All animal care and experimental procedures in this study were reviewed and approved by the Animal Experimentation Ethics Committee of Yanbian University Health Science Center (YBU-2017-2-3). Following acclimatization for 1 week, weight-matched male Sprague-Dawley rats weighing 220 to 240 g were housed in individual cases with a 12-hour artificial light-dark cycle and permitted free access to standard chow and water. The rats were randomly assigned to five groups: Sham operation (Sham, n = 6); UUO 7 days (UUO7; n = 8); UUO + LC7 (n = 8); UUO 14 days (UUO14; n = 8); and UUO + LC14 (n = 8). Induction of UUO was performed as described previously [13]. Briefly, rats were anesthetized with pentobarbital (40 mg/kg) and a flank incision was made. After exposure of the kidneys and ureters, the left ureter was ligated with 4-0 silk, followed by suture of the incision. Sham operation was performed similarly, but without ligation of the left ureter. Administration of LC (200 mg/kg/day intraperitoneal) was started 24 hours later and continued for 14 days, and sham-operated rats were injected intraperitoneally with sterile saline at a dose of 1 mg/mL/day. After 7 or 14 days of treatment, rats were euthanized and the obstructed kidneys were rapidly harvested for further examination.

LC (Sigma Aldrich C0158) was diluted in sterile saline to a final concentration of 200 mg/mL. The dose of LC treatment used in this study was chosen based on that used in previous study [14].

Renal function, including serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C (CysC), was measured by a quantitative enzyme colorimetric method (Roche Cobas 8000 Core ISE, Roche Diagnostics, Mannheim, Germany).

The antibodies used in this study for immunohistochemistry and immunoblotting were as follows: ectodermal dysplasia 1 (ED-1, Serotec Inc., Oxford, UK), osteopontin (OPN, #ab8448, Abcam, Cambridge, UK), transforming growth factor-β1 (TGF-β1, R&D Systems, Minneapolis, MN, USA), connective-tissue growth factor (CTGF, #ab125943, Abcam), matrix metalloproteinase-2 (MMP-2, #ab38929, Abcam), macrophage chemotactic protein-1 (MCP-1, Santa Cruz Biotechnoogy Inc., Santa Cruz, CA, USA), toll-like receptor-2 (TLR-2, Santa Cruz Biotechnoogy Inc.), heme-oxygenase-1 (HO-1, #ab13248, Abcam), superoxide dismutase-1 (SOD1, #ab16831, Abcam), superoxide dismutase-2 (SOD2, #ab13534, Abcam), nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX-2, #ab31092, Abcam), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX-4, #ab109225, Abcam), translocase of the outer mitochondrial membrane 20 (TOMM20, #ab186734, Abcam), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (NDUFA10, #ab96464, Abcam), succinate dehydrogenase complex subunit A (SDHA, #ab66484, Abcam), dynamin-related protein 1 (DLP1/Drp1, BD Transduction Laboratories, Lexington, KY, USA), optic atrophy protein 1 (OPA1, #ab90857, Abcam), B-cell lymphoma-2 (Bcl-2, #ab59348, Abcam), Bcl2-associated X (Bax, #ab32503, Abcam), cleaved caspase-3 (Millipore, Billerica, MA, USA), LC3B (#ab192890, Abcam), Beclin-1 (#ab62557, Abcam), P62 (#ab56416, Abcam), PI3K (#ab180967, Abcam), AKT (protein kinase B, #ab8805, Abcam), phospho-AKT (#ab38449, Abcam), FoxO1a (#ab70382, Abcam), phospho-FoxO1a (#ab131339, Abcam), α-smooth muscle actin (α-SMA, #ab5694, Abcam), β-actin (#ab8226, Abcam).

