Effect of Thymoquinone on Renal Damage Induced by Hyperlipidemia in LDL Receptor-Deficient (LDL-R-/-) Mice

Abstract

Hyperlipidemia is a well-established risk factor for kidney injury, which can lead to chronic kidney disease (CKD). Thymoquinone (TQ) is one of the most active ingredients in Nigella sativa seeds. It has various beneficial properties, including antioxidant and anti-inflammatory activities. TQ also exerts positive effects on doxorubicin- (DOX-) induced nephropathy and ischemia-reperfusion-induced kidney injury in rats. Therefore, in this study, we investigated the possible protective effects of TQ against kidney injury in low-density lipoprotein receptor-deficient (LDL-R^-/^-) mice. Eight-week-old male LDL-R^-/^- mice were randomly divided into the following three groups: normal diet (ND group), high-fat diet (HFD group), and HFD combined with TQ (HFD+TQ group). The mice were fed the same diet for eight weeks. After eight weeks, we performed serological analysis of the mice in all three groups. We histologically analyzed the kidney tissue and also investigated the expression of proinflammatory cytokines in the kidney tissue. Metabolic characteristics, including total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and creatinine (CRE) levels, were lower in the LDL-R^-/^- HFD+TQ mice than in the HFD mice. Periodic acid-Schiff (PAS) and Masson's trichrome staining revealed excessive lipid deposition and collagen accumulation in the kidneys of the LDL-R^-/^- HFD mice, which were significantly reduced in the LDL-R^-/^- HFD+TQ mice. Furthermore, macrophages and levels of proinflammatory cytokines were lower in the kidney tissues of the LDL-R^-/^- HFD+TQ mice than in those of the LDL-R^-/^- HFD mice. Moreover, profibrosis- and oxidative stress-related protein expression was lower in the kidney tissues of the LDL-R^-/^- HFD+TQ mice than in those of the LDL-R^-/^- HFD mice. These results indicate that TQ may be a potential therapeutic agent for kidney damage caused by hyperlipidemia.

Figure 1

Metabolic data from the three mouse groups after eight weeks of consuming different diets. The kidney weight and levels of total cholesterol, low-density lipoprotein, and creatinine of the three mouse groups after eight weeks of different treatments are shown. Data represent the mean ± SEM, n = 3 per group. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone.

Figure 2

(a) H&E staining and immunohistochemical analysis of CD68 in mouse kidney tissues in the three treatment groups. Magnification: 40x. n = 3 per group. For HE staining, arrows indicate inflammatory cell infiltration. And arrows indicate CD68-positive cells for immunohistochemical analysis. (b) Bar graph showing the quantification of H&E staining- and CD68-positive cells. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group. (c) Relative mRNA expression levels of TNF-α, IL-6, and IL-1β in the mouse kidney in the three treatment groups after eight weeks of treatment. Data represent the mean ± SEM; n = 3 per group. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. H&E: hematoxylin and eosin; ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone; TNF-α: tumor necrosis factor α; IL: interleukin.

Figure 3

(a) Periodic acid-Schiff and representative immunohistochemistry staining for CD36 in mouse kidney tissue in the three treatment groups. Magnification: 40x. Arrows indicate positively stained cells. (b) Bar graph showing the quantification of Periodic acid-Schiff staining- and CD36-positive cells. n = 3, ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone.

Figure 4

(a) Masson's trichrome staining and IHC (Collagen I, Collagen III, MMP2, and MMP9) in mouse kidney tissues in the three treatment groups. Blue staining indicates collagen accumulation in Masson's trichrome staining. Magnification: 40x, n = 3. (b) Bar graph showing the quantification of Masson's trichrome staining-, Collagen I-, Collagen III-, MMP2-, and MMP9-positive cells. (c) Immunoblotting for analyzing MMP2 and MMP9 protein expression in renal tissue. (d) Bar graph depicting the semiquantification of MMP2 and MMP9 expressions. Data represent the mean ± SEM; n = 3 per group. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone; MMP: matrix metalloproteinase.

Figure 5

(a) Immunohistochemistry staining for NOX4, NRF2, and HO-1 in mouse kidney tissue in the three treatment groups. Magnification: 40x. Arrows indicate positively stained cells. (b) Bar graph showing the quantification of NOX4-, NRF2-, and HO-1-positive cells. n = 3, ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. (c) Immunoblotting for NOX4, CAT, NRF2, and HO-1 protein expressions in renal tissue. (d) Bar graph depicting the semi-quantification of NOX4, CAT, NRF2, and HO-1 expressions. Data represent the mean ± SEM; n = 3 per group. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group; ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone; NOX4: NADPH oxidase 4; CAT: catalase; NRF2: nuclear factor erythroid 2–related factor 2; HO-1: heme oxygenase-1.

Figure 6

PI3K protein expression in the mouse kidney tissue of the three treatment groups after eight weeks of treatment. (a) Immunoblotting analysis for PI3K protein expression in kidney tissues. (b) Bar graph depicting the semiquantification of PI3K expression. TQ suppressed the expression of PI3K protein in the LDL-R⁻/⁻ HFD mice. Data represent the mean ± SEM; n = 3 per group. ^∗P < 0.05 vs. the LDL-R⁻/⁻ HFD group. ^∗∗P < 0.01 vs. the LDL-R⁻/⁻ HFD group. PI3K: phosphoinositide 3-kinase; ND: normal control; HFD: high-cholesterol diet; TQ: thymoquinone.

Figure 7

Schematic diagram representing how TQ protects against hyperlipidemia-induced renal damage in mice. TQ: thymoquinone; NOX4: NADPH oxidase 4; CAT: catalase; NRF2: nuclear factor erythroid 2–related factor 2; HO-1: heme oxygenase-1; MMP: matrix metalloproteinase; TNF-α: tumor necrosis factor α; IL: interleukin; PI3K: phosphoinositide 3-kinase.