Isorhapontigenin Modulates SOX9/TOLLIP Expression to Attenuate Cell Apoptosis and Oxidative Stress in Paraquat-Induced Acute Kidney Injury

Abstract

Paraquat (PQ) is a widely used herbicide but can be lethal to humans. The kidney is vital for PQ elimination; therefore, explorations for therapeutic approaches for PQ-induced acute kidney injury (AKI) are of great significance. Here, the effects of a natural bioactive polyphenol isorhapontigenin (ISO) on PQ-AKI were investigated. In vitro experiments carried out in PQ-intoxicated rat renal tubular epithelial cells (NRK-52E) showed that ISO treatment inhibited PQ-induced cell apoptosis and oxidative stress, which was evidenced by the decreased proapoptotic proteins [cleaved caspase 3/9 and poly (ADP-ribose) polymerase (PARP)], the reduced oxidative stress indicators [reactive oxygen species (ROS), malondialdehyde (MDA), and lactate dehydrogenase (LDH) leakage], and the increased antioxidants [superoxide dismutase (SOD), nuclear factor E2-related factor 2 (NRF2), and oxygenase-1 (HO-1)]. Furthermore, 50 mg/kg ISO pretreatment before PQ administration significantly attenuated PQ-AKI in rats, as manifested by the improved renal tubule damage, the reduced serum and urine markers of kidney injury, and the inhibited cell apoptosis and oxidative stress in the renal cortex. Furthermore, expression of sex-determining region Y box 9 (SOX9) and Toll-interacting protein (TOLLIP) in NRK-52E cells and the renal cortex was significantly upregulated after ISO treatment. Overexpression of SOX9 increased TOLLIP transcription and attenuated PQ-induced apoptosis and oxidative stress, whereas knockdown of SOX9 impaired the protective effects of ISO on NRK-52E cells against PQ toxicity. In conclusion, the present study demonstrated that ISO modulated SOX9/TOLLIP expression to attenuate cell apoptosis and oxidative stress in PQ-AKI, suggesting the potential of ISO in treating PQ-poisoned patients.

Figure 1

Isorhapontigenin (ISO) alleviates paraquat- (PQ-) induced cell apoptosis. CCK-8 assays showed the cell viability of NRK-52E cells incubated with (a) different concentrations of ISO for 24 h or (b) ISO (5 μM or 10 μM) for 30 min followed by a 24 h coincubation with 300 μM PQ. (c, d) Flow cytometry analysis was performed to determine the percentage of apoptotic cells. (e) Western blot showed the expression of apoptosis-related proteins. (f) Cell death was analyzed via nuclear staining with Hoechst. (g) Quantitative analysis for apoptotic cells. The scale bar represents 50 μm; 400x magnification. Error bars represent standard deviations. ^∗P values < 0.05, ^∗∗P values < 0.01, and ^∗∗∗P values < 0.001.

Figure 2

Isorhapontigenin (ISO) ameliorates the paraquat- (PQ-) induced oxidative stress in NRK-52E cells. NRK-52E cells were pretreated with ISO (5 μM or 10 μM) for 30 min followed by a 24 h coincubation with 300 μM PQ. (a) The malondialdehyde (MDA) contents, (b) superoxide dismutase (SOD) activity, and (c) lactate dehydrogenase (LDH) leakage were determined using the commercially available kits. (d, e) Flow cytometry was performed to determine the reactive oxygen species (ROS) production in cells. The fluorescence intensity of 2′,7′-dichlorofluorescein (DCF) indicated ROS levels. (f) Protein levels of nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) were detected via Western blot. (g) The mRNA levels of HO-1 were measured via quantitative real-time PCR. Error bars represent standard deviations. ^∗∗P values < 0.01; ^∗∗∗P values < 0.001.

