Baicalin alleviates adriamycin-induced focal segmental glomerulosclerosis and proteinuria by inhibiting the Notch1-Snail axis mediated podocyte EMT

doi:10.1016/j.lfs.2020.118010

Life Sciences

Volume 257, 15 September 2020, 118010

https://doi.org/10.1016/j.lfs.2020.118010 Get rights and content

Highlights

•
Baicalin, as a natural bioactive component, is a novel renoprotective agent against FSGS.
•
Baicalin suppresses podocyte EMT accompanied by decreased podocyte injury and cell migration.
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Down-regulating the Notch1-Snail axis is a significant mechanism for baicalin to inhibit podocyte EMT.
•
Baicalin improves renal insufficiency and proteinuria in adriamycin-induced nephropathy.

Abstract

Podocyte injury is an early event and core in the development of focal segmental glomerular sclerosis (FSGS) that induces poor prognosis. Epithelial-mesenchymal transition (EMT) as a response of podocyte to injury leads to podocyte depletion and proteinuria. The abnormally reactivated NOTCH pathway may be involved in podocyte EMT. Baicalin, as a natural flavonoid compound, had significant inhibitory activity on tissue fibrosis and tumor cell invasion. However, its potential role and molecular mechanisms to injured podocyte in FSGS are little known. Here we found that baicalin could inhibit podocyte EMT markers expression and cell migration induced by TGF-β1, accompanied by the up-regulated expression of slit diaphragm (SD) proteins and cell-cell adhesion molecule. Further investigation revealed that EMT inhibition of baicalin on injured podocyte is mainly mediated by the reduction of notch1 activation and its downstream Snail expression. Using the adriamycin-induced FSGS model, we determined that baicalin suppresses the Notch1-Snail axis activation in podocytes, relieves glomerulus structural disruption and dysfunction, and reduces proteinuria. Altogether, these findings suggest that baicalin is a novel renoprotective agent against podocyte EMT in FSGS and indicate its underlying mechanism that involves in negative regulation of the Notch1-Snail axis.

Introduction

FSGS is used to describe a clinicopathological feature with marked proteinuria and podocyte injury in progressive kidney disease. Recent epidemiological studies have shown that the prevalence of FSGS is on the rise worldwide [1]. In primary nephropathy, FSGS is considered as one of the most critical risk factors for progression to end-stage renal disease (ESRD). Unfortunately, adult podocytes cannot adequately proliferate following depletion, which associated with higher albuminuria and decreased renal function. Little is known about the molecular and cellular events in the response of podocyte to injury, leading us to the bottleneck of exploring targeted drugs for FSGS.

Podocyte injury characterized by foot process effacement or shedding is an early event and core link in the development of FSGS. It is noteworthy the fact that shed podocytes can continue to be cultured, which is difficult to explain with apoptosis. Transforming growth factor-β1 (TGF-β1), as a well-known EMT inducer, is highly expressed in injured podocyte and associated with both glomerulosclerosis and podocyte depletion [2,3]. These suggest that the real cause of podocyte shedding might be the beginning of EMT. Notch signaling is essential during the embryonic development of glomerular podocytes and proximal tubules [4]. On the contrary, Notch signaling is not required for podocyte formation beyond the stage of the S-shaped body [4,5]. Especially, Notch1 is reactivated and highly expressed in the injured podocyte. Therefore, blocking Notch activation and its downstream EMT pathways in injured podocyte is a potential therapeutic strategy for FSGS.

Increasing evidence supports the effectiveness of traditional Chinese medicine in nephropathy treatment. Baicalin, as a natural flavonoid compound isolated from the roots of Scutellaria baicalensis Georgi, has shown a positive renal protective potential in kidney diseases related to tubulointerstitial impairment, including alleviating renal tubulointerstitial injury by inhibiting fibrosis and apoptosis, reducing hypoxic and inflammatory damage [[6], [7], [8], [9], [10]]. However, there is little known about the effect and underlying mechanism of baicalin on kidney diseases induced by glomerular impairment, especially FSGS characterized by podocyte injury. The treatment of FSGS in chronic kidney disease (CDK) remains a substantial huge clinical challenge.

