Extracellular Vesicles Facilitate the Crosstalk Between High Glucose-Stimulated Mesangial Cells and Healthy Podocytes to Mediate Injury Responses

TL;DR

The study shows that high glucose levels in mesangial cells lead to extracellular vesicles that harm healthy podocytes, contributing to kidney damage in diabetes.

Contribution

The study reveals a novel mechanism of mesangial-podocyte crosstalk via extracellular vesicles in diabetic nephropathy.

Findings

High glucose increases extracellular vesicle release from mesangial cells without altering their size.

Extracellular vesicles from high glucose-treated mesangial cells induce podocyte dysfunction and EMT markers.

Blocking exosome secretion reduces the harmful effects on podocytes in co-culture.

Abstract

Mesangial cells (MCs) communicate with podocytes and contribute to podocyte damage in diabetes. We hypothesized that intercellular communication plays a critical role in glomerular injury in diabetic nephropathy (DN). This study investigated the role of extracellular vesicles (EVs) secreted by high glucose-treated MCs in podocyte dysfunction. MCs were cultured with normal or high glucose for 24 h, and control EVs (C-EVs) and high-glucose EVs (HG-EVs) were isolated and incubated with healthy podocytes. Immunofluorescence, qRT-PCR, and Western blotting assessed podocyte and profibrotic marker expression. High glucose increased the overall amount of EVs released by MCs, but not their size. HG-EVs induced upregulation of epithelial–mesenchymal transition (EMT) markers, including desmin and TGF-β1, and downregulation of podocyte markers alpha-actinin-4, synaptopodin, and P-cadherin. In a co-culture of high-glucose MCs and podocytes, an exosome secretion inhibitor attenuated these injurious effects. These data suggest that HG-EVs impair podocyte function and mediate communication between MCs and podocytes. Mesangial cell-derived EVs may represent potential therapeutic targets in DN.

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1. Introduction

Diabetic nephropathy is characterized by typical glomerular lesions that lead to pathological quantities of albumin in the urine. It is well established that the excessive transport of glucose and protein through glomerular, vascular, and tubular cells constitutes an essential mechanism for loss of renal function [1]. Mesangial cells (MCs) have a pivotal anatomical position located between the glomerular capillaries and the glomerular basement membrane (GBM) [2]. These cells secrete an extracellular matrix (ECM) and a myriad of essential components for glomerular homeostasis, and during disease pathophysiology, MCs secretions mediate the glomerular response to injury [3].

Podocytes are highly specialized cells of the glomerulus that wrap around glomerular capillaries with their interdigitating foot processes, forming the slit diaphragm [4], creating a crucial component of the glomerular filtration barrier [5]. Injury to MCs and podocytes significantly contributes to diabetic kidney disease, leading to proteinuria and progressive glomerulosclerosis [6]. It has also been demonstrated that the crosstalk between podocyte and MCs may have a role in their functional modulation [6,7,8,9]. Mesangial cells contribute to extracellular matrix remodeling, thereby regulating multiple glomerular processes such as immunity, inflammation, and tissue repair; under pathological conditions, including diabetes, dysregulation of these processes promotes fibrosis and impaired renal function [2].

In response to injury, podocytes can undergo a phenotypic switch characterized by a loss of expression of highly specialized podocyte markers, like podocin, nephrin, a, and synaptopodin [10,11,12]. Moreover, podocyte injury is frequently associated with the activation of epithelial-to-mesenchymal transition (EMT)-like programs, characterized by downregulation of epithelial markers and upregulation of mesenchymal and profibrotic markers, including vimentin, desmin, collagen IV, connective tissue growth factor (CTGF), and transforming growth factor-β1 (TGF-β1) [13,14,15]. Activation of these pathways contributes to cytoskeletal remodeling, loss of slit diaphragm integrity, and progressive glomerulosclerosis. A previous study suggested that an abnormal interaction between glomerular endothelial cells and podocytes may play a critical role in the pathogenesis of diabetic nephropathy. Exosomes released by glomerular endothelial cells treated with high glucose (HG) conditions could transmit information to podocytes and cause them to undergo a pathological epithelial–mesenchymal transition and become dysfunctional [16].

