Poria cocos Polysaccharides Protect Against Contrast‐Induced Renal Injury Through Activating CaMKK2/AMPK/PGC‐1α Signaling Pathway

ABSTRACT

The necessity of iodinated contrast agents in radiographic diagnosis comes with an increased risk of kidney injury. The sclerotium of Poria cocos (Schw.) Wolf, a traditional Chinese medicine, exhibits anti‐inflammatory and diuretic pharmacological properties. Its extracted polysaccharides demonstrate anti‐apoptotic, antioxidant, and anti‐inflammatory activities. This study aimed to investigate the effects and potential mechanisms of Poria cocos polysaccharides (PCP) on contrast‐induced acute kidney injury (CI‐AKI). The purified PCP was characterized using FTIR and monosaccharide analysis. A CI‐AKI mouse model was established to evaluate its preventive effects via oral gavage. Mitochondrial structure, function, and biogenesis were assessed by measuring mitochondrial membrane potential (MMP), ROS, ATP, and mitochondrial DNA copy number. Antioxidant capacity and apoptosis levels were evaluated by detecting 8‐OHdG, caspase 3/7 activity, cleaved‐PARP, MDA, and GSH. Finally, the mechanism of action of PCP was analyzed using qPCR and immunoblotting. The results showed that PCP significantly mitigated CI‐AKI by inhibiting contrast‐induced apoptosis and oxidative stress in kidney tissue. Furthermore, PCP maintained mitochondrial homeostasis in the kidneys by enhancing mitochondrial biogenesis and antioxidant capacity. These findings were consistent with in vitro data. Notably, PGC‐1α was identified as a key factor in the nephroprotective effects of PCP, with its activation attributed to the CaMKK2‐AMPK signaling pathway. These findings suggest that PCP offers significant benefits in mitigating CI‐AKI by enhancing antioxidant defenses and promoting mitochondrial biogenesis through activation of the CaMKK2/AMPK/PGC‐1α signaling pathway in the kidney. Therefore, PCP could be a potential preventive agent for CI‐AKI.

Schematic illustration of the nephroprotective mechanism of PCP via the activation of the CaMKK2/AMPK/PGC‐1α signaling pathway.

Abbreviations

8‐OHdG

8‐hydroxy‐2′‐deoxyguanosine

AMPK

AMP‐activated protein kinase

BUN

blood urea nitrogen

CaMKK2

calcium/calmodulin dependent protein kinase kinase 2

CI‐AKI

contrast‐induced acute kidney injury

CKD

chronic kidney disease

ELISA

enzyme‐linked immunosorbent assay

GPx

glutathione peroxidase

GSH

glutathione

HE

hematoxylin and eosin

IHC

immunohistochemistry

MDA

malondialdehyde

MMP

mitochondrial membrane potential

mtDNA

mitochondrial DNA

mtROS

mitochondrial reactive oxygen species

NGAL

neutrophil gelatinase‐associated lipocalin

PCP

poria cocos polysaccharides

PGC‐1α

peroxisome proliferator‐activated receptor‐γ coactivator 1‐alpha

qPCR

quantitative PCR

ROS

reactive oxygen species

RTECs

renal tubular epithelial cells

SOD

superoxide dismutase

TUNEL

terminal deoxynucleotidyl transferase dUTP nick‐end labeling

1. Introduction

Contrast media play a crucial role in medical imaging procedures; however, their administration carries an elevated risk of acute kidney injury (AKI) (Fahling et al. 2017). Individuals with diabetes, chronic nephropathy, or who are elderly are especially vulnerable to developing contrast‐induced acute kidney injury (CI‐AKI) (Kaliyaperumal et al. 2023). CI‐AKI commonly arises from the rapid infusion of a substantial amount of hypertonic contrast material into the kidneys, contributing to renal ischemia and subsequent hypoxia, ultimately impairing renal function (Chen, Zhao, and Xia 2024). Currently, the main preventive strategies include reducing renal tubular and intravascular viscosity, mitigating oxidative stress and inflammation, preventing local hypoxia, and following hydration and volume expansion protocols (Fahling et al. 2017).

Endothelial damage, oxidative stress, and the cytotoxic effects of vasoconstrictors on renal tubules contribute to contrast agent‐induced renal injury (Kusirisin et al. 2020). Mitochondria are a productive site of intracellular reactive oxygen species (ROS) and serve as the primary organelles targeted by ROS‐mediated actions (Kowaltowski et al. 2009). ROS molecules induce the oxidation of mitochondrial lipids, proteins, and DNA, resulting in mitochondrial impairment (Kowaltowski et al. 2009). Consequently, damaged mitochondria amplify ROS production, establishing a detrimental feedback loop. Cells experiencing mitochondrial impairment can maintain cellular survival by activating mitophagy and mitochondrial biogenesis (Palikaras et al. 2015). Thus, contrast‐induced renal tubular epithelial cells (RTECs) injury may depend on the extent of mitochondrial damage. Augmenting mitochondrial autophagy and biogenesis may represent an effective strategy for the prevention and treatment of CI‐AKI.

AMP‐activated protein kinase (AMPK), a protein kinase responsible for the regulation of energy metabolism, directly phosphorylates mitochondrial fission factor, regulates mitochondrial fission through the dynamin‐related protein 1 (Drp1), and also triggers the activation of unc‐51‐like kinase 1 (ULK1), an upstream kinase involved in autophagy and mitochondrial autophagy (Herzig and Shaw 2018). The process of mitochondrial fission is conducive to the degradation of impaired mitochondria through mitochondrial autophagy (Palikaras et al. 2015). AMPK also activates peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) and intramitochondrial protein kinases to regulate mitochondrial production and oxidative phosphorylation (OXPHOS) complex content and activity (Herzig and Shaw 2018). AMPK activation has been found to lessen injury to tubular cells and preserve renal function (Ma et al. 2022; Zhu et al. 2023). Furthermore, drugs that target AMPK or PGC‐1α have demonstrated efficacy in protecting renal function in preclinical animal models (Dagorn et al. 2023; Song et al. 2020).

