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Calycosin ameliorates albuminuria in nephrotic syndrome by targeting Notch1/Snail pathway

BMC Nephrology volume 26, Article number: 198 (2025) Cite this article

Abstract

Background

Heavy proteinuria is an important hallmark for kidney disease including nephrotic syndrome. Astragali Radix, a traditional Chinese herb, holds the potential to alleviate nephrotic syndrome; however, the underlying mechanism has not been completely clarified. The study aimed to explore the role of calycosin (C16H12O5), a major active component of Astragali Radix, in regulating adriamycin-induced proteinuria.

Methods

A rat model of nephrotic syndrome was established through two adriamycin injections within two weeks (4 mg/kg for the first week and 2 mg/kg for the second week). After the induction of renal injury, 10 mg/kg or 20 mg/kg calycosin was intraperitoneally injected into rats for four weeks. Before euthanasia of rats, urine and blood samples were collected, and body weight was recorded. Then, 24 h urine protein content, kidney index, total cholesterol (TC), triglyceride (TG), as well as renal function indicators including blood urea nitrogen (BUN), serum creatinine (SCR), and urine albumin excretory rate (UAE) were measured. Hematoxylin-eosin staining for renal cortex tissues was performed to evaluate glomerular structural damage. TUNEL assay was performed to evaluate renal cell apoptosis. Western blotting was conducted to measure protein levels of podocyte-specific markers (podocin and nephrin), Notch1, and Snail in rat renal tissues.

Results

Calycosin reversed adriamycin-induced increase in proteinuria content, kidney index, and concentrations of renal function indicators. Calycosin ameliorated glomerular structural damage, inflammatory cell infiltration, and basement membrane thickening in model rats. In addition, calycosin rescued the suppressive impact of adriamycin on renal cell apoptosis and protein levels of podocyte markers. The activated Notch1/Snail signaling in model rats was suppressed by calycosin intervention.

Conclusion

Calycosin exerts a protective role against adriamycin-induced nephrotic syndrome via inhibition of the Notch1/Snail signaling.

Clinical trial details

Not applicable.

Peer Review reports

Introduction

Heavy proteinuria constitutes an important feature for kidney diseases, including nephrotic syndrome [1, 2]. Under normal conditions, less than 150 mg of protein is detected in the urine of human beings per day [1]. The heavy proteinuria in nephrotic syndrome is a result of renal glomerular injury induced by podocyte damage [3].

Glomerular filtration barrier can prevent the leakage of serum proteins into urine and is composed of three layers: podocytes, fenestrated endothelial cells, and the glomerular basement membrane [1, 4]. The fenestrated endothelial cells win the name because these cells have fenestrae with a diameter of 70–100 nm, which are covered by a surface layer containing glycocalyx that is beneficial for the charge-selective barrier [4, 5]. The glomerular basement membrane, an important component of the glomerular capillary wall, is critical for kidney filtration and mainly comprises type IV collagen, laminins, heparan sulfate proteoglycans, and nidogens [6]. Podocytes are separated from endothelial cells by the glomerular basement membrane, which enables small molecules and water to pass into the urinary space and retains macromolecules and cells within the circulation [6]. The response of podocytes to alterations of hemodynamic forces within the glomeruli is achieved by modulating the foot process actin cytoskeleton. It has been reported that foot process fusion reduces the total length of glomerular epithelial slit pores and thereby decreases glomerular capillary permeability to small solutes and water [7].

