Silencing HE4 alleviates the renal fibrosis in lupus nephritis mice by regulating the C3/MMPs/prss axis

Abstract

To explore the regulatory effect of human epididymis protein 4 (HE4) on renal fibrosis in mice with lupus nephritis (LN) and the underlying mechanism. Ten-week old MRL/LPR mice were injected with HE4 shRNA adenovirus vector through the renal pelvis for 5 days. Renal tissues were extracted for HE and Masson staining to evaluate pathological changes and fibrosis in lupus nephritis mice. The level of urine protein was measured using a biochemical analyzer, while the expression level of HE4 and p-NF-κB p65 in renal tissues was visualized using an immunofluorescence assay. The level of β2-microglobulin (β2-MG), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule 1 (Kim-1) was determined by the immunohistochemical assay. Western blotting was used to determine the levels of C3, HE4, matrix metalloproteinase-2 (MMP2), MMP9, p-p65, prss23, and prss35 in renal tissues. Compared to wild-type C57BL/6 mice, MRL/LPR mice showed a marked increase in the number of glomeruli, hyperplasic basement membrane, severe infiltration of inflammatory cells in renal tubules and glomeruli, obvious necrosis in glomeruli, elevated fibrosis levels, and increased levels of urine protein, β2-MG, NGAL, Kim-1, C3, HE4, MMP2, MMP9, and p-p65; and decreased levels of prss23 and prss35 were observed in MRL/LPR mice. After the administration of the HE4 shRNA adenovirus vector, the repaired structure of renal tubules and glomeruli improved infiltration of inflammatory cells, reduced collagen fiber and urine protein, suppressed levels of C3, HE4, MMP2, MMP9, and p-P65, and facilitated the expression of prss23 and prss35 which were observed. Silencing HE4 improved renal fibrosis and inhibited inflammation in mice with lupus nephritis, which may play a role in inhibiting C3/MMPs and promoting prss-related protein expression.

Keywords: Adenovirus; C3; HE4; MMPs; MRL/LPR mice.

Fig. 1

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. The expression level of HE4 in renal tissues was detected by the immunofluorescence assay (n=3, ×400, *p<0.05 vs. control, #p<0.05 vs. model, @p<0.05 vs. M+ NC)

Fig. 2

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. The location and expression of p-NF-κB p65 were determined by the immunofluorescence assay (n=3, ×400, *p<0.05 vs. control, # p<0.05 vs. model, @p<0.05 vs. M+ NC)

Fig. 3.

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. The pathological changes and fibrosis state in renal tissues were evaluated by HE staining and Masson staining assay, respectively (n=3, ×400)

Fig. 4.

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. The expression of β2-MG, NGAL, and Kim-1 in renal tissues was detected using the immunohistochemical assay (n=3, ×400, *p<0.05 vs. control, # p<0.05 vs. model, @p<0.05 vs. M+ NC)

Fig. 5.

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. A The urine protein level on days 0 and 5 was detected using the colorimetric method. B The UPCR value in renal tissues was determined (n=3, *p<0.05 vs. control, # p<0.05 vs. model, @p<0.05 vs. M+ NC)

Fig. 6.

MRL/LPR mice were injected with the adenovirus containing the shRNA-NC or HE4 shRNA. The protein level of C3, HE4, MMP2, MMP9, p-P65, prss23, and prss35 in renal tissues was determined by the Western blotting assay (n=3, *p<0.05 vs. control, #p<0.05 vs. model, @p<0.05 vs. M+ NC)