Poria cocos (Schw.) Wolf has historically been utilized in traditional medicine throughout Asian countries (Sun 2014). It is acknowledged for its dual role as both an edible and medicinal fungus (Guo et al. 2025). Poria cocos, first noted in Shennong Bencao Jing (Shennong's Classic of Materia Medica), is reputed for its various medicinal benefits, such as decreasing swelling, providing diuretic effects, and safeguarding renal health (Guo et al. 2025). Poria cocos is an important part of several well‐known formulas for treating kidney disease, such as Wuling San, Shen Qi Wan (Shenqi Pill), Zhen Wu Tang, and Ji Sheng Shen Qi Wan (Cai et al. 2010; Chen, Yang, et al. 2024; Liu et al. 2024; Ma and Zhou 2024; Yang et al. 2015).

The chemical composition of Poria cocos is predominantly characterized by polysaccharides (Rios 2011). Recent studies have made substantial progress in elucidating the chemical and biological characteristics of Poria cocos polysaccharides (PCP) and their derivatives (Jia et al. 2016; Lv et al. 2024; Tang et al. 2014). PCP demonstrates several beneficial biological activities, such as anticancer, anti‐inflammatory, antioxidant, and antiviral effects (Wan et al. 2024; Wang et al. 2016; Wu et al. 2022; Yu et al. 2022). Consequently, PCP shows significant potential for advancement as therapeutic drugs in cancer treatment, immunomodulation, and antiviral interventions (Qiu et al. 2024; Zhao et al. 2024). Moreover, PCP oral liquid is currently employed in clinical practice as a protective agent for patients receiving chemotherapy in China. It demonstrates significant protective properties against intestinal and immune system impairments caused by different medications (Liu et al. 2021; Wan et al. 2024). However, the protective effect of PCP on renal function remains uncertain. The objective of the current study was to investigate the effects of PCP on renal injury and to elucidate its mechanism of action by establishing a model of CI‐AKI.

2. Materials and Methods

2.1. Cell and Reagents

HK‐2 (CL0144) and TCMK1 (CL0440) cells were obtained from FenghBio (Changsha, China), cultured in minimum essential medium (#41500034, Gibco, Grand Island, USA) containing 10% fetal bovine serum (CTCC‐002‐001, Meisen CTCC, Hangzhou, China) and 1% penicillin and streptomycin (PB180120, Pricella, Wuhan, China). The culture medium powder is dissolved in ultrapure water and then sterilized using a vacuum filter (FPE204000, JET Biofil, Guangzhou, China). Cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C. The authenticity of the cell line was confirmed through short tandem repeat profiling analysis.

Indomethacin (HY‐14397), L‐NAME (HY‐18729A), PGC‐1α inhibitor SR‐18292 (HY‐101491), AMPK inhibitor Compound C (HY‐13418A), and CaMKK2 inhibitor KN‐93 (HY‐15465) were purchased from MCE (Monmouth Junction, USA). Iohexol (Omnipaque 300 mg/mL) was purchased from GE Healthcare (Chicago, IL, USA).

2.2. Preparation of PCP

The medicinal part of Poria cocos (Schw.) Wolf is the dried sclerotium, commonly referred to as Fuling. PCP was extracted using the hot water method and then purified to 90% purity (Batch number: 240701) by Shanxi Tianyu Biological Production Co. Ltd. (Xianyang, Shanxi, China). The extraction process for crude polysaccharide involved several steps. First, fat‐soluble pigments and certain impurities were removed using anhydrous ethanol (material‐to‐liquid ratio, 1:10). Next, the remaining sediment was subjected to extraction with pure water (material‐to‐liquid ratio, 1:20, 60°C, 4 h). After centrifugation (6000 g, 10 min), the supernatant was collected and concentrated through vacuum evaporation, and then anhydrous ethanol (4× volume) was used to facilitate overnight alcoholic precipitation. After centrifugation (8000 g, 10 min), the collected precipitate was dried to gain the crude polysaccharide.

2.3. Fourier Transform Infrared Spectroscopy (FTIR) and Monosaccharide Analysis of PCP

Purified polysaccharide was incubated in a TFA acid solution (2 M, 121°C for 2 h). After a methanol wash, it was dissolved in sterile water and analyzed for monosaccharide fractions using an Ion Chromatography System (ICS 5000+, Thermo, USA) with a liquid column (150 × 3.0 mm, 10 μm, Dionex CarboPac PA20, Thermo, USA) and an electrochemical detector.

Mixture of potassium bromide and polysaccharides was pressed into a tablet of 1 mm thickness, and then analyzed using an FTIR instrument (Nicolet iZ‐10, Thermo, USA) with a scanning range of 4000–450 cm⁻¹ and a total of 32 scans.

2.4. Mouse Model

The Animal Experimentation Ethics Committee at West Anhui University approved all animal procedures (Approval No. 202410001). Jiangsu Huachuang Sino PharmaTech Co. Ltd. (SCXK 2020‐0009, Jiangsu, China) provided 8‐week‐old male C57BL/6J mice. All experimental animals were provided with a standard diet and had free access to water and were housed under the conditions of temperature 22°C–24°C and a 12‐h light/dark cycle. PCP was dissolved in saline at a concentration of 30 mg/mL prior to administration. The mice were randomized according to their weight and divided into six groups (n = 6 per group): the control group, the PCP‐only group (400 mg/kg), the iohexol model group, the iohexol with L‐PCP group (100 mg/kg), the iohexol with M‐PCP group (200 mg/kg), and the iohexol with H‐PCP group (400 mg/kg). Our objective is to investigate the preventive effect of PCP. Therefore, PCP is administered orally once daily, beginning 3 days before modeling, with an additional dose given on the day of modeling. The dosage settings for PCP administration were based on some literature and our preliminary research results (Duan et al. 2023; Yu et al. 2025).