In this study, we used immortalized podocytes and model mice with adriamycin nephropathy to investigate the effect of baicalin on podocytes. Importantly, we demonstrated that baicalin ameliorates FSGS progress by reducing podocyte injury, and revealed its potential mechanism that baicalin inhibits Notch1-Snail axis mediated podocyte EMT. We suggest baicalin as potential therapeutic strategy for FSGS.

Section snippets

Cell culture and treatment

The conditionally immortalized mouse podocyte line MPC-5 was grown in RPMI-1640 medium (Gibco-BRL, China) supplemented with 10% fetal bovine serum (FBS) and recombinant IFN-γ (PeproTech, USA) growth permissive conditions at 33 °C in a 5% CO₂ incubator. Baicalin (Sigma-Aldrich, USA) and recombinant mouse TGF-β1 (Cell Signaling Technology, USA) were dissolved in DMSO and 20 mM sodium citrate (pH 3.0) respectively. Until cells fusion reached 70%, the medium was removed and replaced with complete

Baicalin inhibits TGF-β1-induced podocyte EMT

We firstly evaluated the possible cytotoxicity of baicalin. The MTT results showed that baicalin had no significant effect on cell viability for 72 h at concentrations up to 20 μM. To further identify the roles of baicalin on inhibiting podocyte EMT, MPC-5 was pretreated with baicalin for 48 h and then exposed to 2 ng/ml TGF-β1 for 30 min. Baicalin significantly suppressed Vimentin and increased P-cadherin in a dose-dependent manner, with little negative impact on cell viability. A 10 μM

Discussion

Podocytes are terminally differentiated kidney cells localized to the outer surface of the glomerular basement membrane (GBM) and play a central role in the maintenance of the glomerular filtration. Podocyte EMT has been thought to be a response of podocyte to adverse stimuli and is characterized by cytoskeletal rearrangement and adhesion loss. This process is involved in the induction of the podocyte injury and even shedding [14]. In this study, we demonstrate that baicalin has a

Declaration of competing interests

The authors declare no conflict of interest.

CRediT authorship contribution statement

Yitian Dou: Validation, Formal analysis, Resources, Writing - original draft, Writing - original draft, Supervision, Funding acquisition. Yichun Shang: Validation, Formal analysis, Data curation, Funding acquisition. Yongmei Shen: Data curation, Visualization. Jingtian Qu: Supervision, Funding acquisition. Chunliu Liu: Validation, Writing - original draft. Jiasong Cao: Conceptualization, Methodology, Writing - review & editing, Project administration.

Acknowledgements

This work is supported by grants from the National Natural Science Foundation of China (81403333, 81703968 and 81603648) and Tianjin Municipal Education Commission (2017KJ154). In addition, Jiasong Cao especially wishes to thanks his mother who is suffering from nephrotic syndrome, her encouragement has given him powerful support to this research.

