STAT1-p53-p21axis-dependent stress-induced progression of chronic nephrosis in adriamycin-induced mouse model

Abstract

Background: Chronic nephrosis (CN) is an aging-related disease with high mortality. Signal transduction and transcriptional activator 1 (STAT1) protein promotes senescence in human glomerular mesangial cells (HMCs), but whether it affects the progression of adriamycin (ADR)-induced CN in vivo remains unclear.

Methods: We established an ADR-induced CN mouse model that was completed in wild-type (wt) mice by a single intravenous injection of 10 mg/kg ADR for 2 or 4 weeks. Clinical indexes in each group were determined. Hematoxylin and eosin staining (H&E) was employed to determine renal histopathological damage, SA-β-gal staining was used to evaluate cell senescence phenotype. TUNEL and immunohistochemistry (IHC) staining were used to detect renal apoptosis. Protein levels of Bcl-2, Bax, STAT1, p53 and p21 were measured by Western Blot.

Results: STAT1 intervention ameliorated renal function. H&E staining indicated that STAT1-deficient (stat1^-/- ) improved the renal tubular injury, and stat1^-/- obviously inhibited the apoptosis and Caspase-3⁺ number in kidney tissues. Besides, stat1^-/- decreased proteinuria, and the levels of urea nitrogen and creatinine as well as that of reactive oxygen species induced by ADR. Also, stat1^-/- resulted in the reduced expression of p53 and p21.

Conclusions: Our current study strongly demonstrated the involvement of the STAT1-p53-p21 axis in the regulation of CN and is a potential target for the nephrosis treatment.

Keywords: Adriamycin; STAT1; chronic nephrosis (CN); mitogen-activated protein kinase; senescence.

Figure 1

stat1^−/− ameliorates renal injury in ADR-treated mice. (A) Relative mRNA level of expression of STAT1 was determined by RT-qPCR. (B) Representative kidney sections stained with H&E at the end of 2 weeks (upper) or 4 weeks (lower), original magnification at ×400. proteinuria, (D) serum creatinine and (E) urea nitrogen levels were detected. Data are presented as mean ± SD in three independent experiments. *, P<0.05, **, P<0.01 vs. control group; ^#, P<0.05, ^##, P<0.01 vs. ADR group. Scale bar =20 µm. ADR, normal wt mice treated with ADR; control, normal wt mice treated with normal saline.

Figure 2

stat1^−/− on senescence activation. (A) Senescence-associated β-galactosidase (SA-β-gal) staining of kidney tissues in the three groups (control, ADR and ADR + stat1^−/−). (B) Bar graph shows the percentage of SA-β-gal staining cells post 10 mg/kg ADR treatment. Data are presented as mean ± SDs in three independent experiments. *, P<0.05, **, P<0.01 vs. control group; ^##, P<0.01 vs. control group. Scale bar =20 µm. GSH, glutathione; MDA, malondialdehyde; ADR, normal wt mice treated with ADR; SOD, superoxide dismutase; control, normal wt mice treated with normal saline.

Figure 3

stat1^−/− mediates oxidative stress in ADR-treated mice. Post 10 mg/kg ADR treatment, the relative (A) ROS production, (B) SOD, (C) MDA and (D) GSH concentrations were detected at the end of 2 or 4 weeks. Data are presented as mean ± SD in three independent experiments. *, P<0.05, **, P<0.01 vs. control group; ^##, P<0.01 vs. ADR group. ADR, normal wt mice treated with ADR; control, normal wt mice treated with normal saline.

Figure 4

stat1^−/− aggravates renal apoptosis in ADR-treated mice. (A) Representative photomicrographs of TUNEL staining in the kidney at the end of 2 weeks (upper) and 4 weeks (lower)in control, ADR and ADR + stat1^−/− groups (original magnification at ×400), brown-staining cells were quantified as the apoptosis index. (B) Immunohistochemistry for expression of Caspase-3+ cells. (C) Relative level of protein expressions of Bcl-2 and Bax were determined by western blot. Data are presented as mean ± SD in three independent experiments. *, P<0.05, **, P<0.01 vs. control group; ^#, P<0.05, ^##, P<0.01 vs. ADR group. Scale bar =20 µm. ADR, normal wt mice treated with ADR; control, normal wt mice treated with normal saline.

Figure 5

stat1^−/− regulates the STAT1/p53/p21 axis. (A) Relative mRNA level of expression of STAT1, p53, and p21 were determined by RT-qPCR. β-actin was used as an internal control. Data are presented as the mean ± SD in three independent experiments. (B) Relative level of protein expressions of STAT1, p53, and p21 were determined by western blot. β-actin was used as loading control. Data are presented as mean ± SD in three independent experiments. *, P<0.05, **, P<0.01 vs. control group; ^##, P<0.01 vs. ADR group. ADR, normal wt mice treated with adriamycin; control, normal wt mice treated with normal saline.