To sensitize the kidneys and establish a predisposed state for injury, mice were pre‐treated with the nitric oxide synthase inhibitor L‐NAME and the non‐selective cyclooxygenase inhibitor indomethacin. This combined pre‐treatment effectively blocks key renal vasodilatory pathways mediated by nitric oxide and prostaglandins, thereby impairing renal hemodynamic adaptation. Subsequently, the contrast agent was administered to induce AKI. To establish the CI‐AKI model, mice were administered iohexol according to published methods (Oh et al. 2023). Briefly, mice were intraperitoneally injected with indomethacin (10 mg/kg) and L‐NAME (10 mg/kg) after 24 h of water deprivation. Twenty minutes later, 10 mL/kg of iohexol was administered via tail vein injection. Mice in the control group received saline injections concurrently. Subsequently, PCP was administered orally to the PCP group and the iohexol with PCP group. Twenty‐four hours later, blood and kidney tissues were collected after euthanasia of the mice using 0.2 mL of 1.25% tribromoethanol per 10 g of body weight.

2.5. Analysis of Renal Function and Morphology

Serum blood urea nitrogen (BUN), neutrophil gelatinase‐associated lipocalin (NGAL), and creatinine levels were tested to evaluate renal function. After maintaining the blood samples at 4°C for 2 h, serum was obtained by centrifugation at 5000 rpm for 15 min. Serum BUN (Cat No. E‐BC‐K183‐M), NGAL (Cat No. E‐EL‐M0828), and creatinine (Cat No. E‐BC‐K188‐M) levels were measured using assay kits provided by Elabscience (Wuhan, China).

After fixation in a 4% paraformaldehyde solution, kidney tissues were routinely dehydrated, embedded in paraffin, and sectioned into slices measuring 5 μm. Kidney sections were stained with hematoxylin & eosin (H&E) solutions (G1121, Solarbio, Beijing, China), then observed and photographed under a light microscope. Tissue damage was assessed and quantified according to a previous study (Zhang et al. 2024). All histopathology scoring was performed blinded.

For immunohistochemistry, the procedure was conducted as previously reported (Zhang, Du, Shi, et al. 2022). Briefly, the sections underwent standard dewaxing, rehydration, antigen retrieval, and blocking. Kidney sections were incubated with the KIM1 primary antibody (30948‐1‐AP, Proteintech, Wuhan, China) overnight at 4°C, and then incubated with the secondary antibody for 1 h at room temperature. Subsequently, chromogenic development was performed using DAB (ZLI‐9017, ZSGB‐BIO, Beijing, China) and hematoxylin (I030‐1‐1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) staining solutions, and the sections were visualized under a microscope. Subsequently, a semi‐quantitative analysis of KIM‐1 was conducted based on staining intensity and the proportion of positive cells. Staining intensity was scored as follows: 0, no staining; 1, weak staining (light brown, almost indistinguishable from the background); 2, moderate staining (clear brown, easily recognizable); and 3, strong staining (dark brown, intense signal). The percentage of positive cells was scored as follows: 0, 0% positive cells; 1, ≤ 25% positive cells; 2, 26%–50% positive cells; 3, 51%–75% positive cells; and 4, > 75% positive cells. The overall score was calculated by multiplying the intensity score by the positive cell proportion score.

2.6. Analysis of Oxidative Damage

Detection of oxidative damage in serum and kidney tissues was conducted using the 8‐OHdG assay kit (E‐EL‐0028c, Elabscience, Wuhan, China). The levels of cleaved poly (ADP‐ribose) polymerase (cleaved‐PARP, Asp214) were assessed through an enzyme‐linked immunosorbent assay (ELISA) kit (#7262, CST, USA). The activities of caspase‐3/7 (KTA3027, Abbkine, Wuhan, China), superoxide dismutase (SOD, Cat No. JC0115‐M), glutathione peroxidase (GPx, Cat No. JC1101‐M), glutathione (GSH, Cat No. JC1103‐M), and malondialdehyde (MDA, Cat No. JC0202‐M) in kidney tissues and cells were measured using commercially available kits purchased from JC DETECT Biotechnologies Co. Ltd. (Nanjing, China). The results were normalized to the total protein concentrations in the kidney tissue.

2.7. TUNEL Assay

Apoptosis in kidney tissues was assessed using the terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assay kit (C1098, Beyotime, China) according to the kit manual. Images were captured using a DM2000 microscope (Leica, Germany). The calculation of TUNEL‐positive cells involved determining the percentage of brown‐stained cells out of the total cells that were counterstained blue with hematoxylin.

2.8. Analysis of Intracellular and Mitochondrial ROS

Intracellular reactive oxygen species (ROS) levels were determined using the DCFH‐DA fluorescent probe (S0033, Beyotime, Shanghai, China). Mitochondrial ROS production was assessed with the MitoSOX Red probe (S0061, Beyotime, Shanghai, China). HK‐2 and TCMK1 cells were seeded into 24‐well culture plates (CCP06‐024, Bioland, Hangzhou, China) and treated with 5 μM of the MitoSOX Red probe for 30 min at 37°C. Positive signals were subsequently observed using fluorescence microscopy (Axio Observer 7, Zeiss, Oberkochen, Germany) for imaging or measured with a multimode microplate reader (Varioskan LUX, Thermo). Fluorescence intensity was quantified in relative fluorescence units.

2.9. Analysis of Mitochondrial Damage and Function

The levels of mitochondrial membrane potential (MMP) (Kit number: C2006), ATP (Kit number: C20008), NAD+/NADH (Kit number: S0175), and NADP+/NADPH (Kit number: S0179) were determined using assay kit from Beyotime (Shanghai, China). The test procedure referenced the instruction manual, and positive signals were subsequently measured using a microplate reader (Varioskan LUX, Thermo Fisher).

