Introduction: Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1 (TGF-β1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulphate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress, and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-β1 signalling pathway is still unclear. Methods: In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA by quantitative real-time PCR (qRT-PCR) and Western blotting after siRNA-mediated TGF-β1 silencing, TGF-β1 overexpression, and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. Results: The results indicated that nephrin, synaptopodin, CD2AP, and SRGAP2a expressions were significantly upregulated, and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-β1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-β1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7, and NOX4; and increased ROS production in podocytes. Conclusion: This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP, and SRGAP2a by regulating the ROS and TGF-β1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.

Diabetic kidney disease (DKD) is a disease that exhibits pathological structural and functional changes in the kidney secondary to diabetes mellitus (DM) and has become the leading cause of end-stage kidney disease worldwide [1]. Clinical studies have shown that due to the decreased glomerular filtration rate, DKD leads to the accumulation of a variety of metabolic compounds in the body, causing structural abnormalities and dysfunctions in various tissues and organs of the body and related metabolic disorders and clinical manifestations [2, 3]. Such metabolic compounds are called uraemic toxins. The protein-bound uraemic toxins indoxyl sulphate (IS) and p-cresol sulphate are the two most harmful protein-bound uraemic toxins due to their binding to plasma albumin, which results in residual renal function and routine haemodialysis in patients with DKD [4]. The levels of IS and p-cresyl sulphate in the blood of DKD patients are the key independent factors that promote renal fibrosis and induce the progression of DKD to end-stage renal disease [5]. The occurrence and development of glomerular sclerosis and interstitial fibrosis generally start in the glomerulus, and glomerular podocytes are an important part of the kidney filtration barrier [6]. The glomerular basement membrane (GBM) detaches and is excreted in the urine, resulting in exposure of the GBM and an impaired filtration barrier [7]. Clinically, there may be urinary protein leakage, impaired filtration function, tubular interstitial damage, and fibrosis [8]. Therefore, podocyte injury, foot process changes, and cell shedding are often the key causes of glomerulosclerosis initiation, the promotion of DKD, and its progression under the action of multiple aetiologies.

The protein-binding toxin IS accumulates in the body of DKD patients, which directly damages intrinsic renal cells, such as glomerular podocytes, and induces and promotes the progression of renal fibrosis [9]. Podocytes and foot mutagenesis are mainly regulated by the cytoskeletal microfilaments of the foot processes. The cytoskeletal-related proteins nephrin and synaptopodin, which are specifically expressed in the foot processes of podocytes, are important molecules that regulate cytoskeletal microfilaments [10, 11]. As a result, the podocyte cytoskeletal microfilaments are reduced, and the cytoskeletal structure is destroyed. Eventually, the podocytes are separated and detached from the basement membrane, and the integrity of the podocytes and the hiatus septum is impaired. Studies have shown that podocyte injury is associated with and plays a key role in the progression of nephrotic syndrome, glomerulosclerosis, and a variety of chronic kidney diseases [12]. The slit diaphragm (SD) between the foot processes of the podocytes is a key factor in maintaining the integrity of the filtration barrier and preventing proteinuria. The nephrin, podocin, and CD2AP molecules on the SD constitute a zipper-like protein complex, which is an indispensable molecular structure for maintaining the stability and normal function of the SD [13]. The SD protein complex also mediates the tight connection between the SD and the cytoskeleton-related proteins synaptopodin and a-actinin-4, which play an important role in maintaining the integrity of the actin skeleton and the normal migration of podocytes [14].

New evidence from clinical and experimental studies suggests that IS may play a role in the progression of CVD in CKD patients by enhancing oxidative stress in the myocardium and vascular system [15]. NOX is the main source of reactive oxygen species (ROS), and IS is the strongest ROS producer among many known protein-bound uraemic toxins. In vascular smooth muscle cells, IS enhances the activity of NADPH oxidases such as NOX4; upregulates the expression of osteoblast proteins such as core binding Factor 1 (Cbfa1), alkaline phosphatase, and osteopontin; and promotes ROS in aortic smooth muscle cells. Formation and osteogenesis suggest that IS induces the expression of osteoblast-specific proteins through oxidative stress, leading to arterial calcification [16]. One study showed that IS can also stimulate the production of ROS in human umbilical vein endothelial cells, thereby reducing the production of nitric oxide (NO) and activating the excessive oxidation of endothelial cells. Under the stimulation of TGF-β1, the production of NOX4 is increased by the Smad protein, which increases intracellular ROS, promotes ECM, and inhibits its degradation [17]. Further studies have found that the increase in ROS production may be related to the promotion of the conversion of inactivated TGF-β1 into the active form, an increase in the phosphorylation of Tβ-RI (ALK5), and the promotion of TGF-β/Smad signalling. Recent studies have shown that oxidative stress is a key factor in DKD podocyte damage [18]. Our research elucidates the mechanism of indoxyl sulphate-induced podocyte damage in diabetic nephropathy, which has important guiding significance for the diagnosis and treatment of renal diseases.

