CoQ₁₀ was purchased from Kaneka Nutrients (Pasadena, TX, USA). Trisodium glycyrrhizinate hydrate was purchased from Tokyo Chemical Industry Co. LTD. (Tokyo, Japan). Eicosapentaenoic acid (EPA) was purchased from Phycoil Biotech Korea, Inc. (Seoul, Korea). EPA was used to improve micelle stability because glycyrrhizin alone is not enough to maintain the micelle. In addition, EPA itself reduces the inflammatory response by inhibiting prostaglandin synthesis. To produce a uniform nano-emulsion (Qmicelle), the following pretreatment is essential. Coenzyme Q₁₀, EPA, and trisodium glycyrrhizinate hydrate (at a concentration of 1 mg/mL each) were dispersed in hot water the mixture was pre-homogenized at 10,000 rpm with a homogenizer (Multi-Purpose Homogenizer, YSTRAL, Ballrechten-Dottingen, Germany) until a homogeneous consistency was obtained. An APV2000 microfluidizer processor (SPX Flow, Leeds, UK) was used to produce nano-emulsions (Qmicelle). It has a reservoir capacity of 1,500 mL and can be operated at pressures of up to 2,000 bars. The prepared mixture at 60°C was poured into the fluidizer and Qmicelle was obtained after 10 cycles at a pressure of 1,200 bars. Each component under EPA, CoQ₁₀, and trisodium glycyrrhizinate hydrate, showed single peaks at retention times of 6.0, 6.6, and 15.9 minutes, respectively, and their concentrations were 0.24, 1.0, and 0.6 mg/mL. We detected homogeneity and size distribution by dynamic light scattering (DLS), and conformed that sizes of the Qmicelles were uniform and distributed in a narrow range around 100 nm.

All animal experiments in this study were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, Catholic University of Korea (CUMC-2018-0340-01), and all procedures in this study were performed in accordance with the ethical guidelines for animal studies. Eight-week-old male Sprague Dawley rats (Charles River Technology, Seoul, Korea) that initially weighed 220 to 230 g were housed in cages (Nalge Co., Rochester, NY, USA) in a temperature- and light-controlled environment at the Catholic University of Korea’s animal care facility. The rats received a low-salt diet (0.05% sodium; Teklad Premier, Madison, WI, USA).

The primary goals of this study were to determine if CoQ₁₀-W has better protective effects than CoQ₁₀-L in Tac-induced renal injury. After acclimation for 1 week, weight-matched rats were randomized into four groups, nine per group, as follows.

We decided on the route of administration and the concentration based on previous studies [16,17].

Rats were pair-fed, and their body weight was monitored daily. After the 4-week treatment period, animals were housed individually in metabolic cages, and their water intake and urine volume were measured over 24 hours. On the following day, animals were anesthetized, and blood samples and tissue specimens were obtained for further analysis. Serum creatinine was measured using a quantitative enzyme colorimetric method (Stanbio Laboratory, Boerne, TX, USA).

Tac level in whole-blood was measured using liquid chromatography-tandem mass spectrometry (Abbott Diagnostics, Abbott Park, IL, USA). Content of CoQ₁₀ in plasma was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis [18].

Immunohistochemistry was performed as described previously [2]. Oxidative damage markers (8-hydroxy-2′-deoxyguanosine [8-OHdG] and 4-hydroxy-2-hexenal [4-HHE]), and pro-inflammatory marker (ED-1) were detected in 4-m tissue sections by incubating the cells for 12 hours with specific antibodies against 8-OHdG (JaICA), 4-HHE (JaICA, Shizuoka, Japan), and ED-1 (AbDSerotec, Oxford, UK) at 4°C. The TdT-mediated dUTP nick-end labeling (TUNEL) staining was performed and evaluated using the In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) as per the manufacturer’s protocols. All data were analyzed using a color image analyzer (TDI Scope Eye version 3.6 for Windows, Seoul, Korea). Each section was counted in 20 different fields at 200× magnification.