The level of MMP was detected using the JC‐1 fluorescent probe, following the procedure described below. TCMK1 and HK‐2 cells were digested, resuspended, and incubated with the JC‐1 staining working solution at 37°C in a cell culture incubator for 20 min. Subsequently, the cells were washed twice with staining buffer, resuspended, and analyzed using a multifunctional microplate reader (Varioskan LUX, Thermo Fisher). JC‐1 monomers were detected with an excitation wavelength of 490 nm and an emission wavelength of 530 nm, while JC‐1 aggregates were detected with excitation at 525 nm and emission at 590 nm. The MMP level was determined by calculating the ratio of fluorescence intensities of JC‐1 aggregates to monomers.

2.10. Detection of Intracellular Calcium

HK‐2 cells were plated in all‐black 96‐well plates (CCP06‐096S, Bioland, Hangzhou, China) and subsequently treated with various concentrations of PCP for 6 h. The Fluo‐4 Calcium Assay Kit (Beyotime, S1061S, Shanghai, China) was utilized to measure intracellular calcium ion concentrations. Quantitative detection of fluorescence was performed using a fluorescence enzyme labeler.

2.11. Quantification of Mitochondrial DNA

The total DNA from kidney tissues and cells was isolated using the DNA Easy Blood and Tissue Kit (#69504, Vazyme, Nanjing, China) according to the manual instructions. Mitochondrial DNA (mtDNA) quantification was performed using quantitative PCR (qPCR). The mitochondrial gene NADH–ubiquinone oxidoreductase chain 1 (ND1) was used to represent mtDNA copy number, while beta‐2 microglobulin (B2M) served as the nuclear reference gene. The cycle threshold (Ct) values for both genes were obtained from the fluorescence signal accumulation curves generated by qPCR. The relative mtDNA copy number was then calculated using the 2^−ΔΔCt method. Primers were purchased from Sangon; sequences are listed in Table S1.

2.12. Cell Viability and Apoptosis Assay

HK‐2 and TCMK1 cells were seeded into 96‐well plates (CCP06‐096, Bioland, Hangzhou, China). After the indicated PCP or iohexol treatments, the CCK‐8 assay was performed. Briefly, after adding 10 μL CCK‐8 solution per well (KGA9310, KeyGEN BioTECH, Nanjing, China), cells were incubated for 2 h at 37°C. Following the measurement of absorbance at 450 nm, the cell viability was calculated as a percentage normalized to the control group.

After collecting HK‐2 and TCMK1 cells from six‐well culture plates (CCP06‐006, Bioland, Hangzhou, China) and counting via hemocytometry, 5 × 10⁵ cells were exposed to Annexin V‐PE and 7‐AAD dyes (E‐CK‐A216, Elabscience, Wuhan, China) in binding buffer away from light at room temperature for 20 min. Subsequently, the HK‐2 and TCMK1 cells were analyzed in the flow cytometry system (DxP Athena, Cytek Biosciences, USA) after incubation for 20 min at room temperature away from light.

2.13. Cell Transfections

PGC‐1α, calcium/calmodulin‐dependent protein kinase kinase 2 (CaMKK2), and scrambled control siRNA were purchased from RiboBio (Guangzhou, China). Lipofectamine 3000 transfection reagent (L3000015, Invitrogen, Carlsbad, USA) was used to transfect HK‐2 cells with siRNAs. Sequences were provided in Table S2.

2.14. Quantitative PCR

Using Trizol Reagent (Vazyme, R411‐01, Nanjing, China) and complementary DNA (cDNA) Synthesis Kit (Vazyme, R223‐01, Nanjing, China), RNA was isolated and reverse transcribed into cDNA in a PCR tube (Jet Biofil, PCR410200, Guangzhou, China). SYBR Green qPCR Mix (Vazyme, Q711‐02, Nanjing, China) was used to perform the qPCR amplification reaction on the CFX Opus 96 real‐time PCR system (Bio‐Rad, USA). Using the 2^−ΔΔct method with β‐actin as the internal control, the relative gene expression levels were measured. Primer sequences can be found in Table S1.

2.15. Western Blotting

Renal tissue and cell lysates were prepared in Lysis Buffer (AIWB‐012, Affinibody LifeScience). Sample protein concentrations were analyzed using the BCA protein assay kit (KGP902, KeyGEN BioTECH, Nanjing, China). Proteins were denatured in loading buffer (P0963, Beyotime, Shanghai, China) at 95°C. Western blot protocols were referenced in our previous study (Zhang, Du, Xu, et al. 2022). The gray values of the protein bands were analyzed using ImageJ software (Marlyand, USA). Anti‐AMPK (#10929‐2‐AP, Proteintech, Wuhan, China), anti‐p‐AMPK (#2535, CST, Danvers, USA), anti‐GAPDH (#21612‐2, Signalway Antibody, Greenbelt, USA), and anti‐PGC‐1α (#66369–1‐Ig, Proteintech, Wuhan, China) were used.

2.16. Statistical Analysis

Data analysis and graphing were performed using GraphPad Prism 8.0 software; the results are expressed as mean ± SD. Statistical significance was determined using either Student's t‐test or analysis of variance (ANOVA) with Sidak's multiple comparisons test. p < 0.05 was deemed statistically significant.

3. Results

3.1. PCP Protects Against Iohexol‐Induced Kidney Injury in CI‐AKI Model

After extraction and purification, we obtained PCP samples with 90% purity. PCP sample was characterized by infrared spectroscopy. FT‐IR absorption peaks of PCP correspond to the following groups: 3320 nm⁻¹ (O—H), 2922 cm⁻¹ (C—H), 1651 cm⁻¹ (C=O), 1352 cm⁻¹ (—CH2), 1149 cm⁻¹ (C—O), 1018 cm⁻¹ (C—OH), and 847 cm⁻¹ (β‐type glycosidic linkage) (Figure 1A) (Lv et al. 2024; Wang et al. 2016). The results of monosaccharide analysis showed that PCP is composed of glucose (Figure 1B). Additionally, the NMR structural analysis results of PCP have been reported in our previous article (Yu et al. 2025).