Harvested kidney specimens were rapidly fixed in periodate-lysine-paraformaldehyde solution and embedded in wax. Following dewaxing, 4-μm sections were conducted and stained with Masson’s trichrome and hematoxylin. The quantitative analysis of TIF was performed using a color image auto-analyzer (TDI Scope Eye Version 3.6 for Windows, Olympus, Seoul, Korea). A minimum of 20 fields per section was evaluated by counting the percentage of injured area under × 100 magnification. Histopathological analysis was fulfilled in randomly selected cortical fields of sections by a pathologist blinded to the assignment of the treatment groups.

Immunohistochemistry was performed as described previously [7]. Twenty different fields in each section at × 400 magnification were analyzed using a color image analyzer (TDI Scope Eye Version 3.0 for Windows, Olympus, Tokyo, Japan).

The procedure of transmission electron microscopy was performed as we previously detailed [15]. Kidney tissues were post-fixed with 1% osmium oxide (OsO₄) and embedded in Epon 812 following fixation in 2.5% glutaraldehyde in 0.1 M phosphate buffer. Ultrathin sections were cut and stained with uranyl acetate/lead citrate, and photographed with a JEM-1200EX transmission electron microscope (JEOL Ltd., Tokyo, Japan). Using an autoimage analyzer, the number and size of mitochondria were measured in 20 random unoverlapped proximal tubular cells (TDI Scope Eye Version 3.0 for Windows, Olympus, Tokyo, Japan).

Immunoblotting was performed as described previously [7]. Appropriate kidney specimens were selected to perform immunoblotting analysis according to protein loading lines as follows: sham (n = 6); UUO7 (n = 7); UUO + LC (n = 7); UUO14 (n = 8); UUO + LC14 (n = 8). Images were analyzed with an image analyzer (Quantity One, Bio-Rad Tachnical Service Department, Hercules, CA, USA). Optical densities were obtained using the sham group as 100% reference and normalized with β-actin.

Apoptosis in tissue sections was identified using the ApopTag in situ Apoptosis Detection Kit (Sigma-Aldrich, Millipore). The number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells was counted on 20 different fields in each section at × 400 magnification.

A twenty-four-hour urine for each rat was collected and centrifuged. The supernatants were applied to determine the concentrations of the DNA adduct 8-hydroxy-2'-deoxyguanosine (8-OHdG) using a competitive enzyme-linked immunesorbent assay (Japan Institute for the Control of Aging, Shizuoka, Japan) according to the manufacturer’s instruction. All samples were performed in triplicate and averaged.

HK-2 cells from an immortalized human proximal tubular epithelial cell line were grown in Dulbecco’s modified Eagle’s medium/Nutrient F12 (DMEM/F12; HyClone, GE Healthcare Life Science, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco; Thermo Fisher Scientific Inc.). The cells were cultured in a humidified incubator with 5% CO₂ at 37°C. For some experiments, cells were pretreated with different concentrations of LC (10 or 100 μM) for 1 hour, and then treated with H₂O₂ (500 μM) with or without treatment with LC for 24 hours.

HK-2 cell viability was examined using cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan) according to the manufacture’s instructions. Approximately 1.0 × 10⁴ cells/well were seeded in a 96-well plate. After treatment with H₂O₂ and/or LC, 10 μL of CCK-8 solution were added to each well and incubated at 37°C for 3 hours. Absorbance was measured as the optical density at 450 nm (VersaMax Microplate Reader, Molecular Devices, LLC, Sunnyvale, CA, USA).

The levels of intracellular reactive oxygen species (ROS) were measured using 2',7'-dichlorodihydrofluorescein diacetate (H₂-DCFDA, Invitrogen, Carlsbad, CA, USA) according to the manufacture’s instructions. HK-2 cells were seeded at a density of 2.0 × 10⁵ cells/well in a 6-well plate. After treatment with H₂O₂ or LC for the indicated time, cells were washed with phosphate-buffered saline and then incubated with H₂-DCFDA for 30 minutes. Fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Annexin V-positive HK-2 cells were detected using Annexin V-FITC apoptosis detection kit (Biosharp, Hefei, China) according to the manufacturer’s protocol. Briefly, HK-2 cells were seeded and incubated with 500 μM H₂O₂ with or without LC (100 μM) for 24 hours, and the samples were analyzed using a FACSCalibur flow cytometer (BD Biosciences). Apoptotic cells were examined as a percentage of the total cell count. The percentage of apoptotic cells was calculated as the number of propidium iodide (PI)-positive and Annexin-V positive cells divided by the total number of cells. Three independent experiments were performed.