Primary Podocyte Culture

We isolated two rat kidneys under aseptic conditions and dissected and gently ground the kidney cortex. The tissues were rinsed by sequential passage through 100 and 200 μm sieves at 4°C. Glomeruli were collected on 200 μm sieves, and 2 g/L type IV collagenase (Sigma, China) was properly digested at 37°C for approximately 15 min. Under observation with an inverted microscope, when a few cells were dissociated in the medium, complete Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, USA) was added to terminate the digestion. The cells were centrifuged and washed twice at 1,000 r/min for 10 min, resuspended in DMEM, inoculated into three 75 cm2 culture flasks lined with rat tail collagen, and incubated at 37°C and 5% CO2 for 7–8 days. The majority of cells observed with an electron microscope (Leica TCS SP8 STED, Germany) were primary podocytes.

Immunofluorescence Staining

Primary podocytes were cultured for another 7 days after being trypsinized and subsequently passaged. Podocyte morphology was observed with an inverted phase contrast microscope (Leica TCS SP8 STED, Germany) when the cells covered the bottom of the dish. The cells were fixed with cold acetone and processed for immunofluorescence staining of the podocyte markers nephrin and synaptopodin.

Cell Transfection

All siRNAs used in this study were synthesized by Biostorms (Suzhou, China). The siRNA-TGF-β1 sequence consisted of a 21-nucleotide sense strand (5′-GAC​AAG​UUC​AAG​CAG​AGU​ACA-3′) and an antisense strand (5′-UAC​UCU​GCU​UGA​ACU​UGU​CAU-3′). Podocytes were inoculated in 24-well plates and transfected with the designed siRNAs to mediate TGF-β1 silencing with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions (Thermo Fisher, Waltham, MA, USA). Twenty-four hours after transfection, the podocytes were treated with high glucose (HG) and harvested for subsequent quantitative real-time PCR (qRT-PCR) and Western blotting analyses.

Quantitative Real-Time PCR

Podocyte samples were treated with mannitol (100 mm), glucose (100 mm), glucose (100 mm) plus acetylcysteine (10 μm) or glucose (100 mm) plus IS (0–200 μg/mL, dissolved in DMSO) for 24 h, and samples without additional sugar treatment were used as a negative control [19]. The medium was then removed from the 6-well cell culture plate, and 1 × PBS solution (Sangon Shanghai) was added to gently wash the cells. Next, the 6-well plate was placed on ice, 800 μL of TRIzol reagent (Sigma, USA) was added to each sample, which was repeatedly pipetted to dislodge all adherent cells, and the cells were transferred to 1.5 mL EP tubes. The total RNA of each sample was extracted according to the manufacturer’s instructions and reverse transcribed with the PrimeScript RT reagent kit (TaKaRa, Japan). qRT-PCR was performed with SYBR Green detection mix (TaKaRa, Japan). The relative expression levels of genes in this study were normalized to actin expression, analysed using the 2−ΔΔCt method, and summarized from three separately harvested podocyte samples.