Immunoblot analysis was performed as described previously [19,20]. Using tissue lysates from the renal cortex, E-cadherin (BD Biosciences, San Jose, CA, USA), α-smooth muscle actin (α-SMA), collagen type IV, active caspase-3, BCL2-associated X (BAX), and β-actin were detected by incubating for 12 hours with specific antibodies against E-cadherin (BD Biosciences), α-SMA (Sigma, St Louis, MO, USA), collagen type IV (Abcam, Cambridge, UK), active caspase-3 (Millipore), Bax (Delta Biolabs, Gilroy, CA, USA), and β-actin (Sigma) at 4°C.

Histological assessment of tubule interstitial fibrosis (TIF) in trichrome-stained tissue sections was performed as described previously [21]. The extent of fibrosis was estimated by counting the percentage of injured area per field using a polygon program. A minimum of 20 fields per section was assessed and graded using color image analyzer (TDI Scope Eye version 3.0 for Windows, Olympus, Tokyo, Japan). Histopathological analyses were performed in randomly selected cortical fields of sections by a pathologist blinded to the identity of the treatment groups.

All procedures strictly followed the protocol described in the enzyme linked immunosorbent assay (ELISA) kit (Cell Biolabs, San Diego, CA, USA).

Human kidney 2 (HK-2) cells were seeded into 96-well plates at a density of 2.5 × 10⁴ cells/well. After pre-incubation in an incubator at 37°C for 24 hours, the culture medium was changed to serum-free medium containing Tac (50 μg/mL) and CoQ₁₀-L or CoQ₁₀-W (1, 10, 100 pg/mL; 1, 10, 100 ng/mL; and 1 μg/mL). Cell viability was assayed using a Cell Counting Kit-8 assay kit (Dojin Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol.

Flow cytometry was performed to assess reactive oxygen species (ROS) production. HK-2 cells were seeded into 6-well plates at a density of 2.5 × 10⁵ cells/well. After pre-incubation in an incubator at 37°C for 24 hours, the culture medium was changed to serum-free medium containing Tac (50 μg/mL) and CoQ₁₀-L or CoQ₁₀-W (1, 10, 100 pg/mL; 1, 10, 100 ng/mL; and 1 μg/mL). The evaluation method was performed as described previously [22].

All data are presented as the mean ± standard error (SE) of more than three independent experiments. Multiple comparisons between different groups were identified by one-way analysis of variance with Bonferroni post hoc tests using IBM SPSS Statistics version 24 (IBM, Armonk, NY, USA). Results with p values less than 0.05 were considered significant.

The concentration of CoQ₁₀ in plasma (Fig. 1A) in the VH and Tac groups was 57 ± 19 and 33 ± 10 ng/mL, respectively (p < 0.05). However, the CoQ₁₀ level in the Tac + CoQ₁₀-W group was nearly twice as high as that in the Tac + CoQ₁₀-L group (888 ± 101 ng/mL vs. 463 ± 91 ng/mL, respectively; p < 0.05 vs. Tac + CoQ₁₀-L group). In the kidney tissue (Fig. 1B), the CoQ₁₀ content was lower in the Tac group than in the VH (1,837 ± 196 ng/mL vs. 2,481 ± 78 ng/mL, respectively; p < 0.05 vs. VH group). An increase in CoQ₁₀ level was observed in both Tac + CoQ₁₀-L and Tac + CoQ₁₀-W groups compared with that in the Tac group (2,665 ± 316 ng/mL in the Tac + CoQ₁₀-L group and 2,629 ± 319 ng/mL in the Tac + CoQ10-W, p < 0.05 vs. Tac group) in the kidney. However, there was no significant difference between the two groups.

Table 1 summarizes the characteristics of each group of rats after daily treatment with Tac with/without CoQ₁₀ for 4 weeks. Compared with the VH group, the Tac group had significantly changed body weight, water intake, and urine volume. Tac treatment alone resulted in higher levels of serum creatinine than that in VH-treated rats; however co-treatment with CoQ₁₀-L or CoQ₁₀-W along with Tac normalized kidney function.