Natural extraction can be used to improve renal function and mitigate proteinuria according to published literature. For example, Brazilian green propolis extract significantly reduces proteinuria in patients with diabetic and non-diabetic chronic kidney disease [8]. Ginkgolide B attenuates the progression of diabetic nephropathy by alleviating ferroptosis and oxidative stress via inhibition of GPX4 ubiquitination [9]. Danshen injection ameliorates nephrotic syndrome by inducing podocyte autophagy through inactivation of the PI3K/AKT/mTOR signaling [10]. Calycosin is the main active component of Astragali Radix and has been reported to exert a protective role in many renal diseases. For example, calycosin inhibits inflammatory response dependent on NF-kappaB in renal ischemia/reperfusion injury by increasing PPARγ and reducing EGR1 [11]. Calycosin protects the renal function of rats subjected to a high fat diet and streptozotocin treatment by suppressing fibrosis, oxidative stress, and inflammation [12]. Calycosin reduces levels of serum creatine, blood urea nitrogen, and albuminuria in streptozotocin-treated Sprague-Dawely rats, suggesting that calycosin can improve kidney function in diabetic nephropathy [13]. Calycosin were included in two traditional Chinese medicines, Yiqi Huoxue Decoction and Moshen granule, and the two medicines have been recently reported to alleviate podocyte injury and nephrotic syndrome [14, 15]. However, the functions and mechanism of calycosin in regulating proteinuria in nephrotic syndrome have not been explored.

Notch1 signaling is closely associated with proteinuria and kidney dysfunction as previously reported [16,17,18]. Two other components extracted from Radix Astragali have been reported to regulate notch signaling, namely astragaloside and astragalus polysaccharide RAP [19, 20]. Astragaloside protects against bile duct ligation-induced liver fibrosis by inhibiting the activation of notch signaling [19]. Astragalus polysaccharide RAP activates Notch signaling and thereby induces macrophage polarization to M1 [20]. Calycosin is also an extract from Radix Astragali; however, whether calycosin can control the activation of Notch signaling is unknown.

The study was designed to explore whether calycosin can improve proteinuria and protect renal function in adriamycin-stimulated rats. In addition, the way in which calycosin exerts a renal protective role was also investigated. The study might provide a novel component for the treatment of proteinuria and nephrotic syndrome.

Materials and methods

Animal model establishment

Sprague-Dawley rats (male, 200–220 g) were purchased from Beijing Vital River company (Beijing, China). All rats were kept in an environment with a temperature of 25 ± 2℃, a humidity of 55 ± 5%, and a 12/12 h light/dark cycle. Standard food and water were provided ad libitum. One week later, the rats were adaptive to the environment and then randomly divided into four groups (n = 8/group) using a random number table. The four experimental groups are control group, model group, model + 10 mg/kg calycosin group, and model + 20 mg/kg calycosin group. Calycosin was purchased from Medchem Express (20575-57-9, purity: 99.86%). The concentrations of calycosin used for in vivo experiments were identified according to previous studies, with some appropriate adjustments [11, 13]. Before administration, calycosin was suspended in a mixture consisting of 10% dimethyl sulfoxide and 90% corn oil.

The nephrotic syndrome animal model was established through twice adriamycin injection. In detail, 4 mg/kg adriamycin was injected into the tail veins of the rats in the first week, and injection of 2 mg/kg adriamycin via tail vein was performed in the second week. Meanwhile, the same amount of normal saline was injected to rats in the control group.

After adriamycin intervention, all rats were kept in metabolic cages and fasted before collection of urine samples within 24 h. During the period, drinking water was provided as normal. A biochemical analyzer (Seamaty, Chengdu, China) was adopted to measure 24 h urine protein content. The successful establishment of the nephrotic syndrome model was regarded as a significantly higher 24 h urine protein content in the model group than that in the control group.

After successful modeling, rats in model + calycosin group were intraperitoneally injected with 10 mg/kg or 20 mg/kg calycosin daily for four weeks. The body weight and urine protein of the rats were measured after drug intervention. Subsequently, the rats were anesthetized through intraperitoneal injection of 50 mg/kg sodium pentobarbital to collect blood samples from the abdominal aorta. The collected blood samples were used for biochemical analysis. Finally, the rats were euthanized via intraperitoneal injection of a lethal dose of sodium pentobarbital (120 mg/kg). Renal cortex tissues were then collected for hematoxylin-eosin staining and western blotting.

