oxidative stress, thus exacerbating renal injury.