Data are presented as mean ± SEM. Multiple comparisons between groups were conducted using one-way analysis of variance and the Bonferroni post hoc test using SPSS software version 21.0 (IBM, Armonk, NY, USA). Statistical significance was accepted at p < 0.05.

Table 1 summarizes the renal function in each of the treatment groups. Neither UUO nor LC influenced renal function, as shown by Scr, BUN, and CysC.

Gross examination of the kidneys 7 days after UUO indicated kidney enlargement, hydronephrosis, ureterectasis, and a significant thinning of the renal cortex, changes that had developed further by day 14 (Fig. 1A). Trichrome and hematoxylin-eosin staining demonstrated that rats that had undergone UUO displayed tubular basement membrane thickening, necrosis, vacuolization and atrophy (Fig. 1B and 1C), swelling of the tubular epithelium and interstitium, inflammatory cell infiltration, and mild TIF formation at day 7. After 14 days of UUO, renal histopathology was characterized by extensive tubular vacuolization and atrophy, a massive inflammatory cell influx, and moderate-to-severe TIF (Fig. 2). The sites of severe TIF were notably surrounded by areas of tubular vacuolization and atrophy (Fig. 1B, 1C and Fig. 3B, 3C). As evaluated with our quantitative analysis system, UUO induced a gradual increase in TIF at days 7 (22.10% ± 3.83%/0.5 mm² vs. 2.14% ± 0.95%/0.5 mm², p < 0.01) and 14 (28.47% ± 5.69%/0.5 mm² vs. 2.14% ± 0.95%/0.5 mm², p < 0.01) compared with sham operation, whereas LC treatment decreased TIF formation at both time points compared with UUO alone (17.62% ± 4.98%/0.5 mm² vs. UUO7, p < 0.01; 23.39% ± 3.71%/0.5 mm² vs. UUO14, p < 0.05) (Fig. 1D). At a molecular level, UUO significantly upregulated TGF-β1 and connective-tissue growth factor (CTGF) expression and induced MMP-2 overexpression at day 7 and this upregulation increased at day 14. All changes were reversed by administration of LC (Fig. 4).

Inflammation plays an important role in the renal fibrosis induced by UUO [16,17]. To address the effect of LC on inflammation, we evaluated inflammatory mediators by immunohistochemistry and immunoblotting. As shown in Figs. 2 and 3, constitutive expression of ED-1 and OPN in sham-operated kidneys was low, but was clearly increased in the obstructed kidneys at day 7 (ED-1: 23.44 ± 4.97/0.5 mm² vs. 2.15 ± 0.84/0.5 mm², p < 0.01; OPN: 13.24 ± 2.23/0.5 mm² vs. 3.41 ± 1.17/0.5 mm², p < 0.01), and was further increased at day 14 (ED-1: 31.63 ± 4.68/0.5 mm² vs. 23.44 ± 4.97/0.5 mm², p < 0.01; OPN: 19.63 ± 2.67/0.5 mm² vs. 13.24 ± 2.23/0.5 mm², p < 0.01). Immunoblotting showed that UUO caused a significant upregulation of macrophage chemotactic protein-1 (MCP-1) and toll-like receptor-2 (TLR-2) expression at day 7, and further upregulation was observed at day 14 (Fig. 4). Overexpression of these inflammatory mediators was markedly decreased by LC treatment.

