Interferon-gamma plays protective roles in sodium arsenite-induced renal injury by up-regulating intrarenal multidrug resistance-associated protein 1 expression

Abstract

Subcutaneous injection of sodium arsenite (NaAs, 12.5 mg/kg) into BALB/c [wild-type (WT)] mice causes acute renal dysfunction characterized by severe hemorrhages, acute tubular necrosis, and cast formation, with increases in serum blood urea nitrogen and creatinine levels. Concomitant enhancement in intrarenal interferon (IFN)-gamma expression prompted us to examine its roles in this pathology. IFN-gamma-deficient (IFN-gamma-/-) mice exhibited higher serum blood urea nitrogen and creatinine levels and exaggerated histopathological changes, compared with WT mice. Eventually, IFN-gamma-/- mice exhibited a high mortality (87.5%) within 24 hours after NaAs challenge, whereas most WT mice survived. The intrarenal arsenic concentration was significantly higher in IFN-gamma-/- mice later than 10 hours after NaAs treatment, with attenuated intrarenal expression of multidrug resistance-associated protein (MRP) 1, a main transporter for NaAs efflux, compared with WT mice. NF-E2-related factor (Nrf) 2 protein, a transcription factor crucial for MRP1 gene expression, was similarly increased in the kidneys of both strains of mice after NaAs treatment. In contrast, the absence of IFN-gamma augmented transforming growth factor-beta-Smad3 signal pathway and eventually enhanced the expression of activating transcription factor 3, which is presumed to repress Nrf2-mediated MRP1 gene expression. Thus, IFN-gamma can protect against NaAs-induced acute renal injury, probably by maintaining Nrf2-mediated intrarenal MRP1 gene expression.

FIGURE 1

Analysis on IFN-γ expression in the kidney of WT mice. a: Intrarenal IFN-γ contents were determined at the indicated time intervals after NaAs challenge as described in Materials and Methods. All values represent means ± SEM (n = 6 animals). **P < 0.01, *P < 0.05; NaAs-treated WT mice versus control mice. b: Immunohistochemical detection of IFN-γ in the kidneys of WT mice after NaAs challenge. Representative results from six individual animals are shown here. At 1 hour after NaAs challenge, IFN-γ protein was immunohistochemically detected in most renal tubular cells and some interstitial cells (arrows). c: In situ hybridization analysis to detect IFN-γ mRNA in the kidneys of WT mice at 1 hour after NaAs challenge. In situ hybridization analysis was performed using either anti-sense (c) or sense probe (d) as described in Materials and Methods. Representative results from six independent experiments are shown here. Original magnifications, ×200.

FIGURE 2

Analysis of NaAs-induced renal injury in WT and IFN-γ^−/− mice. a: Survival rates of WT (n = 16 animals) and IFN-γ^−/− mice (n = 16 animals) after administration of NaAs (12.5 mg/kg). b and c: Determination of serum BUN (b) and serum CRE (c) levels in WT (open bars) and IFN-γ^−/− mice (closed bars) at indicated time intervals after NaAs challenge. All values represent means ± SEM (n = 15). *P < 0.05, **P < 0.01 WT versus IFN-γ^−/− mice. d–i: Histopathological observations of the kidneys from WT (d–f) and IFN-γ^−/− mice (g–i). Representative results from six individual animals are shown here. The specimens were obtained from mice at 10 hours after NaAs challenge and were stained with H&E (d, e, g, h) or PAS (f, i). In IFN-γ^−/− mice, massive tubular necrosis with cast formation and severe hemorrhages (g and h), and disappearance of PAS-positive brush borders (i) were observed. In contrast, the histopathological changes were less evident in WT mice, compared with IFN-γ^−/− mice (d, e), and PAS-positive brush borders were retained in WT mice (f).