Animal experiments were performed under the approval of animal ethics and were supervised by the Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd (approval number: HLK-202206112; approval date: June 15, 2022). Efforts were made to minimize the suffering of all rats.

Biochemical of blood and urine

After the collection of blood samples, a biochemical analyzer (Seamaty, Chengdu, China) was used to measure some parameters, including blood urea nitrogen (BUN), serum creatinine (SCR), total cholesterol (TC) and triglyceride (TG). In urine samples, the ratio of urinary albumin to urine creatinine was defined as urine albumin excretory rate (UAE).

Hematoxylin-eosin staining

Renal cortex tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. Then, the samples were cut into 3 μm thick sections and stained with hematoxylin-eosin for 5 min at room temperature. The sections were then dehydrated with alcohol, cleared in xylene, and covered with slips. The pathological changes of glomeruli and renal interstitium were observed under a light microscope (Olympus, Tokyo, Japan). Areas of kidney histology changes were quantified based on representative images. More specifically, ImageJ software (National Institutes of Health, Bethesda, USA) was used to analyze the percentage of tubular injury, including infiltrated inflammatory cells, tubular dilatation, and tubular vacuolization, according to a histological score system. Score 0 represents damage of less than 1%, while score 6 refers to damage of larger than 75%. Score 1: 1–10% damage. Score 2: 11–20% injury. Score 3: 21-40% damage. Score 4: 41-60% injury. Score 5: 61–75% damage.

Transmission electron microscopy

The kidney sections of 1 mm3 were fixed with 2.5% glutaraldehyde for 2 h and washed with phosphate buffer as previously described [21]. Next, the samples were immersed in osmic acid (1%, 2 h), dehydrated using graded alcohols, followed by immersion in an embedding medium overnight. After drying, the sections were cut into 60 nm slices and subjected to staining with lead citrate and uranyl acetate. A transmission electron microscope was utilized to observe the slices. The ultrastructure of the podocyte foot process was examined using the electron microscope, and the average width of the foot process was measured to assess their degree of fusion.

Western blotting

Renal tissues were lysed using a radio immunoprecipitation lysis buffer (#HY-K1001, Medchem Express, Monmouth Junction, USA). Protein extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (#88585, Millipore, Billerica, USA). The membranes were first blocked in skim milk for 2 h at room temperature and then washed thrice using Tris-buffered saline containing 0.1% Tween-20 (TBST). Next, the membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies include anti-Nephrin (#ab216341, 1/1000), anti-Podocin (NPHS2 gene, #ab181143, 1/2000), anti-Notch1 (#ab167441, 1/1000), anti-Snail (#MA5-14801, 1/1000), and anti-β-actin (#ab6276, 1/5000). Then, the membranes were washed three times with TBST and incubated with secondary antibodies at room temperature for 1 h. The bands were visualized using an electrochemiluminescence system, and the intensity of the signals was analyzed using ImageJ software. Original blots are available in the Supplementary file.

TUNEL staining

The TUNEL assay kit (Roche, Basel, Switzerland) was used in line with the instructions to examine renal cell apoptosis in rat renal samples. Rat kidney slices (3 μm) were dewaxed, rehydrated, and incubated with 20 µg/mL protease K without DNase (Roche, Germany) for 30 min at 37℃, followed by washing with phosphorate buffered saline. The sections were then incubated with 50 µL TUNEL reaction mixture at 37℃ for 1 h without light exposure. After the stained tissues were washed with phosphorate buffered saline, a confocal fluorescence microscope (Zeiss, Germany) was used to observe renal cell apoptosis. For each slice, five visual fields were selected at random. The apoptotic rate was calculated according to the formula: apoptotic index = the number of apoptotic cells × 100/total number of nucleated cells.

Statistical analysis

Data analysis was performed using GraphPad Prism (GraphPad software, La Jolla, USA). Data are shown as the mean ± standard deviation. One-way analysis of variance followed by Tukey’s post hoc analysis were used to compare differences among multiple groups. The value of p < 0.05 was the threshold for statistical significance.

