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. 2023 Aug 17;22(17):1884–1899. doi: 10.1080/15384101.2023.2247251

Hydroxycitric acid prevents hyperoxaluric-induced nephrolithiasis and oxidative stress via activation of the Nrf2/Keap1 signaling pathway

Bowei Yang 1, Guang Wang 1, Yuhang Li 1, Tongxin Yang 1, Haixiang Guo 1, Pei Li 1, Jiongming Li 1,✉
PMCID: PMC10599177  PMID: 37592762

ABSTRACT

Nephrolithiasis is a common and frequently-occurring disease in the urinary system with high recurrence. The present study aimed to explore the protective effect and underlying mechanism of hydroxycitric acid (HCA) in hyperoxaluria-induced nephrolithiasis in vitro and in vivo. Crystal deposition and pathophysiological injury in rat models of glyoxylate-induced nephrolithiasis were examined using H&E staining. Cell models of nephrolithiasis were established by oxalate-treated renal tubular epithelial cells. The levels of oxidative stress indexes were determined by ELISA kits. Cell proliferation in vivo and in vitro was evaluated using a cell counting kit-8 (CCK-8) assay and Ki-67 cell proliferation detection kit. Cell apoptosis was measured by flow cytometry and TUNEL staining. The protein levels were examined by western blotting. Our results showed that HCA administration significantly reduced crystal deposition and kidney injury induced by glyoxylate. HCA also alleviated oxidative stress via upregulating the antioxidant enzyme activities of superoxide dismutase (SOD) and catalase (CAT) and reducing the malondialdehyde (MDA) content. Moreover, HCA treatment promoted cell proliferation and inhibited apoptosis of renal tubular epithelial cells exposed to hyperoxaluria. Of note, Nrf2 activator dimethyl fumarate (DMF) exerted the same beneficial effects as HCA in nephrolithiasis. Mechanistically, HCA prevented crystal deposition and oxidative stress induced by hyperoxaluria through targeting the Nrf2/Keap1 antioxidant defense pathway, while knockdown of Nrf2 significantly abrogated these effects. Taken together, HCA exhibited antioxidation and anti-apoptosis activities in nephrolithiasis induced by hyperoxaluria via activating Nrf2/Keap1 pathway, suggesting that it may be an effective therapeutic agent for the prevention and treatment of nephrolithiasis.

KEYWORDS: Nephrolithiasis, hydroxycitric acid, oxidative stress

Introduction

Nephrolithiasis is a common urinary system disease that often causes renal colic, urinary tract infections, and renal function impairment [1], which has become a prominent public health problem. The prevalence of kidney stones in China and worldwide is about 5.8% [2] and 14.80% [3], and the trend is increasing year by year. At the same time, kidney stones are also characterized by a high recurrence rate, with a 10-year recurrence rate of up to 50% [4]. Currently, although extracorporeal shock wave lithotripsy and minimally invasive surgery have achieved remarkable results in the clinical treatment of kidney stones, patients still have a high recurrence rate of secondary stone formation after surgery. Clinical studies have found that the formation of kidney stones is a complex multistep process, and approximately 80% of stones consist of calcium oxalate (CaOx) [5], but the complex formation mechanism and the lack of etiologic prevention and treatment also directly or indirectly contribute to the high incidence and recurrence rate of kidney stones. Therefore, it is of great significance to study the formation mechanism of CaOx stones and seek an effective therapeutic agent for the prevention and treatment of kidney stones.