Oxidative stress caused by UUO is well known to be closely associated with dysregulation of oxidant and antioxidant enzymes. In this study, immunoblotting revealed that LC treatment counteracted oxidative stress injury by upregulating SOD1 and SOD2 expression but downregulating NAPDH oxidase (NOX)-2 and NOX-4 compared with UUO alone (Fig. 5). Interestingly, UUO increased hemoxygenase (HO)-1 protein expression, and this was further increased following LC administration. This is consistent with previous reports by Chen et al. [18] and Qiao et al. [19] suggesting that induction of HO-1 is an adaptive response to UUO injury and that a further increase in HO-1 expression induced by LC plays a protective role. In addition, LC reduced 8-OHdG concentrations in serum (UUO7 + LC vs. UUO7: 46.14 ± 3.39 ng/mL vs. 60.12 ± 7.32 ng/mL; UUO14 + LC vs. UUO14: 50.52 ± 5.19 ng/mL vs. 71.16 ± 8.35 ng/mL, p < 0.05, respectively) (Fig. 6A) and urine (UUO7 + LC vs. UUO7: 131.54 ± 12.31 ng/day vs. 158.26 ± 21.47 ng/day; UUO14 + LC vs. UUO14: 150.38 ± 14.67 ng/day vs. 181.46 ± 23.37 ng/day, p < 0.05, respectively) (Fig. 6B).

Transmission electron microscopy showed that UUO damaged mitochondrial structures, as indicated by swelling of disorganized cristae, fragmented mitochondria (fission), and mitochondrial fussion (Fig. 3H and 3I). By our quantitative analysis system, LC treatment preserved the number and size of mitochondrial structure integrity (Fig. 3). Quantitative immunoblotting analysis indicated that expression of TOMM20, NDUFA10 (one of the subunits of complex I), and SDHA was significantly suppressed in obstructed kidneys compared with sham-operated kidneys, whereas the expression of these molecules was restored by treatment with LC (Fig. 7). Moreover, LC decreased expression of Drp1 (fission protein), but increased expression of both the long form and short form of OPA1 (fussion protein). These findings suggest that LC may improve mitochondrial function in rat kidneys after UUO.

Both of type I (apoptosis) and type II programmed cell death (autophagy) are involved in renal fibrosis of obstructed kidneys. By electron microscopy (Fig. 3E) and TUNEL assay (Fig. 8A), we observed that UUO induced typical apoptotic bodies in renal cells and an increase in the number of TUNEL-positive cells, and this trend was inhibited at both time points by addition of LC. At a molecular level, LC regulated the ratio of expression of Bcl-2 and Bax and suppressed cleaved caspase-3 expression toward levels required for cell survival (Fig. 8B). Additionally, establishment of UUO was associated with excessive autophagy. Using electron microscopy, autophagic components such as lipid droplets, myelinbodies, autolysosomes, and autophagosomes in UUO rat kidneys were observed (Fig. 3G). By immunoblotting analysis, we found that LC decreased expression of autophagy-related genes such as LC3B, Beclin-1, and P62 (Fig. 9). This finding was consistent with our previous data demonstrating the inhibitory effect of LC on autophagy [7].

To explore the mechanism of the effect of LC on renal fibrosis after UUO, we evaluated expression of components of the PI3K/AKT/FoxO1a signaling pathway by immunoblotting. As shown in Fig. 10, UUO led to activation of PI3K/AKT/FoxO1a protein expression at days 7 and 14, whereas their expression was inhibited by LC treatment. Our results indicated that LC treatment attenuates renal fibrosis induced by UUO through the PI3K/AKT/FoxO1a-dependent signaling pathway.

The HK-2 cell line was treated with H₂O₂ to mimic the animal model of UUO-induced hypoxia. Addition of LC inhibited intracellular ROS production (Fig. 11A) and apoptotic cells (Fig. 11B) and dose-dependently improved cell viability in H₂O₂-treated HK-2 cells (Fig. 11C) (p < 0.001). Moreover, LC treatment suppressed expression of profibrotic cytokine TGF-β1, CTGF, and α-SMA (Fig. 12). In parallel with our results in vivo, this in vitro study demonstrated that LC treatment exerts antioxidant effects subsequently inhibit HK-2 cell death.