Results

Calycosin exerts a protective effect on adriamycin-induced proteinuria and renal dysfunction in rats

Two weeks after adriamycin intervention, the 24 h urine protein content of rats was measured. It was found that model rats showed much higher urine protein content compared to the control group (348.5 vs. 12.5 mg) (Fig. 1A). Importantly, the urine protein content in model rats remained at a high level until the end of the experiment, which implied the successful establishment of the modeling. Calycosin treatment significantly reduced the urine protein content in model rats, especially 20 mg/kg calycosin (86.7 mg) (Fig. 1A). Hence, the protective impact of calycosin on proteinuria in nephrotic syndrome can be determined. The influence of calycosin on renal function of model rats was explored by measuring kidney index, triglyceride (TG) and total cholesterol (TC) contents, and levels of renal indicators including blood urea nitrogen (BUN), serum creatinine (SCR) and urine albumin excretory rate (UAE). As shown by Fig. 1B, the ratio of kidney weight and body weight was prominently increased in model rats (57% increase versus control group), and the increase was gradually reduced by calycosin treatment (28% and 35% decrease versus model group). In addition, the induction of renal injury contributes to high values of BUN, SCR, UAE, TG and TC (p < 0.001), and these alterations were countervailed upon calycosin treatment (p < 0.05) (Fig. 1C-G). In summary, results in the subsection revealed that calycosin exerts a protective impact on calycosin-induced proteinuria and renal dysfunction.

Fig. 1
figure 1

Calycosin exerts a protective effect on adriamycin-induced proteinuria and renal dysfunction in rats. Rats were divided into four groups: control group, model group, model + 10 mg/kg calycosin group, and model + 20 mg/kg calycosin group. The following measurement was performed after 4 weeks of indicated treatment. (A) The 24 h urine protein content in four groups of rats was measured using an automatic biochemical analyzer. (B) The kidney index (kidney weight/body weight) of rats in four groups was calculated. (C-D, F-G) Blood samples were collected from abdominal aorta, and the biochemical parameters including blood urea nitrogen (BUN), serum creatinine (SCR), triglyceride (TG), and total cholesterol (TC) in the blood were measured using an automatic biochemical analyzer. (E) After the collection of urine samples, the ratio of urinary albumin to urine creatinine was measured using a biochemical analyzer, and the ratio was defined as the urine albumin excretory rate (UAE). **p < 0.01, ***p < 0.001 versus control group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus model group

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Calycosin alleviates glomerular structural damage and podocyte injury in model rats

Glomerular filtration barrier is a structure that prevents the filtration of large proteins into the urine and is consisted of glomerular basement membrane, fenestrated endothelial cells, and podocytes [1]. Hence, whether calycosin can also alleviate glomerular structural damage in model rats was explored. Renal cortex samples were stained with hematoxylin-eosin and then observed using a light microscope. The glomerular structure in the control group was normal without glomerular atrophy or hypertrophy, and uniform basement membrane thickness was also observed (Fig. 2A-B). Many renal pathological alterations occurred in model rats. Specifically, inflammatory cells were infiltrated, and the basement membrane was thickened. Additionally, glomerular mesangial matrix was proliferated, and immune complexes were deposited in the mesangial region (Fig. 2A-B). Calycosin treatment significantly alleviated the abovementioned features that emerged after renal injury (Fig. 2A-B). As known, podocyte injury can directly cause proteinuria [22]. Transmission electron microscopy was used to observe the morphology of podocytes. As shown by Fig. 2C-D, podocytes in the control group were intact, featured by a clear and orderly arrangement of foot process, a uniform distribution of the basement membrane and endothelial cells, and no obvious podocyte fusion. After modeling, the podocyte structure was destroyed, with a higher fusion rate in the model group compared to that in the control group (20.41 folds) (Fig. 2C-D). Calycosin significantly improved the discordant podocyte structure, and the fusion rate was significantly reduced (Fig. 2C-D). Overall, calycosin alleviates proteinuria by improving glomerular structural damage and podocyte injury.