Previous studies have demonstrated that hyperoxaluria was considered to be a vital risk factor for kidney stone formation [6]. Numerous studies have confirmed that injury and apoptosis of renal tubular epithelial cells caused by high concentrations of oxalic acid are the key steps in the formation of CaOx stone [7,8]. In addition, oxidative damage occurs in the kidney of patients with nephrolithiasis and rat nephrolithiasis model induced by glyoxylate [9,10], as well as oxidative stress contributed to the development and progression of kidney stones [11]. Of note, antioxidative therapy reduced hyper-oxalate-induced cell injury and CaOx crystal deposition [12]. Furthermore, increasing evidence has proved that the nuclear factor E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway serves as a potential preventive and therapeutic target against oxidative stress diseases [13]. Recent studies have found that the activation of the Nrf2/Keap1 pathway significantly restricted CaOx-induced kidney stone formation [14]. These studies suggest that inhibiting oxidative stress mediated by Nrf2/Keap1 signaling pathway may be a potential strategy to ameliorate kidney stones.

Hydroxycitric acid (HCA) is a natural organic acid isolated from the fruit shells of Garcinia cambogia. Previous studies have found that HCA exhibited anti-oxidative stress and anti-inflammation activities in many diseases [15,16]. In recent years, HCA was considered a potential new therapeutic agent for CaOx nephrolithiasis due to its role as an effective calcium ion inhibitor [17]. Meanwhile, HCA treatment significantly reduced CaOx crystal deposition and alleviated oxidative stress induced by glyoxylate in mice [18,19]. Our previous study also proved that HCA exerted protective effects against CaOx crystal formation, kidney damage, and oxidative stress caused by glyoxylate, as well as enhanced Nrf2 expression [20]. However, whether HCA exhibited protective effects on CaOx crystal formation and oxidative stress by regulating the Nrf2/Keap1 pathway has not been confirmed in vitro and in vivo.

In the present study, we aimed to investigate the protective effect and mechanism of HCA for the prevention and treatment of nephrolithiasis. Firstly, we explored the effect of HCA on the CaOx crystal deposition, pathophysiological injury, and oxidative stress induced by glyoxylate in vivo. Moreover, the effect of HCA on the apoptosis and oxidative stress of oxalate-treated renal tubular epithelial cells was examined. Functionally, we verified whether HCA inhibited apoptosis and oxidative stress by regulating the Nrf2/Keap1 pathway in cell models of oxalate-induced nephrolithiasis.

Material and methods

Animal model and treatment

A total of 40 male SD rats (age 8-week-old, bodyweight 180–220 g) were purchased from the Animal Experiment Center of Kunming Medical University and randomly divided into five groups, including the control (Cont) group, solvent control (SC) group, glyoxylic acid-induced nephrolithiasis model group (Gly), HCA treatment group (HCA+Gly), and dimethyl fumarate (DMF, Nrf2 activator) treatment group (DMF+Gly) with 10 rats in each group. Rats in the Cont group were given free drinking of purified water. Rats in the Gly group were intraperitoneally injected with 60 mg/kg glyoxylic acid (Zehao, China) five times a week for four weeks. Rats in the SC group were intraperitoneally injected with 2.5 mL/kg sodium chloride injection (0.9%). Rats in the HCA+Gly group were given 1 g/kg HCA (Zehao, China) gavage based on the modeling group. Rats in the DMF+Gly group were given 25 mg/kg HCA (Sigma-Aldrich, USA) gavage based on the modeling group. The animal experiments were approved by The Second Affiliated Hospital of Kunming Medical University.

Biochemical indicators analysis

On the day after the final treatment (4 weeks), 24 h urine samples and serum samples were collected. The levels of urinary oxalate were analyzed by an ion chromatography system (Thermo Fisher Scientific, USA), and urinary Ca2+ was measured using an AU800 Animal Auto Biochemistry Analyzer (Olympus, Japan). The levels of serum BUN, serum creatinine, and serum UA were measured according to our previous study [20].

Histologic analysis

The kidney tissues of rats in each group were collected after 4 weeks of continuous treatment. Subsequently, hematoxylin and eosin (H&E) staining was performed to evaluate CaOx crystal deposition in kidney tissues according to described previously [14].

Oxidative stress indexes analysis

The renal malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and catalase (CAT) activity in kidney tissues and cells were examined by ELISA kits (CUSABIO, China) according to the manufacturer’s protocols.