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Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease (ESRD). The progression of DKD is often marked by heightened renal fibrosis due to hindered fatty acid oxidation within renal tubules. Baicalin (BA), a naturally derived compound, has exhibited the potential to mitigate the advancement of DKD. Delving deeper into the precise targets and mechanisms of BA's effect on DKD is crucial.
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The results demonstrated that BA notably reduced lipid accumulation and renal fibrosis in db/db mice. Moreover, mRNA sequencing pinpointed a significant downregulation of CPT1α in DKD. In vitro assays revealed that both the overexpression of CPT1α and treatment with BA exerted similar influences on mitochondrial respiration, fatty acid oxidation, and renal fibrosis levels. Given the pronounced downregulation of CPT1α in DKD patients and its substantial correlation with clinical indicators, it was evident that CPT1α could serve as a therapeutic target for BA in addressing DKD.
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Laminaria japonica polysaccharide attenuates podocyte epithelial-mesenchymal transformation via TGF-β1-mediated Smad3 and p38MAPK pathways
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In the present work, we explored the interventional effect and potential mechanism of a purified Laminaria japonica polysaccharide (LJP61A) on podocyte epithelial-mesenchymal transition (EMT) in TGF-β1-induced podocytes and adriamycin-treated mice. Results showed that compared to the model groups, LJP61A significantly up-regulated the levels of epithelial markers (Nephrin, WT-1, podocin) and down-regulated the levels of mesenchymal markers (α-SMA, FN1) in vitro and in vivo, thus preventing EMT-like morphological changes of podocytes, proteinuria and kidney injury. Smad3 and p38MAPK are two central pathways mediating podocyte EMT activated by TGF-β1. We found that LJP61A suppressed TGF-β1-induced activation of Smad3, Smad4 and p38MAPK in vitro and in vivo. Moreover, the inhibitory actions of LJP61A on podocyte EMT were synergistically strengthened by Smad3 inhibitor SIS3 and p38MAPK inhibitor SB203580. Taken together, these findings revealed that LJP61A could prevent podocyte EMT, which might be related to the inhibition of TGF-β1-mediated Smad3 and p38MAPK pathways.
EP300/CBP is crucial for cAMP-PKA pathway to alleviate podocyte dedifferentiation via targeting Notch3 signaling
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Citation Excerpt :
Notch signaling makes a crucial contribution in podocyte development and maintains quiescence after birth [6]. Abnormal activation of Notch signaling has been reported in podocytes in various proteinuric kidney diseases [5,7] and is involved in podocyte dedifferentiation in other words epithelial-mesenchymal transition (EMT) [8,9]. Among the four Notch receptors (Notch1∼4), Notch3 attracts significant attention because of its unique molecular construct and roles in podocyte-injured kidney diseases.
Podocyte injury is the hallmark of proteinuric glomerular diseases. Notch3 is neo-activated simultaneously in damaged podocytes and podocyte's progenitor cells of FSGS, indicating a unique role of Notch3. We previously showed that activation of cAMP-PKA pathway alleviated podocyte injury possibly via inhibiting Notch3 expression. However, the mechanisms are unknown. In the present study, Notch3 signaling was significantly activated in ADR-induced podocytes in vitro and in PAN nephrosis rats and patients with idiopathic FSGS in vivo, concomitantly with podocyte dedifferentiation. In cultured podocytes, pCPT-cAMP, a selective cAMP-PKA activator, dramatically blocked ADR-induced activation of Notch3 signaling as well as inhibition of cAMP-PKA pathway, thus alleviating the decreased cell viability and podocyte dedifferentiation. Bioinformatics analysis revealed EP300/CBP, a transcriptional co-activator, as a central hub for the crosstalk between these two signaling pathways. Additionally, CREB/KLF15 in cAMP-PKA pathway competed with RBP-J the major transcriptional factor of Notch3 signaling for binding to EP300/CBP. EP300/CBP siRNA significantly inhibited these two signaling transduction pathways and disrupted the interactions between the above major transcriptional factors. These data indicate a crucial role of EP300/CBP in regulating the crosstalk between cAMP-PKA pathway and Notch3 signaling and modulating the phenotypic change of podocytes, and enrich the reno-protective mechanisms of cAMP-PKA pathway.
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These results suggested that baicalin was different from baicalein in the regulation of cell apoptosis and inflammation. XO was the key enzyme for UA production (Dou et al., 2020). Studies found that UA and MSU caused the kidney inflammation through TLR2, TLR4, and NLRP3 pathways (Yin et al., 2019; Milanesi et al., 2019).
Oxidative stress and inflammation in kidney are the main causes for hyperuricemic nephropathy (HN). Baicalin and baicalein, two flavonoids, have anti-inflammatory and anti-oxidative effects and they are interconvertible in the body. In this study, both baicalin and baicalein were administered by intragastric administration (i.g.) or intraperitoneal injection (i.p.) at the dose of 50 mg kg⁻¹, once a day for 15 consecutive days to HN mice, a model established by i.g. of yeast extract combined with i.p. of potassium oxonate. In HN mice, baicalin and baicalein reduced serum uric acid (SUA) levels and protected kidneys by anti-inflammatory and anti-oxidative effects. Mechanistically, the effect of baicalin and baicalein on reducing SUA levels might due to their inhibitory effect on xanthine oxidase (XO) activity in vivo and in vitro. Furthermore, the mechanisms of baicalin and baicalein against HN were analyzed with network pharmacology and molecular docking technology. The network pharmacology indicated that the protective effects of baicalin and baicalein against HN were mainly related to their down-regulating effects on TLRs, NF-κB, MAPK, PI3K/AKT and NOD-like receptor signaling pathways. Molecular docking indicated high binding affinity of baicalin/baicalein to targets such as AKT1 and MAPK1. In summary, baicalin and baicalein are promising drug candidates for the treatment of HN by inhibiting XO activity, reducing inflammation and cell apoptosis through down-regulating TLRs/NLRP3/NF-κB, MAPK, PI3K/AKT/NF-κB pathways.
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¹: These authors contributed equally to this paper