Fig. 2
figure 2

Calycosin alleviates glomerular structural damage and podocyte injury. After the euthanasia of rats in four experimental groups, renal cortex tissues were harvested. (A) Hematoxylin-eosin staining was performed to evaluate glomerular structural damage. Yellow arrows: inflammatory cell infiltration. Black arrows: the thickened basement membrane. Blue arrows: mesangial cell expansion. Red arrows: immunological complex deposition. (B) Areas of kidney histology damage was quantified based on the representative images of hematoxylin-eosin staining. (C) Transmission electron microscopy was used to observe podocyte structure. (D) Podocyte foot process was measured using the electron microscope to evaluate the degree of fusion. *p < 0.05, ***p < 0.001 versus control group; ##p < 0.01, ###p < 0.001 versus model group

Full size image

Calycosin alleviates podocyte injury and renal cell apoptosis in adriamycin-administered rats

Podocytes are important parts of glomerular filtration barrier. Podocyte injury is the major cause of proteinuria and is even regarded as the clinical symptom of proteinuria [23, 24]. In this study, podocyte-specific markers (nephrin and podocin) were subjected to western blotting to further explore the effect of adriamycin and/or calycosin on podocyte structural changes and glomerular filtration membrane. As shown by Fig. 3A-C, nephrin and podocin levels were markedly reduced after the induction of renal injury in rats (p < 0.001), implying the damage of glomerular filtration membrane and podocyte structure. Calycosin at the concentration of 20 mg/kg showed the most significant rescue effect on nephrin and podocin protein levels in model rats (p < 0.001) (Fig. 3A-C). The alterations of podocyte structural proteins further reflect the damage of podocytes and glomerular filtration membranes, which are responsible for proteinuria. As shown by Fig. 3D-E, renal cell apoptosis was enhanced in model rats compared that that in the control group (31.1% versus 7.2%). Additionally, calycosin intervention (20 mg/kg) effectively reduced the percentage of apoptotic renal cells to 15.6% (Fig. 3D-E).

Fig. 3
figure 3

Calycosin alleviates podocyte injury and renal cell apoptosis in adriamycin-administered rats. Renal tissues were collected from rats in control group, model group, model + 10 mg/kg calycosin group, and model + 20 mg/kg calycosin group. (A) Westen blot analysis was performed to measure levels of podocyte structural proteins in renal tissues and thereby assess podocyte injury. (B-C) Protein levels of nephrin and podocin were quantified based on the visualized bands with normalization to β-actin. (D) Kidney tissues were subjected to TUNEL staining to assess renal cell apoptosis. (E) The fluorescence intensity of TUNEL in each group was quantified and presented as a bar graph. ***p < 0.001 versus control group; ##p < 0.01, ###p < 0.001 versus model group

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Calycosin inactivates the Notch1/Snail signaling pathway in adriamycin-treated rats

Previous literature revealed that Notch signaling is critical for the formation of renal glomeruli during embryonic development, and the aberrant Notch1 expression can lead to proteinuria after kidney mature [25, 26]. Activation of Notch signaling can lead to the apoptosis of renal cells such as podocytes [27]. In addition, Notch1 can collaborate with Snail to participate in the progression of many diseases [18, 28]. Therefore, the Notch1/Snail signaling was investigated in this study. It was found that Notch1 and Snail protein levels were increased in renal tissues after adriamycin intervention (p < 0.001), and the trend was gradually reduced by calycosin treatment (p < 0.01) (Fig. 4A-C). Moreover, vimentin and α-SMA protein levels were also elevated by adriamycin stimulation (p < 0.001), and the changes were suppressed by calycosin administration (p < 0.01) (Fig. 4D-F). The findings suggested the inhibitory of calycosin on the Notch1/Snail signaling and epithelial-mesenchymal transition biomarkers (vimentin and α-SMA).