Cell culture and treatment

Human (HK-2) and rat (NRK-52E) renal tubular epithelial cells were achieved from the Chinese Academy of Science Cell Bank (Shanghai, China). Cells were cultured in Dulbecco’s modified Eagle medium-F/12 (DMEM-F/12; Gibco, USA) supplied with 10% fetal bovine serum (FBS; Gibco, USA) at 37°C and 5% CO2. After 3–5 passages, cells were treated with/without 20 mM sodium oxalate (NaOx) [21] for 12 h, followed by HCA (100 nM) or DMF (10 μM) treatment for 24 h.

Cell transfection

For cell transfection, lentiviral Nrf2 siRNA (si-Nrf2) and negative control (si-NC) were sourced from GeneChem company (Shanghai, China). The vectors above were introduced into both HK-2 and NRK-52E cells with the help of a Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA). After the 24 h cell transfection, western blotting was performed to estimate the efficiency of the transfections in each group.

Cell apoptosis and proliferation

Cell apoptosis was examined by TUNEL assay kits (Roche, Shanghai, China) in accordance with the manufacturer’s instructions. Moreover, cell proliferation was analyzed using the Ki-67 cell proliferation Detection kit (KeyGEN BioTECH, Jiangsu, China) and the CCK-8 assay kit according to the manufacturer’s instructions.

Quantitative real-time reverse transcription PCR (qRT-PCR)

Total RNA was extracted from frozen kidneys using Trizol reagent (Invitrogen) and cDNA synthesis was performed using the Prime Script RT reagent Kit (Takara, China) according to the manufacturer’s instructions. Quantitative PCR was performed on a Bio-Rad CFX384 Touch detection system using the SYBR Green qPCR Master Mix kit (Qiagen, Germany). All the reactions were repeated three times. GAPDH is used as an internal control for RNA integrity and load normalization. mRNA level of HO-1, NQO1, and SOD1 was calculated using the 2−ΔΔCt method. The primer sequences of target genes were as follows: HO-1 forward 5” -ACGCGTTGTAATTAAGCCTCGCAC-3‘, reverse 5’-TTCCGCTGGTCATTAAGGCTGAGT-3‘; NQO1 forward 5’-AAGGATGGAAGAAACGCCTGGAGA-3‘, reverse 5’-GGCCCACAGAAAGGCCAAATTTCT-3‘; SOD1 forward 5’-GCAGGGCATCATCAATTTCGAGCA-3‘,reverse5’-TGCAGGCCTTCAGTCAGTCCTTTA-3‘; GAPDH forward 5’-TCGACAGTCAGCCGCATCTTCTTT-3‘;reverse5’-ACCAAATCCGTTGACTCCGACCTT-3”.

Western blotting analysis

All the proteins from tissues and cell lines were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene fluoride membranes (Millipore, Germany). The membranes were blocked with 5% nonfat milk and then incubated with the following primary antibodies against Nrf2 (1:1,000; Abcam, USA), Keap1 (1:1,000; Abcam, USA), histone 3 (1:1,000; Abcam, USA), cleaved caspase-3 (1:1,000; Abcam, USA), Bcl-2 (1:1,000; Abcam, USA), and β-actin (1:1,000; Abcam, USA) overnight at 4°C. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody to IgG (1:3,000; Abcam, USA). Protein bands were analyzed using ImageJ software.

Statistical analysis

Statistical results were shown as mean ± standard deviation. The student’s t-test was utilized to evaluate the comparisons in two groups, and a one-way analysis of variance (ANOVA) followed by Tukey’s test was used to assess the differences in multiple groups. The differences were considered statistically significant at P < 0.05.