Figure 3

SRY-box transcription factor 9 (SOX9) regulates Toll-interacting protein (TOLLIP) expression and attenuates paraquat (PQ) toxicity to NRK-52E cells. NRK-52E cells were pretreated with ISO (5 μM or 10 μM) for 30 min followed by a 24 h coincubation with 300 μM PQ. The expression of SOX9 and TOLLIP was evaluated at (a, b) protein and (c) mRNA levels using Western blot and quantitative real-time PCR (qPCR). (d) A dual-luciferase reporter assay was performed to explore the effects of SOX9 on the activities of the TOLLIP promoter. (e, f) Verification of adenovirus- (Ad-) mediated overexpression of SOX9 in NRK-52E cells using Western blot and qPCR. Forty-eight hours after Ad transduction, cells were incubated with 300 μM PQ for 24 h. (g, h) Flow cytometry was performed to determine the cell apoptosis. Levels of (i) malondialdehyde (MDA) and (j) lactate dehydrogenase (LDH) were measured using the commercially available kits. TOLLIP expression was determined at the (k) protein and (l) mRNA levels using Western blot and qPCR. Error bars represent standard deviations. ^∗P values < 0.05, ^∗∗P values < 0.01, and ^∗∗∗P values < 0.001.

Figure 4

SRY-box transcription factor 9 (SOX9) and Toll-interacting protein (TOLLIP) participate in the pharmacological effects of isorhapontigenin (ISO). (a, b) Verification of adenovirus- (Ad-) mediated knockdown of SOX9 in NRK-52E cells using Western blot and quantitative real-time PCR (qPCR). Forty-eight hours after Ad transduction, cells were incubated with 300 μM PQ for 24 h. (c, d) Flow cytometry was performed to determine the cell apoptosis. Levels of (e) malondialdehyde (MDA) and (f) lactate dehydrogenase (LDH) were measured using commercially available kits. TOLLIP expression was determined at the (g) protein and (h) mRNA levels using Western blot and qPCR. Error bars represent standard deviations. ^∗P values < 0.05, ^∗∗P values < 0.01, and ^∗∗∗P values < 0.001.

Figure 5

Isorhapontigenin (ISO) attenuates renal injury in paraquat- (PQ-) acute kidney injury (AKI) rats. Rats received an intraperitoneal injection of 25 mg/kg or 50 mg/kg ISO once a day for 7 days before injection of PQ. (a) Hematoxylin and eosin (H&E) staining and (b) periodic acid-Schiff (PAS) staining showed pathological alterations in the renal cortex of rats. Scale bars represent 200 μm or 50 μm; 100x or 400x magnification. (c) Serum creatinine (Scr), (d) blood urea nitrogen (BUN), (e) urinary neutrophil gelatinase-associated lipocalin (NGAL), and (f) urinary proteins were evaluated using commercially available detection kits. Eight rats in each group. Error bars represent standard deviations. ^∗∗P values < 0.01; ^∗∗∗P values < 0.001.

Figure 6

Isorhapontigenin (ISO) alleviates cell apoptosis and oxidative stress in the renal cortex of paraquat- (PQ-) acute kidney injury (AKI) rats. (a) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining showed the apoptotic cells in the renal cortex. Scale bars represent 200 μm or 50 μm; 100x or 400x magnification. (b) Expression of cell apoptosis-related proteins in the renal cortex was detected using Western blot. (c) Malondialdehyde (MDA) content in the renal cortex was measured using the commercially available kit. (d) Protein levels of nuclear factor erythroid 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) were detected using Western blot. (e) The mRNA levels of HO-1 were measured using quantitative real-time PCR (qPCR). Eight rats in each group. Error bars represent standard deviations. ^∗∗∗P values < 0.001.

Figure 7

Isorhapontigenin (ISO) upregulates expression levels of SRY-box transcription factor 9 (SOX9) and Toll-interacting protein (TOLLIP) in the renal cortex of paraquat- (PQ-) acute kidney injury (AKI) rats. Immunohistochemistry staining for (a) SOX9 and (b) TOLLIP in the renal cortex. The scale bar represents 50 μm; 400x magnification. Expression levels of SOX9 and TOLLIP in the renal cortex were determined at (c, d) protein and (e) mRNA levels using Western blot and quantitative real-time PCR (qPCR). Error bars represent standard deviations. Eight rats in each group. ^∗∗P values < 0.01; ^∗∗∗P values < 0.001.