Western Blot Analysis

Podocyte samples were treated as described above for qRT-PCR. Total protein was extracted from each sample to prepare cell lysates using RIPA lysis buffer (Sangon, Shanghai, China) and stored at −20°C. The bicinchoninic acid (BCA) protein quantification method was used to ensure that the concentration of each sample was basically equal. Protein samples were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes within electrophoresis systems (Tanon VE180 and Tanon VE186, Shanghai, China). The PVDF membranes were blocked with 5% (w/v) skimmed milk powder for 2 h and incubated at 4°C overnight with the following primary antibodies: nephrin (Abcam ab216341, diluted 1:1,000), synaptopodin (Abcam ab259976, diluted 1:1,000), CD2AP (CST #5478S, diluted 1:1,000), SRGAP2a (GeneTex GTX130797, diluted 1:500), α-SMA (Abcam ab150301, diluted 1:1,000), decapentaplegic (Smad)2/3 (Abcam ab202445, diluted 1:1,000), phosphorylated (p)-Smad2/3 (Abcam ab280888, diluted 1:500), Smad7 (Abcam ab216428, diluted 1:500), and NAD(P)H oxidase 4 (NOX4) (Abcam ab133303, diluted 1:2,000). After being washed with 1 × PBS solution (Sangon, Shanghai, China) three times, the membranes were incubated with HRP-labelled goat anti-rabbit IgG secondary antibodies (Abcam ab205718, diluted to 1:10,000). Immunoreactivity was determined with enhanced chemiluminescence reagent (Thermo Fisher). A gel imaging system (BIO-RAD Gel DocXR +, US) and software (BIO-RAD Image Lab Software, Version 5.1 and SPSS 20.0) were used for imaging and statistical analysis. GAPDH was used as an internal control to ensure equal protein loading.

Analysis of Intracellular ROS Generation

We measured intracellular ROS generation with the fluorescent probe DCFH-DA, which is hydrolysed and generates nonfluorescent DCFH after passing through the cell membrane. Intracellular ROS oxidizes DCFH to produce fluorescent DCF. The intensity of DCF fluorescence indicates the level of intracellular ROS. Podocyte samples were treated as described above for qRT-PCR and Western blotting, washed with cold 1 × PBS solution once, resuspended in serum-free DMEM, and incubated with DCFH-DA (10 μm) for 30 min at 37°C. The samples were mixed every 3–5 min to ensure good contact between the probes and podocytes. The podocytes were then washed with serum-free DMEM three times and resuspended in 1 × PBS solution. Finally, the podocyte samples were analysed by flow cytometry (Life Attune NxT, US). FlowJo 10 software (BD, USA) was used for data analysis.

Statistical Analysis

SigmaPlot 12.0 and SPSS 20.0 were used for statistical analysis. All data are presented as the mean ± SD. Independent group comparisons were performed using a Student’s t test or one-way ANOVA with Bonferroni’s post hoc test. A value of p < 0.05 was considered statistically significant.

IS Altered the Expression of Differentiation Markers in Rat Podocytes in vitro

Podocytes are important target cells for various kidney diseases. Therefore, we selected podocytes as the experimental material in this study. To identify the podocytes, we used synaptopodin (Fig. 1a) and nephrin (Fig. 1b), which are specific markers for differentiated podocytes, for immunofluorescence labelling, and co-localization of the two markers through double immunostaining (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000538858). Furthermore, cell viability decreased in a dose-dependent manner (Fig. 1c). The expression of podocyte markers Nephrin and Synaptopodin were significantly decreased by IS exposure in a dose-dependent fashion for 24 h (Fig. 1d). The results indicated that the cultured differentiated rat podocytes exposed to 200 μg/mL IS were sufficiently pure for further study.

Fig. 1.

Immunofluorescence staining to label synaptopodin (a) and nephrin (b) in cultured differentiated rat podocytes. c The viability of differentiated podocytes was assessed using an MTT assay under IS (0–200 μg/mL) treatment for 24 h. d Podocyte marker mRNA expression was assessed by qRT-PCR in differentiated rat podocytes after IS (0–200 μg/mL) treatment for 24 h.

Fig. 1.

Immunofluorescence staining to label synaptopodin (a) and nephrin (b) in cultured differentiated rat podocytes. c The viability of differentiated podocytes was assessed using an MTT assay under IS (0–200 μg/mL) treatment for 24 h. d Podocyte marker mRNA expression was assessed by qRT-PCR in differentiated rat podocytes after IS (0–200 μg/mL) treatment for 24 h.