Results

HCA reduced crystal deposition and oxidative stress in rats with glyoxylate-induced nephrolithiasis

To verify the inhibitor effect of HCA on glyoxylate-induced CaOx crystal deposition in the kidney. H&E staining showed that abundant CaOx crystal deposition was observed in the kidney tissue of rats treated with glyoxylate compared to rats in the control group (Figure 1a, b), but HCA treatment significantly reduced CaOx crystal precipitation in the kidney of glyoxylate-induced rats. In addition, the protective effect of HCA against glyoxylate-induced pathophysiological changes was investigated. The 24-h urine volume was lower in the Gly group than in the control group (Figure 1c), while it increased in the Gly group after HCA treatment. The level of urinary oxalate (Figure1d), serum BUN (Figure 1e), serum creatinine (Figure 1f), and serum UA (Figure 1G) in the Gly group were significantly higher than those in the control group, whereas HCA administration significantly reversed these results in the Gly group. Furthermore, previous studies have confirmed that oxidative stress serves a key role in the development and progression of nephrolithiasis [22]. In the present study, HCA administration significantly reduced renal MDA levels compared to the Gly group (Figure 1h), and treatment of glyoxylate-induced rats with HCA caused a dramatic increase in levels of SOD (Figure1i) and CAT (Figure1j). Taken together, HCA exhibited inhibitory effects on the formation of CaOx crystals and oxidative stress in the rat model of glyoxylate-induced nephrolithiasis.

Figure 1.

Figure 1.

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Effect of HCA on CaOx crystal deposition, pathophysiological, and oxidative stress induced by glyoxylate in rats. a and b: Representative images of kidney sections stained with hematoxylin and eosin (H&E; scale bar = 100 μm); analysis of biochemical results of urinary volume (a) and urinary oxalate (d); the levels of blood urea nitrogen (BUN) (e), creatinine (f), and uric acid (UA) (g); h-j: The oxidative stress indexes in kidney tissues were examined by ELISA kits. HCA: hydroxycitric acid; MDA: malondialdehyde; SOD: superoxide dismutase; CAT: catalase. ***P < 0.001, compared with the solvent control (SC) group; ##P < 0.01, ###P < 0.001, compared with the Gly group.

HCA inhibited apoptosis induced by glyoxylate in rat

Previous studies have demonstrated that apoptosis was involved in nephrolithiasis progression [23], we explored the effect of HCA on apoptosis in the rat model of glyoxylate-induced nephrolithiasis. Immunohistochemical staining revealed that glyoxylate significantly reduced Ki-67 expression in the kidney tissues compared with the control group (Figure 2a), but treatment with HCA improved this effect. Moreover, TUNEL staining showed that the number of TUNEL-positive cells was significantly higher in the Gly group than that in the Gly+HCA group (Figure 2b). In addition, the pro-apoptotic protein (cleaved-caspase-3) was reduced in Gly+HCA compared with the single Gly group (Figure 2c), but the anti-apoptotic protein (Bcl-2) was increased in Gly+HCA group (Figure 2c). Collectively, HCA repressed apoptosis in the rat model of nephrolithiasis induced by glyoxylate.

Figure 2.

Figure 2.

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Effect of HCA on cell proliferation and apoptosis in glyoxylate-induced nephrolithiasis. a: Immunohistochemical staining of Ki-67, the level of Ki-67 in the kidney tissue sections was determined using ImageJ software; scale bars = 100 μm; b: TUNEL staining of renal apoptosis, scale bars = 100 μm; c: Western blotting was used to detect the expression of cleaved caspase-3 and Bcl-2 protein in kidney tissues. ***P < 0.001, compared with the SC group; ###P < 0.001, compared with the Gly group.

HCA facilitated proliferation and repressed apoptosis in oxalate-treated renal tubular epithelial cells

To further explore the impact of HCA on the proliferation and apoptosis of renal tubular epithelial cells exposed to oxalate. CCK-8 assay showed that the concentration of HCA less than 100 nM had no significant effect on both HK-2 and NRK-52E cell viability (Supplementary Figure 1A, B), and a concentration of 100 nM HCA was used for the performance of followed-up experiments. Immunofluorescence revealed that Ki-67 expression was decreased in the NaOx group in contrast to the NC group (Figure 3a, b), and its expression was enhanced in both HK-2 and NRK-52E cells after HCA treatment. Similarly, TUNEL staining showed that the ratio of apoptosis in oxalate-induced both HK-2 and NRK-52E cells was higher than that in the NC group (Figure 3c, d), and HCA treatment reversed this effect. Moreover, HCA significantly reduced the expression of Cleaved caspase-3 in both HK-2 and NRK-52E treated with NaOx (Figure3e, f), while enhancing the expression of Bcl-2 (Figure 3e, f). Collectively, HCA inhibited apoptosis in both HK-2 and NRK-52E cells exposed to oxalate.