Fig. 4
figure 4

Calycosin inactivates the Notch1/Snail signaling pathway in adriamycin-treated rats. (A) Protein levels of Notch1 and Snail in renal tissues of rats (control group, model group, model + 10 mg/kg calycosin group, and model + 20 mg/kg calycosin group) were quantified by western blotting. (B-C) Based on the visualized bands, Notch1 and Snail levels were calculated and normalized to β-actin. (D) Western blotting was performed to measure protein levels of EMT markers (Vimentin and α-SMA) in each group. (E-F) Quantification of Vimentin and α-SMA levels in each group. ***p < 0.001 versus control group; ###p < 0.001 versus model group

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Discussion

Nephrotic syndrome is a prevalent clinical glomerular disease with massive proteinuria as the main clinical manifestation [29]. It is globally acknowledged that nephrotic syndrome is the leading cause of developing end-stage renal disease with severe renal dysfunction [30]. The current study revealed that after the induction of renal injury in adriamycin-stimulated rats, the 24 h urine protein content, kidney weight, and levels of renal function indicators were significantly increased. Glomerular structural damage, including inflammatory cell infiltration, thickening of the glomerular basement membrane, and glomerular mesangial matrix proliferation were presented in renal samples of model rats. Moreover, the loss of podocyte structural proteins reflects the induction of podocyte injury. The abovementioned changes in adriamycin-stimulated rats were all ameliorated by calycosin treatment. Despite the current adriamycin-induced nephrotic syndrome rat model, experimental animal models for nephrotic syndrome can be established using puromycin aminonucleoside and doxorubicin in similar articles [31, 32].

Nephrin and podocin are two proteins specific to podocytes [33]. Nephrin is a glomerular adhesion protein, and its abnormality mediated by mutations in the NPHS1 gene is related to nephrotic syndrome [34]. Podocin has a hairpin-like structure with cytoplasmic C- and N-terminal domains. It helps to form tight junctions between neighboring podocytes by clustering tight junction proteins such as zonula occludens-1 as well as coxsackievirus and adenovirus receptor [1]. Other common podocyte-specific markers include synaptopodin and podocalyxin, which work in concert and contribute to cytoskeleton disorder, foot process fusion, and proteinuria production [33, 35]. The prevention of podocyte injury is a feasible strategy for kidney disease treatment, and targeting these podocyte cytoskeletal proteins is a favorable choice.

The Notch pathway is a highly-conserved signaling that contributes to the transmission of short-range signals between adjacent cells [36]. It is critical during the embryonic development of proximal tubules and glomerular podocytes. In the early kidney development, Notch signaling modulates podocyte fate determination, and the silencing of the signaling leads to podocyte depletion. However, the signaling remains inactive in normal adult kidneys since the signaling is not necessary for podocyte formation beyond the stage of the S-shaped body [36]. Previous studies validated that the Notch signaling is reactivated in podocytes collected from subjects with kidney diseases [16]. Especially, Notch1 shows high expression in the injured podocytes [18]. The upregulation of Notch1 signaling in podocytes correlates to foot process effacement, progressive glomerulosclerosis, and severe proteinuria [36]. In the current study, the Notch1 signaling is activated in rats with nephrotic syndrome, which is consistent with previous evidence. In addition, the activation of Notch signaling is parallel to the EMT process and silencing the Notch1/Snail pathway can obstruct EMT in podocytes [18]. Snail can not only regulate the EMT process but also influence cell survival [37]. Activation of Notch signaling can induce podocyte apoptosis [27]. The present study disclosed the existence of a considerable number of apoptotic kidney cells and the upregulation of EMT markers (Vimentin and α-SMA) in glomerulus of model rats, which might be accountable for the reactivation of the Notch signaling.