FIGURE 1.

(A) FT‐IR spectrum and (B) monosaccharides analysis of PCP.

To explore the effect of PCP in mice, a CI‐AKI model was established through the administration of iohexol (Figure 2A). PCP was administered orally as a pre‐treatment for three days, followed by another dose before modeling, to investigate its potential preventive effects (Figure 2A). Kidney and body weight (KW/BW) ratios were first analyzed. Mice in the iohexol group exhibited a higher KW/BW ratio compared to the control group, whereas PCP administration led to a decrease in the KW/BW ratio (Figure 2B). Renal function analysis indicated that the levels of serum creatinine and BUN were elevated in the iohexol group in comparison to the control group (Figure 2C). Furthermore, NGAL, a marker for kidney injury, showed a much higher expression in the iohexol group than in the control group (Figure 2D). Compared to the iohexol group, PCP treatment resulted in a notable improvement (Figure 2D). H&E staining revealed that mice in the iohexol group exhibited characteristic pathological alterations indicative of renal injury (Figure 2E). However, PCP administration resulted in a notable alleviation of these pathological changes. IHC staining demonstrated PCP treatment significantly suppressed the iohexol‐induced increase in KIM‐1 levels in kidneys (Figure 2F). Collectively, these results indicate that PCP protects against iohexol‐induced AKI.

FIGURE 2.

PCP protects against iohexol‐induced AKI. (A) The schematic representation of the animal model and PCP treatment (n = 6). (B) The kidney weight/body weight ratio. (C, D) Assay of BUN, creatinine, and NGAL in serum (n = 6). (E) H&E staining and renal injury score (n = 6). (F) IHC staining and quantification of KIM‐1 in the kidney (n = 6). Scale bar 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.2. PCP Inhibits Iohexol‐Induced DNA Damage and Apoptosis in CI‐AKI Model

To investigate the effect of PCP on apoptosis in renal tissues, we performed TUNEL apoptosis assay on renal tissues. PCP significantly diminished apoptosis induced by iohexol in kidney (Figure 3A). Furthermore, significant elevations in 8‐OHdG, cleaved PARP, and caspase 3/7 activity were observed in the renal tissues of mice from the iohexol group compared to those from the control group (Figure 3B–D). The administration of PCP suppressed the iohexol‐induced elevation of 8‐OHdG, caspase 3/7 activity, and cleaved PARP levels (Figure 3B–D). qPCR assay demonstrated that PCP treatment suppressed the iohexol‐induced elevation of mRNA levels of Bax and Bak, while also increasing the mRNA expression of Bcl‐2 and Bcl‐xL (Figure 3E). These results suggest that PCP treatment can mitigate iohexol‐induced cell damage in the kidney.

FIGURE 3.

PCP inhibits apoptosis in iohexol‐induced AKI mice. (A) TUNEL staining of kidney and quantification of apoptotic positive cells with the arrows indicating apoptotic cells (n = 6). Scale bar 25 μm. (B–D) Measurement of 8‐OHdG, cleaved‐PARP, and Caspase 3/7 levels in serum and kidney (n = 6). (E) qPCR analysis of renal mRNA expression of Bax, Bak, Bcl‐2, and Bcl‐xL (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.3. PCP Enhances Antioxidant Capacity and Mitochondrial Function in CI‐AKI Model

Oxidative stress is a primary pathological mechanism that contributes to contrast‐induced acute kidney injury (Kusirisin et al. 2020). Mitochondria is a primary organelle in the cellular modulation of oxidative stress and apoptosis (Kowaltowski et al. 2009). We analyzed the levels of MDA and ROS to assess mitochondrial function and oxidative stress. Compared to the control group, mice exposed to iohexol showed a significant elevation in MDA and ROS levels (Figure 4A,B), suggesting that severe oxidative stress was induced by iohexol in the kidneys. The oxidative stress levels described above were significantly reduced following the administration of PCP pretreatment (Figure 4A,B). Additionally, the antioxidant capacity of mice in the iohexol group was significantly diminished, as evidenced by a reduction in GSH, SOD and GPx levels compared with the control group (Figure 4C–E). In contrast, the application of PCP pretreatment caused a substantial increase in GSH levels, as well as the activities of the antioxidant enzymes SOD and GPx (Figure 4C–E).

FIGURE 4.

PCP decreases oxidative damage in iohexol‐induced AKI mice. (A, B) Determination of renal MDA and ROS (n = 6). (C–E) Determination of renal GPx, SOD, and GSH (n = 6). (F–H) Determination of renal NAD+/NADH, NADP+/NADPH, and ATP (n = 6). (I) Mitochondrial content was measured by qRT‐PCR of mitochondrial DNA. (J) qPCR analysis of NRF1, NRF2, and TFAM in kidney tissues (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

We further examined the effect of PCP on mitochondrial function and biogenesis. PCP pretreatment suppressed the iohexol‐induced elevation in NADP+/NADPH levels, a reduction in NAD+/NADH and ATP levels (Figure 4F–H). Furthermore, the assay of mitochondrial DNA copy number demonstrated a significant increase in mitochondrial DNA content in the PCP‐only group relative to the control group (Figure 4I). The mRNA levels of NRF1, NRF2, and TFAM were found to be significantly higher in the PCP‐only group than in the control group, as revealed by the qPCR assay (Figure 4J). Moreover, PCP pretreatment significantly attenuated the iohexol‐induced decline in mitochondrial DNA copy number and reversed the downregulation of mitochondrial biogenesis‐related gene expression in mouse renal tissue (Figure 4I,J). Collectively, these findings suggest that PCP treatment mitigates oxidative stress, promotes mitochondrial biogenesis, and enhances antioxidant capacity, thereby preventing contrast‐induced kidney injury.