Figure 3.

Figure 3.

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Effect of HCA on proliferation and apoptosis in human renal tubular epithelial cells (HK-2 and NRK-52E) exposed to oxalate. a and b: Immunofluorescence staining of Ki-67 in HK-2 and NRK-52E cells, scale bars = 100 μm; c and d: TUNEL staining of cell apoptosis in HK-2 and NRK-52E cells, scale bars = 100 μm; e and f: Western blotting was used to detect the expression of cleaved caspase-3 and Bcl-2 protein. ***P < 0.001, compared with the NC group; ###P < 0.001, compared with the NaOx group.

HCA reduced oxidative stress and inactivation of the Nrf2/Keap1 signaling pathway in oxalate-treated renal tubular epithelial cells

Next, to investigate the impact of HCA on oxidative stress in renal tubular epithelial cells exposed to oxalate. Compared with the NC group, MDA content was greatly increased in both HK-2 and NRK-52E cells after NaOx exposure (Figure4a), but SOD activity (Figure 4b) and CAT activity (Figure 4c) were significantly decreased. However, HCA treatment significantly elevated the levels of SOD and CAT in oxalate-induced renal tubular epithelial cells, and reduced the MDA content. Moreover, numerous studies have illustrated that Nrf2/Keap1 signaling pathway serves as an important pathway for anti-oxidative stress in the progression of many diseases [24], including nephrolithiasis. Our results showed that oxalate exposure significantly reduced the protein level of nuclear Nrf2 in both HK-2 and NRK-52E cells compared with the control group (Figure 4d), but upregulated the protein levels of Nrf2 and Keap1 in the cytoplasm (Figure 4e, f). In addition, the mRNA expression of HO-1, NQO1, and SOD1 in the NaOx group was significantly lower than that in the NC group (Figure4g, h). Importantly, treatment with HCA significantly abrogated these changes in both HK-2 and NRK-52E cells exposed to oxalate (Figure 4d-f). Taken together, HCA exhibited anti-oxidative stress via activation of the Nrf2/Keap1 pathway in oxalate-induced renal tubular epithelial cells.

Figure 4.

Figure 4.

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Effect of HCA on oxidative stress and Nrf2/Keap1 pathway in oxalate-treated renal tubular epithelial cells. A-C: The MDA content, SOD activity, and CAT activity in both HK-2 and NRK-52E cells were measured by ELISA kits; E and F: Western blotting was performed to examine the protein levels of Nrf2 and Keap1; G and H: qRT-PCR was used to detect the mRNA expression of HO-1, NQO1, and SOD1. ***P < 0.001, compared with the NC group; ###P < 0.001, compared with the NaOx group.

Knockdown of Nrf2 ameliorated the preventive effect of HCA on oxalate-induced renal tubular epithelial cell injury in vitro