View full text

Baicalin alleviates adriamycin-induced focal segmental glomerulosclerosis and proteinuria by inhibiting the Notch1-Snail axis mediated podocyte EMT

Highlights

Abstract

Introduction

Section snippets

Cell culture and treatment

Baicalin inhibits TGF-β1-induced podocyte EMT

Discussion

Declaration of competing interests

CRediT authorship contribution statement

Acknowledgements

Focal segmental glomerulosclerosis

Clin. J. Am. Soc. Nephrol.

Activation of the TGF-beta/Smad signaling pathway in focal segmental glomerulosclerosis

Kidney Int.

Low TGFbeta1 expression prevents and high expression exacerbates diabetic nephropathy in mice

Proc. Natl. Acad. Sci. U. S. A.

The role of Notch signaling in specification of podocyte and proximal tubules within the developing mouse kidney

Kidney Int.

The role of Notch signaling in kidney podocytes

Clin. Exp. Nephrol.

Baicalin and baicalein attenuate renal fibrosis in vitro via inhibition of the TGF-beta1 signaling pathway

Exp Ther Med

Baicalin ameliorates H2O2 induced cytotoxicity in HK-2 cells through the inhibition of ER stress and the activation of Nrf2 signaling

Int. J. Mol. Sci.

A potential role of Baicalin to inhibit apoptosis and protect against acute liver and kidney injury in rat preeclampsia model

Biomed. Pharmacother.

The protective effect of baicalin against renal ischemia-reperfusion injury through inhibition of inflammation and apoptosis

BMC Complement. Altern. Med.

PPARgamma activation by baicalin suppresses NF-kappaB-mediated inflammation in aged rat kidney

Biogerontology

Adriamycin nephropathy: a model of focal segmental glomerulosclerosis

Nephrology (Carlton)

Trpc6 inactivation confers protection in a model of severe nephrosis in rats

J Mol Med (Berl)

Estrogen receptor alpha-NOTCH1 axis enhances basal stem-like cells and epithelial-mesenchymal transition phenotypes in prostate cancer

Cell Commun Signal

Progression of glomerular diseases: is the podocyte the culprit?

Kidney Int.

Wnt/beta-catenin signaling promotes podocyte dysfunction and albuminuria

J. Am. Soc. Nephrol.

Disease-causing mutation in alpha-actinin-4 promotes podocyte detachment through maladaptation to periodic stretch

Proc. Natl. Acad. Sci. U. S. A.