3.4. PCP Protects Against Iohexol‐Induced Renal Tubular Injury In Vitro

To provide additional evidence supporting the nephroprotective functions of PCP, in vitro experiments will be performed. TCMK1 and HK‐2 cell viability decreased with increasing concentrations of iohexol treatment compared to untreated cells (Figure 5A), while PCP enhanced cell viability after iohexol exposure (Figure 5B). Additionally, the administration of iohexol not only induced apoptosis, as indicated by flow cytometry results, but also increased the levels of 8‐OHdG and caspase‐3/7 activity (Figure 5C–E). In contrast, treatment with PCP effectively mitigated the adverse effects of iohexol (Figure 5C–E). qPCR analysis revealed that treatment with PCP reversed the iohexol‐induced downregulation of Bcl‐xL and Bcl‐2 expression, as well as the upregulation of Bak and Bax expression (Figure 5F). These results demonstrate that PCP can protect renal tubular epithelial cells (RTECs) from iohexol exposure in vitro.

FIGURE 5.

PCP protects renal tubular cells from iohexol in vitro. (A) Assessment of the viability of HK‐2 and TCMK1 cells exposed to gradient concentrations of iohexol (0, 5, 10, 20, 40, 80 mg/mL) for 24 h (n = 6). (B) Cell viability was evaluated in cells exposed to iohexol (40 mg/mL) after pretreatment with gradient concentrations of PCP (0, 25, 50, 100 μg/mL) (n = 6). (C) Assessment of the apoptotic cells (n = 5). (D, E) Determination of 8‐OHdG and caspase 3/7 levels (n = 6). (F) Determination of Bak, Bax, Bcl‐xL, and Bcl‐2 (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.5. PCP Enhances Antioxidant Capacity and Mitochondrial Function in RTECs

We further evaluated the levels of oxidative stress following iohexol administration in HK‐2 and TCMK1 cells. The levels of mitochondrial reactive oxygen species (mtROS) increased after iohexol administration; treatment with PCP abolished this effect (Figure 6A). PCP treatment inhibited the decrease in MMP and the production of MDA induced by iohexol (Figure 6B,C). Additionally, it was observed that the levels of GSH, the NAD+/NADH ratio, ATP levels, and superoxide dismutase (SOD) activity in iohexol‐treated cells were restored following PCP treatment (Figure 6D–G). Notably, PCP rescued the expression of mitochondrial DNA, which was downregulated by iohexol treatment in HK‐2 cells (Figure 6H). qPCR analysis indicated that PCP reversed the iohexol‐induced reduction of NRF1, NRF2, and TFAM expression (Figure 6I). These results demonstrate the protective effect of PCP on mitochondrial biogenesis and physiological functions in HK‐2 cells treated with iohexol.

FIGURE 6.

PCP enhances mitochondrial function and antioxidant capacity in tubular cells. (A) mtROS was detected with MitoSOX in cells exposed to iohexol (40 mgI/mL) for 6 h and pretreated with PCP (100 μg/mL) (n = 5). Scale bar 50 μm. (B) MMP was detected with JC‐1 in HK‐2 cells exposed to iohexol (40 mgI/mL) for 12 h and pretreated with PCP (100 μg/mL) (n = 5). (C–G) The levels of MDA, GSH, NAD+/NADH, SOD, and ATP were determined in HK‐2 cells exposed to iohexol (40 mgI/mL) for 24 h and pretreated with PCP (100 μg/mL) (n = 5). (H) qPCR analysis of mitochondrial DNA (n = 3). (I) Determination of NRF1, NRF2, and TFAM mRNA levels (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.6. PCP Inhibits Iohexol Induced Oxidative Damage by Activating PGC‐1α Signaling Pathway

To examine the effect of PCP on mitochondrial biogenesis, we analyzed the levels of PGC‐1α protein, a key regulatory protein involved in intracellular mitochondrial generation. In HK‐2 cells, PCP treatment raised the protein levels of PGC‐1α (Figure 7A). Pretreatment with the PGC‐1α inhibitor SR‐18292 blocked the PCP‐induced mRNA expression of NRF1, NRF2, and TFAM, which are genes associated with mitochondrial biogenesis (Figure 7B). Additionally, the inhibition of PGC‐1α reduced the protective effects of PCP on the cells, preventing PCP from elevating the mtROS and amount of mitochondrial DNA (Figure 7C). Similarly, the knockdown of PGC‐1α using siRNA yielded identical results, as it abolished the PCP‐induced mRNA expression of NRF1, NRF2, and TFAM (Figure 7D). PCP could not inhibit iohexol‐induced mtROS production, nor could it reverse the iohexol‐induced reductions in SOD activity, GSH levels, NAD+/NADH, and ATP levels following PGC‐1α knockdown (Figure 7E–I). Additionally, PCP‐induced mitochondrial production was also obstructed after PGC‐1α knockdown (Figure 7J). These findings indicate that the PGC‐1α signaling pathway is involved in the cytoprotective effect of PCP in HK‐2 cells.

FIGURE 7.

PCP protects HK‐2 cells from iohexol through the PGC‐1α signaling pathway. (A) Determination of PGC‐1α protein levels in HK‐2 cells exposed to iohexol and treated with or without PCP (100 μg/mL). (B) qPCR analysis of NRF1, NRF2, and TFAM in HK‐2 cells exposed to PCP (100 μg/mL) and treated with or without SR‐18292 (n = 3). (C) Cell viability, mtROS, and mtDNA copy number were detected (n = 5). (D) qPCR analysis of NRF1, NRF2, and TFAM in PGC‐1α knockdown HK‐2 cells exposed to iohexol and treated with or without PCP (100 μg/mL) for 24 h (n = 3). (E–J) The levels of mtROS, SOD, GSH, NAD+/NADH, ATP, and mitochondrial content were determined (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.7. PCP Activates the PGC‐1α Through CaMKK2‐AMPK Signaling Pathway

Next, we evaluated the levels of AMPK, which serves as an upstream regulatory factor for PGC‐1α. Immunoblotting results showed PCP treatment increased the levels of p‐AMPK protein (Figure 8A). To confirm that PCP induces an increase in PGC‐1α through the activation of AMPK, cells were pretreated with the AMPK inhibitor ComC. Immunoblotting results showed that the presence of ComC inhibited the PCP‐induced elevation of PGC‐1α protein levels (Figure 8B). Additionally, the PCP‐induced mRNA expression of NRF1, NRF2, and TFAM was diminished upon co‐administration of ComC (Figure 8C). Subsequently, we examined the effect of blocking AMPK signaling on the nephroprotective functions of PCP. AMPK signaling inhibition diminished the protective effects of PCP in the iohexol‐induced cell damage, prevented the PCP‐induced reduction in mtROS, the increase in SOD enzyme activity, and the production of mitochondrial DNA (Figure 8D). Collectively, the findings suggest that PCP protects the kidneys from contrast‐induced damage by activating AMPK signaling pathways.