To further validate whether HCA exerts a protective effect against oxalate-induced cell injury via the Nrf2/Keap1 pathway in vitro, Nrf2 siRNA was transfected into both HK-2 and NRK-52E cells, and the transfection efficiency of Nrf2 was examined by western blotting (Figure 5a). As expected, knockdown of Nrf2 reduced the Ki-67 protein level in both HK-2 and NRK-52E cells after oxalate plus HCA treatment compared with the NaOx+HCA group (Figure 5b, d). Moreover, the number of TUNEL-positive cells in the NaOx+HCA+si-Nrf2 group was higher than that in the NaOx+HCA group (Figure5c, e), and no significant difference in cell apoptosis between the NaOx group and NaOx+HCA+si-Nrf2 group. Furthermore, knockdown of Nrf2 with siRNA significantly downregulated the inhibitory effect of HCA on MDA content (Figure 5f) in both HK-2 and NRK-52E cells after oxalate exposure, as well as the promotion effect of HCA on the activities of SOD (Figure 5g) and CAT (Figure 5h) was weakened due to Nrf2 deficiency. In conclusion, HCA prevented oxidative stress and apoptosis induced by oxalate via activation of the Nrf2/Keap1 pathway in vitro.

Figure 5.

Figure 5.

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Effect of HCA on apoptosis and oxidative stress induced by oxalate in both HK-2 and NRK-52E cells via regulating of Nrf2/keap1 pathway. a: Both HK-2 and NRK-52E cells were transfected with Nrf2 siRNA, and western blotting was used to examine the protein level of Nrf2; b and d: Immunofluorescence staining of Ki-67 in HK-2 and NRK-52E cells, scale bars = 100 μm; c and e: TUNEL staining of cell apoptosis in HK-2 and NRK-52E cells, scale bars = 100 μm; f-h: The MDA content, SOD activity, and CAT activity in both HK-2 and NRK-52E cells were measured by ELISA kits. $$$P < 0.001, compared with the si-NC group; ***P < 0.001, compared with the NC group; ###P < 0.001, compared with the NaOx group; &&&P < 0.001, compared with the NaOx+HCA group.

Upregulation of Nrf2 promoted cell viability and reduced oxidative damage in oxalate-treated renal tubular epithelial cells

CCK-8 assay was performed to explore the effect of DMF (Nrf2 activator) on cell viability and oxidative stress in oxalate-treated renal tubular epithelial cells. The concentration of DMF less than 10 μM had no significant effect on both HK-2 and NRK-52E cell viability (Supplementary Figure 1C, D), and a concentration of 10 μM DMF was used for the performance of followed-up experiments. Subsequently, the effect of DMF on the nuclear translocation and expression of Nrf2 in renal tubular epithelial cells exposed to oxalate was determined by western blotting. As anticipated, DMF significantly promoted Nrf2 translocation from cytoplasm to nucleus by increasing nuclear Nrf2 expression in oxalate-induced renal tubular epithelial cells (Figure 6a), but cytoplasmic Nrf2 protein decreased and cytoplasmic Keap1 protein enhanced (Figure 6b, c). Meanwhile, DMF treatment notably enhanced the mRNA expression of HO-1, NQO1, and SOD1 in oxalate-induced renal tubular epithelial cells (Figure 6d, e). In addition, DMF reduced the cytotoxicity in renal tubular epithelial cells induced by oxalate (Figure 6f). Flow cytometry assays also showed that DMF significantly reduced the early and late apoptosis of HK-2 and NRK-52E cells after exposure to oxalate (Figure 6g). Furthermore, DMF effectively reduced the MDA content (Figure 6h) and enhanced SOD activity (Figure 6i) and CAT activity (Figure 6j) compared with the NaOx group. Of note, upregulation of Nrf2 pathway by DMF has a similar effect of HCA treatment on cell viability and oxidative stress in renal tubular epithelial cells exposed to oxalate. Taken together, overexpression of Nrf2 increased viability and reduced oxidative stress in renal tubular epithelial cells treated with oxalate.

Figure 6.

Figure 6.

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Effect of Nrf2 overexpression on viability and oxidative damage in oxalate-treated renal tubular epithelial cells. The protein level of nuclear (a) and cytoplasmic Nrf2 (b, c), and cytoplasmic Keap1 (b, c) in both HK-2 and NRK-52E cells stimulated by oxalate and treated with DMF or HCA was determined by western blotting; d and e: The mRNA expression of HO-1, NQO1, and SOD1 in both HK-2 and NRK-52E cells was detected by qRT-PCR; f: Cell viability of HK-2 and NRK-52E cells was evaluated by CCK-8; g: Flow cytometry was applied to measure cell apoptosis; the MDA content (h), SOD activity (i), and CAT activity (j) in both HK-2 and NRK-52E cells were determined by ELISA kits. **P < 0.01, ***P < 0.001, compared with the NC group; #P < 0.05, ##P < 0.01, ###P < 0.001, compared with the NaOx group.