We investigated the infiltration of ED-1 positive cells in kidney tissue to evaluate the effect of CoQ₁₀-L and CoQ₁₀-W on Tac-induced inflammatory processes (Fig. 2). As shown in Fig. 2A, the number of ED-1-positive cells was decreased in the VH group. However, Tac treatment markedly increased these numbers, and this increase was markedly attenuated by treatment with CoQ₁₀. Interestingly, addition of CoQ₁₀-W showed a much reduced number of ED-1-positive cells compared to that with CoQ₁₀-L during Tac treatment. Quantitative analysis of Fig. 2A results demonstrate that there was a great contrast in the number of ED-1 positive cells between the VH and Tac groups (432 ± 3 mm²/10⁴ vs. 3 ± 0.3 mm²/10⁴, respectively; p < 0.05 vs. VH group). However, there was a marked reduction in ED-1 positive cells in the Tac + CoQ₁₀-W group compared with that in the Tac + CoQ₁₀-L group (9.2 ± 0.4 mm²/10⁴ vs. 23.5 ± 1.8 mm²/10⁴, respectively; p < 0.05 vs. Tac + CoQ₁₀-L group).

Fig. 3 shows that co-treatment with CoQ₁₀-W or CoQ₁₀-L had an anti-fibrotic effect in Tac-induced nephropathy. Fig. 3A shows that Tac treatment alone increased extensive tubule interstitial fibrosis compared with that in the VH group (16.3% ± 0.4% vs. 4.5% ± 0.1%, respectively; p < 0.05 vs. VH group). Concurrent treatment with CoQ₁₀ decreased the tubule interstitial fibrosis level compared to that with Tac treatment; however, CoQ₁₀-W attenuated the tubule interstitial fibrosis level further than that by CoQ₁₀-L (11% ± 0.4% vs. 13% ± 0.3%, respectively; p < 0.05 vs. Tac + CoQ₁₀-L group). At the molecular level, the expression levels of E-cadherin were lower (62% ± 3% vs. 100% ± 0.1%, p < 0.05 vs. VH group) and those of α-SMA (130% ± 5.5% vs. 100% ± 0.1%, p < 0.05 vs. VH group, p < 0.05), collagen IV (231.9% ± 19.4% vs. 100% ± 0.1%, p < 0.05 vs. VH group, p < 0.05) after Tac treatment than in the VH group. These levels were restored by co-treatment with CoQ₁₀-L or CoQ₁₀-W. Especially, treatment with CoQ₁₀-W significantly changed the E-cadherin (89% ± 2.8% vs. 75.5% ± 2.7%, p < 0.05 vs. CoQ₁₀-L group), α-SMA (121% ± 11% vs. 148% ± 6%, p < 0.05 vs. CoQ₁₀-L group) and collagen IV (165.3% ± 6.4% vs. 181.6% ± 7.5%, p < 0.05 vs. CoQ₁₀-L group, p < 0.05) levels compared to those by CoQ₁₀-L (Fig. 3C–3E).

Fig. 4 shows the results for 8-OHdG in the kidney tissues and serum and 24-hour urinary excretion level. The expression of 8-OHdG was significantly higher in the Tac group than in the VH group (288 ± 16 mm²/10⁴ vs. 122 ± 10 mm²/10⁴, respectively; p < 0.05 vs. VH group), and decreased by co-treatment with CoQ₁₀-L (231 ± 10 mm²/10⁴; p < 0.05 vs. Tac group) or CoQ₁₀-W (142 ± 5 mm²/10⁴; p < 0.05 vs. Tac group). Higher levels of 8-OHdG in serum and urine were also detected in the Tac group than those in the VH group (serum, 9.4 ± 0.5 ng/mL vs. 7.8 ± 0.5 ng/mL; p < 0.05 vs. VH group; urine, 90.3 ± 12.1 ng/mL/day vs. 39.8 ± 10.8 ng/mL/day; p < 0.05 vs. VH group) however, significant changes were only observed in the CoQ₁₀-L (serum, 8.3 ± 0.4 ng/mL; urine, 59 ± 8.6 ng/mL/day; p < 0.05 vs. Tac group) and CoQ₁₀-W (serum, 7 ± 0.4 ng/mL vs. urine, 32.4 ± 13.7 ng/mL/day; p < 0.05 vs. Tac group) groups (Fig. 4C and 4D). Tac + CoQ₁₀-W reduced 8-OHdG expression further than that by Tac + CoQ₁₀-L in the kidney tissue, serum, and 24-urine during treatment with Tac. Tac-induced 4-HHE expression also showed results consistent with those of 8-OHdG as shown in Fig. 5 (Tac + CoQ₁₀-L, 21.5 ± 0.7 mm²/10¹⁰ vs. Tac + CoQ₁₀-W, 7.1 ± 0.4 mm²/10⁴; p < 0.05).

