Resveratrol alleviates the cytotoxicity induced by the radiocontrast agent, ioxitalamate, by reducing the production of reactive oxygen species in HK-2 human renal proximal tubule epithelial cells in vitro

Abstract

Radiocontrast-induced nephropathy (RIN) is one of the leading causes of hospital-acquired acute kidney injury (AKI). The clinical strategies currently available for the prevention of RIN are insufficient. In this study, we aimed to determine whether resveratrol, a polyphenol phytoalexin, can be used to prevent RIN. For this purpose, in vitro experiments were performed using a human renal proximal tubule epithelial cell line (HK-2 cells). Following treatment for 48 h, the highly toxic radiocontrast agent, ioxitalamate, exerted cytotoxic effects on the HK-2 cells in a concentration-dependent manner, as shown by MTT assay. The half maximal inhibitory concentration (IC50) was found to be approximately 30 mg/ml. Flow cytometry also revealed a marked increase in the number of apoptotic cells following exposure to ioxitalamate. In addition, the number of necrotic, but not necroptotic cells was increased. However, treatment with resveratrol (12.5 µM) for 48 h significantly alleviated ioxitalamate (30 mg/ml)-induced cytotoxicity, by reducing cytosolic DNA fragmentation, increasing the expression of the anti-apoptotic protein, Bcl-2 (B-cell lymphoma 2), and survivin, activating caspase-3, preventing autophagic death and suppressing the production of reactive oxygen species (ROS). Resveratrol also suppressed the ioxitalamate-induced formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage. N-acetylcysteine (NAC), a ROS scavenger commonly used to prevent RIN, also reduced ioxitalamate-induced cytotoxicity, but at a high concentration of 1 mM. Sirtuin (SIRT)1 and SIRT3 were not found to play a role in these effects. Overall, our findings suggest that resveratrol may prove to be an effective adjuvant therapy for the prevention of RIN.

Figure 1

Cytotoxic effects of ioxitalamate on HK-2 cells. The viability of HK-2 cells was measured by MTT assay following treatment with ioxitalamate for 48 h. ^***p<0.001.

Figure 2

Ioxitalamate-induced cell death patterns in HK-2 cells. Following 48 h of treatment with 30 mg/ml ioxitalamate, cell death patterns were determined by various methods. (A) PI and Annexin V staining of HK-2 cells was detected by flow cytometry. Ratio of (B) Annexin V-positive and (C) PI-positive cells by flow cytometry. (D) Representative western blots showing the expression levels of receptor-interacting protein kinase 3 (RIP3), a marker of necroptosis, examined following treatment with ioxitalamate for 24 and 48 h. (E) Compared to treatment with ioxitalamate alone, changes in cell viability were determined following 48 h of treatment with ioxitalamate plus necroptosis inhibitor necrostatin-1 (Nec-1) by MTT assay. (F) Autophagic flux by Cyto-ID^® dye was measured by flow cytometry. Immunofluorescent distribution shown by the solid and dashed line represents treatment with ioxitalamate and the control, respectively. (G) Compared to treatment with ioxitalamate alone, cell viability following 48 h of treatment with ioxitalamate plus the autophagy inducers, sirolimus or everolimus, was measured by MTT assay. ^*p<0.05, ^**p<0.01 and ^***p<0.005.

Figure 3

Resveratrol alleviates ioxitalamate-induced cytotoxicity in HK-2 cells. Changes in the viability of HK-2 cells following 48 h of treatment with ioxitalamate (30 mg/ml) plus resveratrol at 6.25, 12.5 or 25 μM were measured by MTT assay. ^**p<0.01 and ^***p<0.005.

Figure 4

Resveratrol suppresses ioxitalamate-induced apoptosis and autophagy in HK-2 cells. Following treatment with 30 mg/ml ioxitalamate for 48 h, the supernatants of both the culture medium and the cytoplasmic fraction of HK-2 cells were collected. (A) Increased expression in the cytoplasm alone indicates apoptosis, while (B) increased expression of DNA fragments in the medium indicates necrosis due to membrane rupture. (C) Representative western blots of caspase-3 in HK-2 cells showing the expression levels of caspase-3 examined following treatment with ioxitalamate (30 mg/ml) and/or resveratrol (12.5 μM) for 24 and 48 h. (D) Immunoreactivity of cleaved caspase-3 was increased following treatment with ioxitalamate for 24 and 48 h, and resveratrol suppressed caspase-3 expression. (E) Representative western blots of LC3B expression levels examined following treatment with ioxitalamate (30 mg/ml) and/or resvera-trol (12.5 μM) for 24 and 48 h. (E) Immunoreactivity of LC3B-II, the active form of autophagy, was increased following 48 h of treatment with ioxitalamate, and resveratrol diminished its expression. ^*p<0.05, ^**p<0.01 and ^***p<0.005.

Figure 5

Expression of anti-apoptotic proteins in HK-2 cells following treatment with ioxitalamate and/or resveratrol. (A) Representative western blots of survivin, Bcl-2, cIAP1, cIAP2 and Bcl-xL expression examined following 48 h of treatment with ioxitalamate and/or resveratrol. (B) Immunoreactivity of Bcl-2 was decreased 48 h following treatment with ioxitalamate, and resve-ratrol reversed its expression. (C) Immunoreactivity of survivin was decreased following treatment with ioxitalamate for 24 and 48 h, and resveratrol reversed its expression after 48 h of treatment. ^**p<0.01 and ^***p<0.005.

Figure 6

Resveratrol suppresses ioxitalamate-induced reactive oxygen species (ROS) production. (A) Expression of 2′,7′-dichlorodihydrofluores-cein (DCF), proportional to the ROS level within the cell cytosol, following 48 h of treatment with ioxitalamate and/or resveratrol (magnification, ×400). (B) Formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a biomarker of oxidative DNA damage following treatment with ioxitalamate and/or resveratrol. (C) Viability of HK-2 cells was measured by MTT assay following 48 h of treatment with ioxitalamate plus N-acetylcysteine (NAC). ^*p<0.05 and ^***p<0.005.

Figure 7

Role of sirtuin (SIRT)1 and SIRT3 in ioxitalamate-induced cytotoxicity. (A) Representative western blots of SIRT1 and phospho-SIRT1 were examined following 48 h of treatment with ioxitalamate and/or resveratrol. (B) Compared to treatment with ioxitalamate alone, changes in cell viability were examined following 48 h of treatment with ioxitalamate plus the SIRT1 inhibitor, EX-527, or the SIRT1 activator, SRT-1720, by MTT assay. ^**p<0.01 and ^***p<0.005.