Upregulation of Nrf2 improved renal function and reduced glyoxylate-induced apoptosis and oxidative stress in rats

To verify whether Nrf2 upregulation possessed protective effects on renal injury, apoptosis, and oxidative stress caused by glyoxylate in rats. H&E staining showed that DMF treatment improved glyoxylate-induced renal histological injury, including glomerular edema, focal tubular necrosis, and crystal deposition (Figure 7a). The levels of BUN (Figure 7b), serum creatinine (Figure 7c), and serum UA (Figure 7d) in the Gly+DMF group were significantly lower than those in the Gly group. Moreover, DMF significantly reduced renal MDA content (Figure 7e) and increased the activities of SOD (Figure 7f) and CAT (Figure 7g) in rats treated with glyoxylate. Immunohistochemistry staining revealed that DMF treatment resulted in significantly increased pro-proliferation protein Ki-67 expression compared with the Gly group (Figure 7h). Furthermore, western blotting showed that DMF treatment notably increased the Nrf2 protein level and reduced the Keap1 protein level in the renal tissues of the glyoxylate-induced nephrolithiasis rat model (Figure 7i, j), as well as decreased cleaved caspase-3 protein level (Figure 7i, k). Intriguing, HCA has the same beneficial effects as DMF in nephrolithiasis. Collectively, upregulation of Nrf2 exhibited a protective effect against glyoxylate-induced nephrolithiasis.

Figure 7.

Figure 7.

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Effect of Nrf2 overexpression on renal function, apoptosis, oxidative stress caused by glyoxylate in rats. a: H&e staining checked the pathological conditions of all rats’ kidney tissues. The BUN (b), creatinine (c), and UA (d) in each group were determined by spectrophotometry-based methods using commercially available kits, scale bars = 50 μm; the MDA content (e), SOD activity (f), and CAT activity (g) in kidney tissues were detected by ELISA kits; H: Immunochemistry examined Ki-67 protein level in kidney tissues, scale bars = 100 μm; I-K: Western blotting was performed to examine the protein levels of Nrf2, Keap1, and cleaved caspase-3 in kidney tissues. **P < 0.01, ***P < 0.001, compared with the Cont group; ##P < 0.01, compared with the Gly group.

Discussion

Nephrolithiasis is a common and frequently-occurring disease worldwide and can seriously damage kidney function and threaten human health. Previous studies have found that hyperoxaluria-induced renal tubular epithelial cell injury including oxidative stress and apoptosis, which facilitates the development and progression of kidney stones [25,26]. In recent years, there have been great advances in the treatment of nephrolithiasis, but the treatment protocols mainly revolve around stones. As the understanding of the pathogenesis of nephrolithiasis is still limited, effective interventions on the formation and development of nephrolithiasis at the etiological level cannot be performed. Therefore, it is important to investigate the pathogenesis of nephrolithiasis, which contributed to developing novel and effective drugs for the prevention and treatment of nephrolithiasis. In the present study, our results showed that HCA significantly reduced CaOx crystal deposition and kidney injury induced by glyoxylate in vivo. Moreover, HCA inhibited apoptosis and oxidative stress in rat models of glyoxylate-induced nephrolithiasis and cell models of oxalate-treated renal tubular epithelial cells. Functionally, HCA treatment exhibited beneficial effects on nephrolithiasis induced by hyperoxaluria via activating the Nrf2/Keap1 pathway (Figure 8). These results indicate HCA serves as an effective therapeutic agent to prevent nephrolithiasis progression via reducing CaOx crystal formation and oxidative stress induced by hyperoxaluria.