We evaluated the effects of CoQ₁₀-L and CoQ₁₀-W on Tac-induced apoptosis using TUNEL assay in tissue sections (Fig. 6A and 6B). The maximum number of TUNEL-positive cells was observed in the Tac group and the minimum in the VH group (15.2 ± 1.2 vs. 5 ± 0.1, respectively; p < 0.05 vs. VH group). As expected, the number of TUNEL-positive cells was reduced by co-treatment with CoQ₁₀-L or CoQ₁₀-W compared to that in the Tac group; however, Tac + CoQ₁₀-W attenuated the number of TUNEL-positive cells much more than that by Tac + CoQ₁₀-L (8.1 ± 0.3 mm²/10⁴ vs. 11.7 ± 1.6 mm²/10⁴, respectively; p < 0.05 vs. Tac + CoQ₁₀-L group). At the molecular level, the expression levels of active caspase-3 and BAX were higher with Tac treatment than in the VH group as shown in Fig. 6C–6E. These levels were restored by co-treatment with CoQ₁₀-L or CoQ10-W. Especially, CoQ₁₀-W significantly reduced the expression of these proteins compared to that by CoQ₁₀-L (active caspase-3, 112% ± 6% vs. 137% ± 9%, p < 0.05 vs. Tac + CoQ₁₀-L group; BAX, 114% ± 7% vs. 158% ± 17%, p < 0.05 vs. Tac + CoQ₁₀-L group).

We evaluated the effect of CoQ₁₀ administration on Tac-induced mitochondrial ultrastructural changes in kidney tissue using electron microscopy to assess the size and the number of mitochondria (Fig. 7). Tac treatment decreased both average mitochondrial size (0.35 ± 0.01 μm² vs. 0.57 ± 0.02 μm², p < 0.05 vs. VH group) and number (0.46 ± 0.02 vs. 0.79 ± 0.04, p < 0.05 vs. VH group). However, co-treatment with CoQ₁₀-L (size, 0.46 ± 0.02 μm²; number, 0.7 ± 0.04 μm²; p < 0.05 vs. Tac group) and CoQ₁₀-W (size, 0.49 ± 0.01 μm²; number, 0.73 ± 0.09 μm²_; p < 0.05 vs. Tac group) restored these changes compared with those by Tac alone (Fig. 7B and 7C).

Fig. 8 demonstrates the protective effect of CoQ₁₀-L and CoQ₁₀-W against cell injury during Tac treatment in the renal proximal tubule cell line, HK-2 cells. The different doses of Tac + CoQ₁₀-L and Tac + CoQ₁₀-W increased the cell viability of HK-2 cells by approximately 2.8 times compared with that in the Tac group. However, there was no significant difference in cell viability between the Tac + CoQ₁₀-L and the Tac + CoQ₁₀-W groups at the indicated doses.

Co-treatment with CoQ₁₀-L or CoQ₁₀-W reduced the Tac-induced MitoSOX Red fluorescence level, which is a marker for mitochondrial ROS; however, as shown in Fig. 9, no difference was observed in the protective level between CoQ₁₀-L and CoQ₁₀-W.