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Nephrin, Synaptopodin, CD2AP, SRGAP2a, and α-SMA Are Involved in the TGF-β1 and ROS Signalling Pathways in Rat Podocytes

Recent studies have demonstrated that oxidative stress is a key factor in the damage of DKD podocytes. TGF-β1 stimulates the expression of NOX4 mediated by Smad protein in cells and induces an increase in ROS levels. Thus, we focused on the expression levels of the podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA in the TGF-β1 and ROS signalling pathways. siRNA-mediated silencing of TGF-β1 and TGF-β1 overexpression was performed in rat podocytes. Based on the results from qRT-PCR and Western blotting, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a increased in the TGF-β1 siRNA group but decreased in the TGF-β1-OE group compared with those in the control without TGF-β1 silencing under HG conditions (Fig. 2a–d). However, the expression of α-SMA was decreased in the TGF-β1 siRNA group but increased in the TGF-β1-OE group compared with that in the NC group (Fig. 2e). The Western blot results were consistent with the qRT-PCR assay results at the protein level (Fig. 2f–k). In addition, when the ROS inhibitor acetylcysteine was added to the HG solutions, the mRNA levels of nephrin, synaptopodin, CD2AP, and SRGAP2a were obviously increased, but α-SMA was decreased compared with those in the HG group (Fig. 3a–e). The Western blot results were consistent with the qRT-PCR findings (Fig. 3f–k). These results indicate that the podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA are involved in TGF-β1 and the ROS signalling pathways in rat podocytes.

Fig. 2.

The podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA were involved in the TGF-β1 signalling pathways in rat podocytes. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA in TGF-β1-silenced + HG treatment and TGF-β1-OE + HG treatment. f The Western blot results showed the nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA levels in TGF-β1-silenced + HG treatment and TGF-β1-OE + HG treatment. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01.

Fig. 2.

The podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA were involved in the TGF-β1 signalling pathways in rat podocytes. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA in TGF-β1-silenced + HG treatment and TGF-β1-OE + HG treatment. f The Western blot results showed the nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA levels in TGF-β1-silenced + HG treatment and TGF-β1-OE + HG treatment. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01.

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Fig. 3.

The podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA were involved in the ROS signalling pathways in rat podocytes. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA after inhibiting ROS generation. f The Western blot results showed the nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA levels under ROS generation inhibition. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01. NC, negative controls without additional treatment.

Fig. 3.

The podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA were involved in the ROS signalling pathways in rat podocytes. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA after inhibiting ROS generation. f The Western blot results showed the nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA levels under ROS generation inhibition. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01. NC, negative controls without additional treatment.

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IS Inhibited Nephrin, Synaptopodin, CD2AP, and SRGAP2a but Promoted α-SMA Levels in HG-Induced Rat Podocytes

To examine the effects of IS on podocyte injury markers, qRT-PCR and Western blot analysis were performed in rat podocytes. The qRT-PCR results showed that the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were decreased when the cells were treated with HG compared with those of the negative control or mannitol group and further obviously reduced after the addition of IS to the HG solutions (Fig. 4a–d). The expression of α-SMA showed the opposite results (Fig. 4e). Furthermore, Western blot analysis showed that nephrin, synaptopodin, CD2AP, and SRGAP2a protein levels decreased significantly with HG treatment and further decreased significantly after IS supplementation in the HG solution (Fig. 4f–k, which confirmed the qRT-PCR results.

Fig. 4.

The expression of the podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA following HG and HG plus IS treatment. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA mRNA with HG and HG plus IS treatments. f The Western blot results showed the protein levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA with HG and HG plus IS treatments. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01.

Fig. 4.

The expression of the podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA following HG and HG plus IS treatment. a-e The qRT-PCR results showed the expressions of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA mRNA with HG and HG plus IS treatments. f The Western blot results showed the protein levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA with HG and HG plus IS treatments. g-k Quantitative analysis of the protein levels in (f). All data are the mean ± SD, (n = 5); *p < 0.05, **p < 0.01.

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IS Regulated the Expression of Key Factors in the TGF-β1/Smad and ROS/NOX4 Signalling Pathways

To determine whether IS aggravates kidney injury by regulating TGF-β1 and ROS signalling in podocytes, we measured the expression of some key factors in the pathways. The expressions of Smad2/3, Smad7, and NOX4 by Western blot were quantified, which showed that the protein levels of Smad2/3, p-Smad2/3, and NOX4 significantly increased in response to HG treatments compared with those of the NC groups and further increased significantly after the addition of IS to the HG solution (Fig. 5a–c, e). Conversely, the levels of Smad7 decreased significantly after HG treatment and decreased more after IS intervention (Fig. 5d). In addition, ROS generation was measured by flow cytometry, and the results showed that ROS generation was increased by approximately two times with HG treatment. Moreover, the ROS levels significantly increased after IS intervention (Fig. 6a–e). These results demonstrated that IS could regulate the expression of key factors in the TGF-β1/Smad and ROS/NOX4 pathways in podocytes.