Figure 8.

Figure 8.

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Schematic illustration of HCA exhibited a beneficial effect on hyperoxaluric-induced nephrolithiasis.

Previous studies have demonstrated that the main component of nephrolithiasis was CaOx, and abundant reactive oxygen species (ROS) secretion [27] and oxidative stress [28] play an important role in the formation and development process of renal CaOx stones. Patel et al. [29] showed that excessive ROS induced by oxalate can inhibit crystalline clearance by macrophages, ultimately facilitating stone formation. Recently, antioxidant agents (such as peppermint oil [30], quercetin [31], and hyperoside [32]) possessed inhibitory effects on CaOx crystal deposition and oxidative stress induced by hyperoxaluria in vivo and in vitro experiments. Similarly, our results showed that HCA administration significantly attenuated crystal deposition and kidney injury induced by glyoxylate in the rat. Moreover, the serum levels of oxidative stress indexes including CAT and SOD activities were reduced in glyoxylate-induced rats, and enhancing serum MDA content [4]. Of note, our results showed that treatment with HCA significantly ameliorated the inhibitory effect of hyperoxaluria on the activities of SOD and CAT, as well as reduced MDA content, suggesting that HCA alleviated nephrolithiasis via inhibiting oxidative stress induced by glyoxylate.

Increasing evidence has proved that hyperoxaluria and/or CaOx crystal-induced renal tubular epithelial cell injury was a key step in the process of stone formation [33]. Meanwhile, hyperoxaluria caused renal tubular epithelial cell injury contributed to triggering oxidative stress in mitochondria, and led to the activation of the apoptotic signaling pathway [34]. For example, the level of ROS in HK-2 cells exposed to CaOx crystal was upregulated, initiating cell apoptosis [35]. Moreover, hyperoxaluria caused oxidative damage to HK-2 cells enhancing crystallite adhesion and formation [36]. In this study, HCA treatment remarkably reduced apoptosis and oxidative damage of both HK-2 and NRK-52E cells induced by oxalate. Functionally, HCA exhibited anti-oxidative and anti-apoptosis activities in renal tubular epithelial cells exposed to hyperoxaluric via activation of the Nrf2/Keap1 pathway. Of note, previous studies have demonstrated that targeting the Nrf2/Keap1 pathway can maintain cellular homeostasis in many oxidative stress-related diseases [37,38]. Several studies proved that upregulation of Nrf2 by active ingredients of traditional Chinese medicines alleviated tubular epithelial cell apoptosis and oxidative damage induced by oxalate [32,39]. Zhu et al. [14] proved that treatment with Nrf2 activator dimethyl fumarate notably inhibited oxidative stress and apoptosis in oxalate-induced nephrolithiasis. Consistent with previous studies, the present study confirmed that knockdown of Nrf2 reversed the inhibitory effect of HCA on hyperoxaluric-induced apoptosis and oxidative stress, indicating Nrf2/Keap1 pathway was a key mechanism for protecting cells from oxidative damage and apoptosis.

In conclusion, the results of this study confirmed the beneficial effect of HCA on hyperoxaluric-induced nephrolithiasis in vivo and in vitro. Mechanistically, HCA treatment reduced CaOx crystal deposition, oxidative stress, and apoptosis by activating Nrf2/Keap1 pathway. Therefore, HCA might be an effective therapeutic agent to prevent the formation of hyperoxaluric-induced nephrolithiasis.

Supplementary Material

Supplemental Material
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Funding Statement

The work was supported financially by the National Natural Science Foundation of China [NO: 82260155], the Applied Basic Research Key Project of Yunnan Province [NO: 202001AY070001-065], Yunnan Fundamental Research Projects [NO: 202301AT070327], and Yunnan Provincial Science and Technology Department Zhangqun Ye Expert’s Workstation [NO. 202105AF150063].

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15384101.2023.2247251

Availability of data and material

Data and materials are available upon request.

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