The DJ-1-Binding Compound Exerts a Protective Effect in Both In Vitro and In Vivo Models of Sepsis-Induced Acute Kidney Injury

Abstract

Although sepsis-induced acute kidney injury (AKI) is associated with significant morbidity and mortality, its treatment remains unresolved. Oxidative stress and inflammation are key elements in the pathomechanism of AKI. Therefore, in the present study, we investigated the role of DJ-1 protein, known for its antioxidant and anti-inflammatory properties in an animal model of lipopolysaccharide (LPS)-induced AKI. The presence of DJ-1 was detected by immunofluorescence staining in mice kidney samples, human embryonic kidney cells (HEK-293), and peripheral blood mononuclear cells (PBMCs). To investigate DJ-1 functions, Compound-23, a specific DJ-1-binding and preserving compound (CAS: 724737-74-0), was used in vitro and in vivo. Compound-23 reduced the H₂O₂-induced reactive oxygen species (ROS) production of the HEK-293 cells, and their LPS- or H₂O₂-induced death, as well. In accordance, Compound-23 decreased the mRNA expression of the oxidative stress markers NAD(P)H quinone dehydrogenase 1 (NQO1) and glutamate-cysteine ligase (GCLC) in the LPS-treated, and NQO1 in the H₂O₂-treated cells. Moreover, Compound-23 reduced the H₂O₂- and LPS-induced mRNA expression of inflammatory cytokine interleukin 6 (IL6) in both HEK-293 and PBMCs. Using the mice model of LPS-induced AKI, we demonstrated that Compound-23 treatment improved the renal functions of the mice. In addition, Compound-23 decreased the renal mRNA expression of kidney injury molecule 1 (Kim1), neutrophil gelatinase-associated lipocalin (Ngal), Nqo1, Gclc, and Il6 in the LPS-treated mice. Our study revealed that compounds protecting DJ-1 functions may protect the kidney from LPS-induced damage, suggesting that DJ-1 could be a potential drug target for sepsis-induced AKI therapy.

Keywords: DJ-1; PARK7; acute kidney injury; antioxidant; drug development; inflammation; oxidative stress; sepsis.

Figure 1

Presence of DJ-1 in the mice kidney samples, HEK-293 cells, and PBMCs. The presence of DJ-1 (green) was determined by immunofluorescent staining of the control kidney, human embryonic kidney cell line (HEK-293), and peripheral blood mononuclear cells (PBMCs). Cell nuclei were counterstained with Hoechst 33,342 (blue). Scale bar: 100 μm.

Figure 2

The effect of DJ-1-binding Compound-23 on oxidative-stress-induced cytotoxicity of HEK-293 cells. The effect of Compound-23 on the proliferation was investigated by MTT treated with Compound-23 (0.001 μM–1 μM) (A) for 24 h. HEK-293 cells were treated with LPS (1 µg/mL) (B,D,F,H,J) or H₂O₂ (100µM) (C,E,G,I,K) in the absence or presence of Compound-23 (0.1 μM) for 24 h. The effect of Compound-23 on intracellular reactive oxygen production was determined by DCFDA assay (B,C). The effect of Compound-23 on the viability of the HEK-293 cells was determined by LDH assay (D,E). The effect of Compound-23 on the mRNA expression of NQO1, GCLC, and IL6 was determined by real-time PCR relative to with GAPDH expression (F–K). Data were normalized and presented as the ratio of the mean values of the control group. Values were expressed as mean ± SD. Dots represent individual values (n = 5–6). ^# p < 0.05 vs. control; ^## p < 0.05 vs. LPS or H₂O₂.

Figure 3

The effect of DJ-1-binding Compound-23 on proinflammatory cytokine production of PBMCs. PBMCs were treated with LPS (0.01 ng/mL) (A) or H₂O₂ (100 µM) (B) in the absence or presence of Compound-23 (0.1 μM) for 24 h. The effect of Compound-23 on the mRNA expression of IL6 was determined by real-time PCR (n = 5–6) relative to GAPDH expression. Data were normalized and presented as the ratio of the mean values of the control group. ^# p < 0.05 vs. control; ^## p < 0.05 vs. LPS or H₂O₂.

Figure 4

Effect of DJ-1-binding Compound-23 (20 mg/kg) on the LPS (5 mg/kg)-induced acute kidney injury (AKI). The protein level (A) of DJ-1 (20 kDa) in the kidney was determined by Western blot in comparison with GAPDH (36 kDa). The serum level of creatinine (B) and blood urea nitrogen (BUN) (C) were determined by enzyme assays. The renal mRNA expression of Kim1 (D), Ngal (E) was determined by real-time PCR relative to Gapdh expression. Data were normalized and presented as the ratio of the mean values of the control group. Values were expressed as mean ± SD. Dots represent individual values in each group (n = 3–8); ^# p < 0.05 vs. Control; ^## p < 0.05 vs. LPS.

Figure 5

The effect of DJ-1-binding Compound-23 (20 mg/kg) on antioxidant and proinflammatory markers of kidney injury in the LPS (5 mg/kg)-induced acute kidney injury (AKI) model. The renal mRNA expression of Nqo1 (A), Gclc (B), and Il6 (C) was determined by real-time PCR relative to Gapdh expression. Data were normalized and presented as the ratio of the mean values of the control group. Values were expressed as mean ± SD. Dots represent individual values in each group (n = 6–8); ^# p < 0.05 vs. Control; ^## p < 0.05 vs. LPS.

Figure 6

Compound-23 protects the antioxidant function of DJ-1 in the kidney, thereby reducing reactive oxygen species (ROS) formation. Reduction in ROS indirectly prevents the activation of the NRF2 pathway, as ROS are the primary inducers of NRF2 activation. Consequently, without NRF2 activation, the expression of the antioxidant genes NQO1 and GCLC remains lower. Moreover, treatment with Compound-23 reduces ROS-and LPS-induced apoptosis and IL-6 production in renal epithelial cells. The protective effect of Compound-23 on DJ-1 leads to less severe kidney damage in sepsis-induced acute kidney injury. Abbreviations: DJ-1: PARK7/Protein Deglycase DJ-1; ROS: reactive oxygen species; NRF2: NF-E2 related factor-2; NQO1: NAD(P)H quinone oxidoreductase 1 (NQO1); GCLC: γ-glutamylcysteine synthetase (GCLC); IL-6: interleukin-6.