FIGURE 8.

PCP induced PGC‐1α signal by activating the AMPK pathway. (A) Determination of p‐AMPK protein levels in HK‐2 cells exposed to iohexol and treated with or without PCP (100 μg/mL). (B) Determination of PGC‐1α protein levels in HK‐2 cells exposed to PCP (100 μg/mL) and treated with or without 10 μM ComC. (C) qPCR assay of the mRNA expression of NRF1, NRF2, and TFAM (n = 3). (D) The levels of mtROS, SOD, and mtDNA were determined (n = 5). **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

Next, we further explored the upstream signaling pathways involved in PCP‐induced AMPK activation. LKB1 and CaMKK2 are the two primary upstream kinases responsible for phosphorylating the Thr172 site of AMPK. To assess their roles in PCP‐induced AMPK phosphorylation, we utilized siRNA to downregulate their expression. The knockdown of CaMKK2, but not LKB1, abolished the effect of PCP on AMPKα phosphorylation, indicating the critical role of CaMKK2 in PCP‐induced AMPK activation (Figure 9A,B). Additionally, we observed an increase in intracellular calcium ion levels after PCP treatment (Figure 9C). Subsequent investigations were conducted to assess the impact of inhibiting CaMKK2 signaling using the inhibitor KN‐93 and siRNA. The findings revealed that the blockade of CaMKK2 signaling suppressed the inhibitory effects of PCP on iohexol‐induced mtROS, thereby impeding the PCP‐induced enhancements in SOD enzyme activity and mitochondrial DNA (Figure 9D,E). Furthermore, the knockdown of CaMKK2 also inhibited the PCP‐induced elevation of NRF‐1, NRF‐2, and TFAM mRNA levels (Figure 9F). Collectively, these results suggest that PCP promotes AMPK phosphorylation activation through CaMKK2 signaling.

FIGURE 9.

PCP promotes AMPK phosphorylation activation through CaMKK2 signaling. (A, B) Determination of p‐AMPK protein levels in HK‐2 cells transfected with CaMKK2 or LKB1 siRNA. (C) Detection of intracellular calcium ions using fluorescent probes (n = 5). (D) The levels of mtROS, SOD, and mtDNA were determined in HK‐2 cells pretreated with KN‐93 (n = 5). (E) The levels of mtROS, SOD, and mtDNA were determined in HK‐2 cells treated with PCP (100 μg/mL) for 24 h following transfection with CaMKK2 siRNA (n = 5). (F) Determination of NRF1, NRF2, and TFAM mRNA levels (n = 3). **p < 0.01, ***p < 0.001. All these experiments were performed in triplicates.

3.8. Blockade of AMPK Signaling Inhibits the Nephroprotective Effect of PCP In Vivo

To provide in vivo evidence that the nephroprotective functions of PCP rely on the AMPK signaling, the specific AMPK inhibitor ComC was utilized. ComC was administered via intraperitoneal injection to inhibit AMPK signaling activation in the kidneys 1 h prior to each PCP administration. The results indicated that pretreatment with ComC did not affect iohexol‐induced kidney toxicity; however, it significantly abolished the nephroprotective function of PCP in mice (Figure 10A). PCP exhibited a diminished capacity to inhibit the elevation of BUN, creatinine, and NGAL levels in serum following the blockade of AMPK signaling (Figure 10A,B). HE staining showed that ComC blocked the protection of PCP against iohexol‐induced kidney tissue damage (Figure 10C). Additionally, IHC staining revealed that PCP was ineffective in reducing the expression level of KIM‐1 after ComC pretreatment (Figure 10D). Collectively, the findings indicate that PCP provides protection against contrast‐induced kidney injury through the activation of AMPK signaling pathways.

FIGURE 10.

Blockade of AMPK signaling inhibits the nephroprotective effect of PCP in mice. (A, B) Determination of BUN, creatinine, and NGAL in serum (n = 6). (C) H&E staining and injury scores of the kidneys (n = 6). (D) IHC staining and quantification of KIM‐1 in kidneys (n = 6). Scale bar 50 μm. ***p < 0.001. All these experiments were performed in triplicates.

4. Discussion

In this study, our results suggest that PCP effectively preserves renal function in the presence of contrast treatment. Firstly, the prophylactic administration of PCP demonstrates efficacy in preserving renal function and ameliorating renal structural and functional damage. Secondly, the nephroprotective effect of PCP was achieved by inhibiting apoptosis, enhancing antioxidants, and improving mitochondrial function through the promotion of mitochondrial biosynthesis. Lastly, PCP demonstrates protection against contrast‐induced renal injury by activating the CaMKK2/AMPK/PGC‐1α signaling pathway.