FIGURE 3

Arsenic contents in the kidney after NaAs challenge. Arsenic contents in the kidneys from WT (open bars) and IFN-γ^−/− (filled bars) mice were analyzed by atomic absorption spectrometry at 3, 6, 10, 18, and 24 hours after NaAs challenge (12.5 mg/kg) and expressed as the arsenic amount (μg) divided by the sample weight (g). All values represent means ± SEM (n = 6 animals). **P < 0.01, WT versus IFN-γ^−/− mice.

FIGURE 4

a and b: MRP1 mRNA expression in the kidneys of WT (open bars) and IFN-γ^−/− mice (filled bars) were determined by RT-PCR at 3, 6, and 10 hours after NaAs challenge. Representative results from six independent experiments are shown in a. The ratios of MRP1 to β-actin were calculated and are shown in b (open bars, WT; filled bars, IFN-γ^−/−). All values represent means ± SEM (n = 6 animals). *P < 0.05, WT versus IFN-γ^−/− mice. c and d: MRP1 protein expression in kidneys of WT and IFN-γ^−/− mice. Western blotting analysis was performed on the membrane-rich fraction obtained from the kidney tissues as described in Materials and Methods. Under the used condition, MRP1 protein was faintly detected in the kidneys of untreated WT and IFN-γ^−/− mice. Western blotting analysis using anti-β-actin antibody confirmed that an equal amount of protein was loaded onto each lane. Representative results from six individual animals in each group are shown in c. The ratios of MRP1 protein to β-actin protein were calculated and are shown in d (open bars, WT; filled bars, IFN-γ^−/−). All values represent means ± SEM (n = 6 animals). *P < 0.05, WT versus IFN-γ^−/− mice. e: Immunohistochemical detection of MRP1 protein in the kidneys before, at 6 hours, or 10 hours after NaAs challenge. Representative results from six individual animals in each group are shown here. Original magnifications, ×200.

FIGURE 5

a–e: Western blotting analysis to detect TGF-β, phosphorylated Smad3, ATF3, and Nrf2 proteins in the kidney. Under the conditions used, these molecules were detected faintly but to a similar extent, in the kidneys of untreated WT and IFN-γ ^−/− mice. Western blotting analysis using anti-β-actin antibody confirmed that an equal amount of protein was loaded onto each lane. Representative results from six individual animals in each group are shown in a. The ratios of each molecule to β-actin were calculated and are shown in b to e (open bars, WT; filled bars, IFN-γ^−/−). All values represent means ± SEM (n = 6 animals). **P < 0.01; *P < 0.05, WT versus IFN-γ^−/− mice. f: The association of ATF3 protein with Nrf2 proteins. Cell extracts of the kidneys were immunoprecipitated with anti-Nrf2 antibody, followed by Western blotting analysis using anti-ATF3 antibody. Representative results from six individual animals are shown here (6 hours after NaAs challenge).

FIGURE 6

Effects of the various combinations of NaAs, IFN-γ, and TGF-β on MRP1, ATF3, and Nrf2 protein expression in mProx24 cells. The membrane-rich fractions were obtained from mProx24 cells at 6 hours after the treatment. Western blotting analysis was performed as described in Materials and Methods. Western blotting analysis using anti-β-actin antibody confirmed that an equal amount of protein was loaded onto each lane. Representative results from six independent experiments are shown here.

FIGURE 7

The effects of anti-IFN-γ antibody (a, b) or exogenous IFN-γ (c, d) on serum BUN levels and intrarenal MRP1 expression. WT mice were treated with anti-IFN-γ antibody or recombinant IFN-γ, as described in Materials and Methods. a and c: Serum BUN levels were determined at 6 and 10 hours after NaAs challenge. All values represent means ± SEM (n = 8). *P < 0.05, control IgG versus anti-IFN-γ antibody, **P < 0.01; vehicle versus recombinant IFN-γ. b and d: Intrarenal MRP1 expression was examined at 10 hours after NaAs challenge, as described in Materials and Methods. Western blotting analysis using anti-β-actin antibody confirmed that an equal amount of protein was loaded onto each lane. Representative results from six independent experiments are shown here.