Fig. 5.

IS regulated the expression of Smad2/3, p-Smad2/3, Smad7, and NOX4. a The Western blot results showed that the protein levels of Smad2/3, p-Smad2/3, Smad7, and NOX4 changed with HG and HG plus IS treatments compared with those of the NC groups. b–e Quantitative analysis of the results from (a). *p < 0.05, **p < 0.01.

Fig. 5.

IS regulated the expression of Smad2/3, p-Smad2/3, Smad7, and NOX4. a The Western blot results showed that the protein levels of Smad2/3, p-Smad2/3, Smad7, and NOX4 changed with HG and HG plus IS treatments compared with those of the NC groups. b–e Quantitative analysis of the results from (a). *p < 0.05, **p < 0.01.

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Fig. 6.

ROS generation with HG and HG plus IS treatments. A flow cytometry analysis showed ROS generation in the NC group (a), mannitol group (b), HG group (c), and HG plus IS group (d). e Statistical analysis of ROS generation, as determined by flow cytometry. All data are the mean ± SD, (n = 3); **p < 0.01.

Fig. 6.

ROS generation with HG and HG plus IS treatments. A flow cytometry analysis showed ROS generation in the NC group (a), mannitol group (b), HG group (c), and HG plus IS group (d). e Statistical analysis of ROS generation, as determined by flow cytometry. All data are the mean ± SD, (n = 3); **p < 0.01.

Close modal

Podocytes are an important component of the glomerular filtration barrier. SD is a key structure of podocytes that maintains the structural and functional integrity of the glomerular filtration barrier [20]. In recent years, several SD molecules expressed by podocytes have been discovered such as nephrin, podocin, CD2AP, synaptopodin, TrpC6, densin, and SRGAP2a [19, 21‒23]. Currently, these podocyte structural molecules are classified into four categories, i.e., SD membrane proteins represented by nephrin, podocyte cytoskeletal proteins represented by synaptopodin, basement membrane-podocyte junctions represented by α3β1-integrin, and membrane proteins and podocyte apical membrane proteins represented by podocalyxin [24]. Podocyte injury is accompanied by changes in the cytoskeleton and/or SD of the podocytes and the loss or reduction of epithelial cell-specific markers such as FSP-1 and α-SMA [25].

All factors that can cause podocyte injuries such as immune injury, oxidative stress, drugs and toxins, cytokines and chemokines, metabolic factors, and infection can cause podocyte transdifferentiation [26‒28]. When podocytes undergo EMT, they lose the specific markers of mature podocyte epithelial cells such as nephrin, ZO-1, FAT, Nephl-3, α-SMA, ILK, vimentin, type I collagen, and matrix metalloenzyme 9 (MMP-9) [29‒31]. The results of Rossini et al. [32] showed that there were no apoptotic podocytes in the urine of 95% of diabetic patients, and 86% of the podocytes expressed FSP-1. The clinical symptoms and pathology of diabetic nephropathy in diabetic patients with FSP-1-positive podocytes showed severe changes, which may be related to the shedding of podocytes from the GBM after transdifferentiation but not to apoptosis of the podocytes. Zou et al. [33] found that nestin, vimentin, and desmin were expressed in glomeruli in a rat model of puromycin nephropathy, particularly in podocytes. The results of Kang et al. [34] showed that similar to renal tubular epithelial cells, podocytes can undergo EMT under the stimulation of TGF-β1, and the expressions of the epithelial cell markers P-cadherin, ZO-1, and nephrin are reduced, resulting in transdifferentiation. The markers FSP-1, desmin, type I collagen, fibronectin, and SD were extensively destroyed. When renal tubular epithelial cells undergo EMT, they are transformed into fibroblasts and/or myofibroblasts, thereby secreting extracellular matrix and promoting renal fibrosis. Unlike renal tubular epithelial cells, podocytes have a limited proliferative capacity. The EMT of podocytes is caused by shedding and apoptosis of the GBM, which inevitably leads to the loss of podocytes and a reduction in podocyte density [35]. This results in proteinuria due to the destruction of the filtration barrier. Nakano et al. [36] found that in injured kidneys, IS activated mTORC1 to induce renal fibrosis, and administration of mTORC1-targeted rapamycin alleviated renal fibrosis in adenine-induced CKD mice. IS activates NADPH oxidase to produce excessive ROS, leading to renal tubular interstitial injury, which in turn induces the expression of TGF-β1 in damaged renal tubular epithelial cells. Myofibroblasts promote the production of proinflammatory cytokines in macrophages and their differentiation into proinflammatory macrophages, thereby promoting the deposition of extracellular matrix and ultimately leading to renal fibrosis [37]. In this study, we found that after TGF-β1 siRNA and ROS inhibitor treatment of HG-induced podocytes, the expressions of the podocyte injury markers nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly increased, and the expression of α-SMA was significantly decreased. After the addition of IS, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a in the HG-induced podocytes were significantly reduced, and the expression of α-SMA was significantly increased. These results are consistent with the findings of a significant increase in IS levels in DN patients. These findings indicate that IS promotes the production of ROS through TGF-β1, which further aggravates podocyte damage in diabetic nephropathy.