In recent decades, PCP has garnered more attention. Numerous studies have clearly demonstrated its pharmacological and biological activities, such as anti‐tumor, anti‐oxidant, immunomodulatory, hepatoprotection, and anti‐inflammatory (Liu et al. 2021; Lv et al. 2024; Qiu et al. 2024; Wu et al. 2022; Zhao et al. 2024). PCP oral solution can significantly improve the immune function and reduce the adverse effects in patients receiving radiotherapy and chemotherapy. Additionally, poricoic acid derived from Poria cocos has been reported to protect against ischemia–reperfusion renal injury and diabetic podocyte injury (Wu et al. 2023; Younis et al. 2022). In addition, several evidences have shown that PCP exhibits potential for treating kidney diseases. The extracts of Poria cocos may reverse chronic kidney disease (CKD)'s pathological process by adjusting the altered adenine and amino acid metabolic pathways (Zhao et al. 2013). Studies have shown that PCP can improve CKD, and exhibit nephroprotective effects in hyperuricemic mice (Fu et al. 2021; Liang et al. 2021). In this study, a reliable AKI model induced by iohexol was established in mice and HK‐2 cells. Our findings demonstrated that PCP protected against iohexol‐induced nephrotoxicity by effectively mitigating elevated levels of BUN, creatinine, NGAL, and KIM‐1 expression, as well as safeguarding RTECs from contrast‐induced cell damage.

Oxidative stress and apoptosis are crucial factors in the emergence and progression of CI‐AKI (Deng et al. 2024; Kusirisin et al. 2020). PCP exhibit good antioxidant activity by scavenging free radicals, alleviating oxidative damage and regulating antioxidant enzymes (Tang et al. 2014). Treatment with PCP‐Cs reduced the levels of ROS, MDA and 8‐OHdG, increased SOD activity, and rendered RETCs resistant to oxidative damage (Li et al. 2021). Our findings demonstrated that PCP significantly heightened the antioxidant capacity of RTECs cells by elevating SOD activity and GSH levels. PCP provided protection against contrast‐induced apoptosis in RETCs by reducing the expression of Bax and Bak while increasing the expression of Bcl‐2 and Bcl‐xL.

Mitochondrial dysregulation is a common characteristic of renal injury, prompting research into nephroprotective interventions that focus on mitochondria. As a central regulator of mitochondrial biogenesis, PGC‐1α is regarded as a promising target for kidney disease therapies (Lee et al. 2019). Studies have shown PGC‐1α and its targeted genes are diminished in preclinical models of nephrotoxicity, and ischemia–reperfusion (IR), as well as in both experimental and human CKD and diabetic nephropathy (DN) (Han et al. 2017; Kwon et al. 2017; Morigi et al. 2015; Tran et al. 2016; Yuan et al. 2018; Zhang et al. 2018). In experimental DN, endogenous expression of PGC‐1α suppress oxidative stress, glomerulosclerosis, and renal tubular interstitial fibrosis (Han et al. 2017). Additionally, the protective effects of PGC‐1α have been observed in various models of renal injury induced by glycerol, IR, cisplatin, and lipopolysaccharide (Lee et al. 2019). In animal models, AMPK activators have been shown to effectively increase renal PGC‐1α activity (Fontecha‐Barriuso et al. 2020). Our results showed that PCP protects the kidney by activating PGC‐1α activation. Our results suggest that PCP can protect the kidney from contrast‐induced injury by activating PGC‐1α induced mitochondrial biogenesis. In addition, we also demonstrate that PCP‐activated CAMKK2‐AMPK signaling is responsible for PGC‐1α activation (Figure 11).

FIGURE 11.

The schematic representation illustrates the nephroprotective effect of PCP through activating CaMKK2/AMPK/PGC‐1α signaling pathways.

Currently, there are no therapeutic drugs specifically approved for CI‐AKI in clinical practice. Treatment primarily relies on hydration after onset to alleviate symptoms (Kaliyaperumal et al. 2023). Additionally, PCP has been used as an active pharmaceutical ingredient, such as in PCP oral liquid, in clinical settings to reduce the side effects of radiotherapy and chemotherapy, with an established safety profile. This study expands its indications to include CI‐AKI. The oral administration route is highly convenient for preventive medication prior to contrast procedures, facilitating patient acceptance and ease of promotion. Based on equivalent dose conversion according to body surface area (BSA) (Nair and Jacob 2016), the projected clinical doses of PCP are 8.1 mg/kg for a low dose, 16.2 mg/kg for a medium dose, and 32.4 mg/kg for a high dose.

Despite the promising findings, our study has several limitations. One limitation is the absence of a positive control group. Although there are currently no clinical drugs specifically approved for CI‐AKI, future studies could include a clinically relevant or mechanistically established nephroprotective agent (e.g., N‐acetylcysteine) as a positive control. Additionally, a comprehensive toxicological profile following PCP administration is lacking. Moreover, the long‐term safety of PCP with repeated dosing remains to be evaluated. Future studies focusing on both acute and chronic toxicity, including determination of the maximum tolerated dose and the no‐observed‐adverse‐effect level (NOAEL), is essential to fully establish the safety of PCP before clinical translation.

5. Conclusion

In summary, our findings suggest that PCP could reduce CI‐AKI by inhibiting apoptosis and enhancing antioxidant activity. Furthermore, PCP could preserve mitochondrial function and promote mitochondrial biogenesis in RTECs by activating the CaMKK2/AMPK/PGC‐1α signaling pathway. Consequently, these results offer novel perspectives on the considerable potential of PCP in ameliorating CI‐AKI.

Author Contributions

Yinuo Gong: investigation. Kai Ge: investigation, visualization. Daolun Yu: investigation, funding acquisition, visualization, formal analysis, writing – original draft. Hangbo Xu: investigation. Naidong Chen: validation, project administration. Zongmeng Zhang: conceptualization, visualization, writing – review and editing.

Funding

This work was supported by the Natural Science Research Project for Anhui Universities (2024AH052002, 2023AH052652), the High‐Level Talent Research Start‐Up Funds of West Anhui University (WGKQ2021031), and the Anhui Provincial Science and Technology Commissioner Empowers the “Straw to Meat” and Beef Cattle Revitalization Project (202513b10050012).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Sequences of primer.

Table S2: Sequences of small interfering RNA used in transfection.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.