Doxepin as OCT2 inhibitor ameliorates inflammatory response and modulates PI3K/Akt signaling associated with cisplatin-induced nephrotoxicity in rats

Abstract

Organic cationic transporter 2 (OCT2) was identified as the main transporter involved in the accumulation of cisplatin (CP) in the proximal tubular renal cells, resulting in nephrotoxicity. Doxepin (DOX) is a tricyclic agent with an inhibitory effect on OCT2. This study aimed to explore the possible mechanisms of the renoprotective role of DOX toward CP-induced nephrotoxicity. Rats were randomly divided into six groups: group 1, control; group 2, CP; groups 3, 4, and 5 were treated with graded doses of DOX (5, 10, and 20 mg/kg, respectively) intraperitoneally (ip) once daily for 10 consecutive days and group 6 was treated only with DOX (20 mg/kg). On the seventh day, a single injected dose of CP (10 mg/kg, ip) was given to the rats in groups 2-5. Seventy-two hours after CP injection, rats were sacrificed, and the kidneys were removed for histological and biochemical measurements. DOX ameliorated the CP-induced histopathological alterations. DOX significantly reduced the expression of OCT2, lipid peroxidation marker (MDA), and inflammatory cytokines, including TNF-α, IL-6, IL-1, IL-2, and IL-1β, and increased the activity of antioxidant enzymes. In addition, pre- and co-treatment with DOX significantly reduced the CP-mediated apoptotic effect by reducing the renal tissue expression of BAX and caspase-3 levels, upregulating the expression of Bcl-2, and modulating the phosphorylation of PI3K/Akt signaling cascade. DOX exerts a nephroprotective impact against CP-mediated nephrotoxicity via the inhibition of OCT2, suppression of inflammation, oxidative stress, and apoptotic markers, and modulation of PI3K/Akt signaling cascade.

Keywords: Apoptosis; Cisplatin; Doxepin; OCT2; PI3K/Akt.

Fig. 1

Microscopic examination of renal tissue specimens stained with hematoxylin and eosin. A Section taken from a rat in the CON group showing normal histological features of renal parenchyma (black star), different nephron segments with intact tubular epithelium lining (arrow), and intact vasculatures. B Section taken from a rat in the CP group showing diffuse necrotic and degenerative changes, including nephron tubular segments (red arrow) with many figures of nuclear pyknosis, occasional intraluminal eosinophilic casts (dashed arrow), moderate tubular dilatation (star), and focal interstitial inflammatory cell infiltrates (yellow arrow). C Section taken from a rat in the CP + DOX5 group showing minimal protective efficacy, with persistent records of degenerative changes in the tubular epithelium (red arrow), mild tubular dilatation (black star), and many congested and dilated intratubular BVs (red star). D Section taken from a rat in the CP + DOX10 group showing a better organization of histological features of renal parenchyma, with mild tubular degenerative changes (red arrow), many apparent intact nephron segments (black arrow), mild tubular dilatation (black star), and minimal interstitial inflammatory cell infiltrates. However, many congested and dilated intratubular BVs persisted (red star). E Section taken from a rat in the CP + DOX20 group showing more significant protective efficacy, with abundant records of almost intact renal parenchyma, apparent intact nephron segments (black arrow), mild focal records of degenerative changes and apoptotic body formation (red arrow), interstitial tissue, and normal vasculatures. CON, control; CP, cisplatin; DOX, doxepin (scale bar = 50µ = 40 ×)

Fig. 2

Effect of doxepin on organic cationic transporter 2 expression. A Protein expression of OCT2 transporters. B Quantitative analysis of OCT2 transporters. Data are presented as mean ± S.D. (n = 3). a, b, c, or d: significant difference between the CON, CP, DOX5, or DOX10 groups, respectively, at p < 0.05 using ANOVA, followed by the Tukey–Kramer test as a post-hoc test. CON, control; CP, cisplatin; DOX, doxepin; OCT2, organic cationic transporter 2

Fig. 3

Effect of doxepin on the phosphorylation of the PI3K/Akt signaling cascade against the nephrotoxic impact induced by cisplatin. A Protein expression of PI3K/Akt. B Quantitative analysis of PI3K/Akt. Data are presented as mean ± S.D. (n = 3). a, b, c, or d: Significant difference between the CON, CP, DOX5, or DOX10 groups, respectively, at p < 0.05 using ANOVA, followed by the Tukey–Kramer test as a post-hoc test. CON, control; CP, cisplatin; DOX, doxepin; Akt, protein kinase B; p-Akt, phosphor-Akt; PI3K, phosphatidylinositol 3-kinase

Fig. 4

Immunohistochemical staining of the effects of doxepin on cisplatin-induced apoptotic effects via the alteration of the expression of Bcl-2 family members. BAX expression was enhanced by the administration of CP (intense brown color), but it was reduced by the concomitant treatment with DOX, which is shown as a mild to moderate reduction in brown color (I). Conflicting findings about the Bcl-2 expression were revealed (II). The BAX/Bcl-2 ratio was increased in the CP group, and this rise was amended in DOX-co-treated groups (III). A Section taken from the CON group. B Section taken from the CP group. C Section taken from rats administrated with CP and co-treated with 5 mg/kg DOX. D Section taken from rats administrated with CP and co-treated with 10 mg/kg DOX. E Section taken from rats administrated with CP and co-treated with 20 mg/kg DOX. F Area percentage of immunostaining in the renal tissues of the different studied groups. Data are presented as mean ± S.D. (n = 5). a, b, c, or d: Significant difference between the CON, CP, DOX5, or DOX10 groups, respectively, at p < 0.05 using ANOVA, followed by the Tukey–Kramer test as a post-hoc test. CON, control; CP, cisplatin; DOX, doxepin; BAX, bcl-2-like protein 4; Bcl2, B-cell lymphoma

Fig. 5

Immunohistochemical staining of the effects of doxepin on cisplatin-mediated caspase-3 activation. A Section taken from the CON group showing minimal immunostaining. B Section taken from the CP group showing extensive immunostaining (brown color). C Section taken from rats administrated with CP and co-treated with 5 mg/kg DOX showing moderate immunostaining. D Section taken from rats administrated with CP and co-treated with 10 mg/kg DOX showing minimal immunostaining. E Section taken from rats administrated with CP and co-treated with 20 mg/kg DOX showing limited immunostaining. F Area percentage of immunostaining in the renal tissues of the different studied groups. Data are presented as mean ± S.D. (n = 5). a, b, c, or d: Significant difference between the CON, CP, DOX5, or DOX10 groups, respectively, at p < 0.05 using ANOVA, followed by the Tukey–Kramer test as a post-hoc test. CON, control; CP, cisplatin; DOX, doxepin