NOX is the main source of ROS, and NOX4-derived ROS mediate TGF-β1/Smad signalling in many disease processes [38]. IS is the strongest ROS producer among many protein-bound uraemic toxins. In podocytes, IS enhances the activity of NADPH oxidases such as NOX4; upregulates the expression of renal fibrotic proteins such as TGF-β1, alkaline phosphatase, and α-SMA; and promotes ROS production and EMT in podocytes suggesting that IS induces the expression of the TGF-β1/Smad signalling pathway through oxidative stress, resulting in podocyte damage [39]. Our study found that the expressions of the key factors Smad2/3, p-Smad2/3, Smad7, and NOX4 in the TGF-β1/Smad/ROS signalling pathway changed after IS administration. After HG treatment, the expressions of NOX4 and Smad2/3 were significantly increased, but when IS was added to HG treatment, the expressions were further increased. However, the expression of Smad7 showed a trend opposite to those of Smad2/3 and NOX4. Smad7 is an inhibitory Smad and is a competitor of Smad receptor regulatory proteins (such as Smad2/3). In the presence of HG and IS, the protein level of p-Smad2/3 showed the same trend as the protein levels of Smad2/3 and NOX4. These results indicate that the injury of podocytes by IS is closely related to the TGF-β1/Smad/ROS signalling of SRGAP2a. In addition, in this study, ROS production was analysed by flow cytometry. ROS production was enhanced under HG conditions but was further enhanced after IS administration. In summary, exogenous IS increased the production of ROS and caused further damage to DN podocytes. Therefore, IS may be involved in podocyte injury in diabetic nephropathy through the regulation of the expression of the TGF-β1/Smad signalling pathway and podocyte injury-related factors.

In summary, our findings indicate that IS can interfere with key molecules in the signal transduction of the podocyte injury markers nephrin, synaptopodin, CD2AP, SRGAP2a, α-SMA, and TGF-β1/Smad/ROS and aggravate podocyte damage under HG conditions. Further study is necessary to accurately understand the mechanism by which IS damages podocytes. As this study only includes the rat podocytes, future studies on human nephropathy are needed to address the species-specific differences and make the findings more clinically relevant. Anyway, this study provides theoretical support for IS as a novel treatment target for DN patients.

This study was approved by the Ethics Committee of People’s Hospital of Suzhou New District (Approved No. 551998). All methods were performed in accordance with the NIH guidelines for the care and use of laboratory animals (8th edition, NIH). Consent to participant is not applicable in this case.

The authors declare that there are no relevant conflicts of interest.

This study was supported by research grants from Collaborative Fund of Hospital Management Foundation of Suzhou New District People’s Hospital (No. SGY2021D01), Suzhou Gusu Health Talent Research Project (Grant No. GSWS2022133) and The Basic Research on Medical and Health Application of The Suzhou Minsheng Technology (Grant No. SKYD2022093).

M.J. conceived and designed the study. L.H.L. and K.X. analysed the data. D.M.L., W.J.W., and S.B.S. conducted the experiments. H.Q. and D.H.J. wrote the manuscript. The final manuscript was read and approved by all the authors.

All data and original images generated or analysed during this study are included in this article or supplementary material. Further enquiries can be directed to the corresponding author.

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