The Effects of Sidt2 on the Inflammatory Pathway in Mouse Mesangial Cells

Abstract

In patients with chronic kidney disease, the abnormal activation of inflammatory pathways is usually an important factor leading to renal fibrosis and further deterioration of renal function. Finding effective intervention targets of the inflammatory signaling pathway is an important way to treat chronic kidney disease. As a newly discovered lysosomal membrane protein, the correlation between SID1 transmembrane family member 2 (Sidt2) and the inflammatory signaling pathway has not been reported. The aim of this study was to investigate the effect of Sidt2 on inflammation by inhibiting the expression of the Sidt2 gene in a mouse mesangial cell line mediated by a lentiviral CRISPR/Cas9 vector. Hematoxylin and eosin staining and microscopy found that the mesangial cells lost their normal morphology after inhibiting the expression of Sidt2, showing that the cell body became smaller, the edge between the cells was unclear, and part of the nucleus was pyknotic and fragmented, appearing blue-black. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 ^-/- group were higher than those of the Sidt2 ^+/+ group. p-Jak2 and IL6 increased in the Jak/Stat pathway, and p-ERK and p-P38 increased in the MAPK pathway. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the NF-κB pathway of the Sidt2 ^+/++LPS group were significantly higher than those in the Sidt2 ^+/+ group. The expressions of IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^-/- group. The expressions of p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^+/++LPS group. In the Jak/Stat pathway, the protein expressions of p-Jak2 and IL6 in the Sidt2 ^+/++LPS group were higher than those in the Sidt2 ^+/+ group. The expressions of p-Jak2 and IL6 in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^-/- group. The expressions of p-Jak2 and IL6 in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^+/++LPS group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the MAPK pathway in the Sidt2 ^+/++LPS group were higher than those in the Sidt2 ^+/+ group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^-/- group. The expressions of p-JNK, p-ERK, p-P38, and ERK in the Sidt2 ^-/-+LPS group were higher than those in the Sidt2 ^+/++LPS group. These data suggested that deletion of the Sidt2 gene changed the three inflammatory signal pathways, eventually leading to the damage of glomerular mesangial cells in mice.

Figure 1

Establishment of a Sidt2 knockout cell model and morphological observation. (a) The level of mRNA in the Sidt2^−/− group was significantly lower than that in the Sidt2^+/+ group. (b c) The expression of Sidt2 in the Sidt2^−/− group was significantly decreased by western blotting compared with that in the Sidt2^+/+ group. (d) Hematoxylin and eosin staining revealed different cellular states between the Sidt2^−/− group and the Sidt2^+/+ group. The cell body of the Sidt2^−/− group was significantly smaller than that of the Sidt2^+/+ group (arrow), and the nuclear pyknosis and fragmentation were blue-black (triangle). n ≥ 3, compared with the Sidt2^+/+ group, ∗p < 0.05 and ∗∗p < 0.01.

Figure 2

Inhibition of Sidt2 gene expression in mouse glomerular mesangial cells increased the level of inflammation. (a, b) Compared with the Sidt2^+/+ group, the protein expression of the NF-κB pathway including IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2^−/− group increased significantly. (c, d) The protein expression of the JAK/STAT pathway including p-Jak2 and IL6 in the Sidt2^−/− group was significantly higher compared with that in the Sidt2^+/+ group. (e, f) The protein expression of the MAPK pathway including p-ERK and p-p38 in the Sidt2^−/− group was significantly higher than that in the Sidt2^+/+ group. n ≥ 3, ∗p < 0.05, ∗∗p < 0.01.

Figure 3

SV40 MES 13 cells were treated with different concentrations of LPS; after treatment, western blot analysis was performed to measure the protein expression levels of NF-κB p65. n ≥ 3, ∗p < 0.05.

Figure 4

Under the stimulation of LPS, the deletion of Sidt2 gene leads to the activation of inflammatory pathway in SV40 MES 13 cells. (a, b) After being treated with LPS for 12 hours, the protein expressions of the NF-κB pathway including IKK β, p-IKK α/β, NF-κB p65, p-NF-κB p65, p-IκBα, IκBα, and TNF-α in the Sidt2^+/+ group, the Sidt2^+/++LPS group, the Sidt2^−/− group, and the Sidt2^−/−+LPS group were detected by western blotting. (c, d) After being treated with LPS for 12 hours, the protein expressions of the JAK/STAT pathway including p-Jak2, p-STAT3, and IL6 in the Sidt2^+/+ group, the Sidt2^+/++LPS group, the Sidt2^−/− group, and the Sidt2^−/−+LPS group were detected by western blotting. (e, f) After being treated with LPS for 12 hours, the protein expressions of the MAPK pathway including p-JNK, p-ERK, p-P38, and ERK in the Sidt2^+/+ group, the Sidt2^+/++LPS group, the Sidt2^−/− group, and the Sidt2^−/−+LPS group were detected by western blotting. n ≥ 3, ∗p < 0.05, ∗∗p < 0.01.