Previous literature indicates that Notch2 is involved in the prevention of podocyte loss and nephrosis [38]. Tanaka et al. discovered that Notch2 agonist alleviates proteinuria and glomerulosclerosis in mice [38]. However, Sweetwyne et al. pointed out that genetic deletion or transgenic overexpression of Notch2 did not affect albuminuria, mesangial expansion or induce phenotypic changes [39]. This contradiction demonstrates that the role of Notch2 in podocytes is indistinct. Consistent with the role of Notch1, Notch3 is related to renal epithelium and podocyte injury [40]. Overactivation of Notch3 and inflammation in podocytes and renal endothelial cells lead to acute kidney injury [40]. Puri et al. reported that Notch4 activation leads to inflammation in human immunodeficiency virus-associated nephropathy [41]. Nevertheless, the roles of Notch3 and Notch4 in albuminuria and nephrotic syndrome are unclear and require further investigation in the future.

It has been reported that calycosin also exerted an ameliorating role in nephrectomy-induced chronic kidney disease by restraining skeletal muscle atrophy via inhibition of autophagy and oxidative stress [42]. Moreover, calycosin can amplify the antifibrotic activity of mesenchymal stem cells in unilateral ureteral obstruction-induced chronic kidney disease [43]. Consistently, the current study demonstrated the suppressive effect of calycosin on proteinuria, renal dysfunction, glomerular structural damage, podocyte injury, and renal cell apoptosis in adriamycin-induced nephrotic syndrome rat model. In the future, the functions of calycosin in other models of chronic kidney disease may require further exploration.

Off-target effects can affect therapeutic efficacy and give rise to side effects and toxicity [44]. Identification of targets for natural components plays a crucial role in the translation and development of medicines, as it is linked to off-target side effects and toxicity. At present, omics-based techniques such as bioinformatics, genomics, metabolomics, proteomics, and transcriptomics are known as effective tools for the discovery of natural product targets [44]. In this study, the off-target effect of calycosin was not explored, constituting a limitation of this investigation. Another limitation of the study is that the mechanism was preliminary investigated, and the Notch activator or inhibitor was not used for further exploration. Additionally, only the in vivo animal model was established, and podocyte cell line was not used for in vitro experiments in the study.

In summary, the study validated that calycosin exerts a protective effect on adriamycin-induced proteinuria in rats by mitigating glomerular structural damage and podocyte injury via inhibition of the Notch1/Snail signaling. The study enriches the role of calycosin in kidney diseases and may be beneficial for the development of multiple therapeutical options for proteinuria. For future work, the safe dose of calycosin and its side effects should be determined and clarified.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Acknowledgements

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Funding

This work was supported by 2021 Shenzhen Baoan District Basic Research (Medical and Health) Project (Shenzhen Baoan District Science and Technology Innovation Bureau) (No. 2021JD164).

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Authors and Affiliations

  1. Department of Nephrology, Shenzhen Bao’an Authentic TCM Therapy Hospital, Room 1703, Block G, Jiazhou Business Center, Baomin 1 Road, Xin ’an Street, Bao ’an District, Shenzhen, Guangdong, 518100, China

    Xiaohong Ma

  2. Department of Internal Medicine, Shenzhen Bao’an Authentic TCM Therapy Hospital, Shenzhen, 518100, China

    Binghe Guan & Linrong Pang

Authors
  1. Xiaohong Ma
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  2. Binghe Guan
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  3. Linrong Pang
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Contributions

Xiaohong Ma was the main designer of this study. Xiaohong Ma, Binghe Guan and Linrong Pang performed the experiments and analyzed the data. Xiaohong Ma drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xiaohong Ma.

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Animal experiments were performed under the approval of animal ethics and supervised by the Ethics Committee of Wuhan Myhalic Biotechnology Co., Ltd (approval number: HLK-202206112; approval date: June 15, 2022). We confirm that the study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

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Ma, X., Guan, B. & Pang, L. Calycosin ameliorates albuminuria in nephrotic syndrome by targeting Notch1/Snail pathway. BMC Nephrol 26, 198 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12882-025-04113-3

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12882-025-04113-3

Keywords

BMC Nephrology

ISSN: 1471-2369

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