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released from cells that lack the capacity for self-replication [17,18]. These include vesicles originating from the endosomal system, commonly referred to as exosomes, which are released upon fusion of multivesicular bodies with the plasma membrane, as well as vesicles generated by outward budding of the plasma membrane, often termed ectosomes. Unless biogenesis is experimentally demonstrated, isolated vesicles are collectively referred to as EVs [18,19]. EVs vary in composition based on the physiological state of their source cells, including proteins, nucleic acids, and membrane components. These variations influence EV biogenesis, uptake, and target specificity in recipient cells [20,21,22]. EVs have emerged as crucial mediators in various pathophysiological processes, including cell death, inflammation, angiogenesis, and cancer progression [23,24,25]. However, the role of these vesicles and their content in effecting downstream effects in the glomerulus still needs to be established.

MCs and podocytes play vital roles in maintaining glomerular filtration barrier integrity, and their interaction is essential for the homeostasis of the glomerulus [26]. Disruption in this communication, especially under conditions of hyperglycemia, can lead to podocyte dysfunction, mesangial expansion, and, ultimately, glomerulosclerosis. Multiple signaling pathways mediate interactions between cell types in the glomerulus. Glomerular endothelial cells (GECs) influence podocytes and MCs through TGF-β signaling, while MCs similarly impact GECs and podocytes via TGF-β pathways. These pathways may offer more potential for targeted therapies than alternative signaling mechanisms [26].

Investigating the molecular pathways and signaling mechanisms that govern mesangial-podocyte crosstalk, mainly through the transfer of extracellular vesicles, may offer valuable insights into the early stages of diabetic nephropathy and potential therapeutic targets for halting disease progression. This study investigated whether EVs secreted by HG-stimulated MCs induce dysfunction in healthy podocytes.

2. Results

2.1. Characterization of EVs from MCs

EVs secreted by MCs under control (5 mM) or HG concentrations (30 mM) were characterized as shown in Figure 1. The stimuli with HG increased the number of vesicles but did not modify the size (Figure 1B). Nanoparticle tracking analysis revealed that EVs from both experimental groups displayed similar size distributions, with a mean diameter of approximately 155 nm and a size range of 40–200 nm, aligning with the expected exosome size range [27,28,29]. In addition, the expression of exosomal markers, CD63 and CD81 tetraspanins, was confirmed through Western blot analysis in EVs derived from normal MCs, HG-stimulated MCs (30 mM), and Mannitol-treated MC (30 mM) (Figure 1C). As expected, the protein expression of calnexin (an endoplasmic reticulum marker) was not observed, further confirming the specificity of the obtained vesicles. To assess the capacity of podocytes to uptake the exogenous MC EVs, EVs were labeled with CellTracker Red CMTPX fluorescent dye, a lipid marker. Confocal laser microscopy analysis revealed the presence of EVs within the cytoplasm of MCs after 1 h of incubation (Figure 1D), indicating the internalization of EVs by podocytes. These findings show the uptake of EVs and their potential as a vehicle for delivering cargo to target cells.

2.2. Gene Expression Changes in Podocytes

As shown in Figure 2, the HG did not significantly affect the gene expression of the studied markers except for connective tissue growth factor (CTGF), a profibrotic marker (p < 0.05). HG alone did not induce a statistically significant change in alpha-actinin-4 mRNA expression (Figure 2). C-EVs did not influence gene expressions. On the other hand, exposure to HG-EVs induced a phenotypic and epithelial–mesenchymal transition in podocytes process. This was demonstrated by the downregulation of alpha-actinin-4, p-cadherin, and synaptopodin and the upregulation of desmin, collagen IV, CTGF, and TGF-β1. These results indicate that high-glucose conditions could not induce phenotypic transition in the podocytes in vitro, while HG-EVs induced podocyte changes. The downregulation of alpha-actinin-4, p-cadherin, and synaptopodin suggests a loss of highly specialized podocyte markers associated with the epithelial phenotype. At the same time, the upregulation of desmin, collagen IV, TGF-β1, and CTGF indicates the acquisition of mesenchymal characteristics.

2.3. Protein Expression Changes in Podocytes

The loss of epithelial markers (nephrin and podocin) and gain of mesenchymal markers (α-SMA, desmin, and vimentin) are hallmarks of podocyte dysfunction. To investigate the effect of EVs on the podocyte phenotype, the protein expression levels of nephrin, podocin, WT1, desmin, and vimentin were examined by Western blot (Figure 3). Western blot analysis showed that high-glucose conditions did not induce statistically significant changes in the protein expression of the analyzed markers in podocytes. although a decreasing trend for nephrin and podocin was observed. In contrast, HG-EVs decreased the protein expression of nephrin, podocin, and WT1, which are critical in maintaining podocytes’ specialized functions and the integrity of the glomerular filtration barrier.

We also observed an increase in the expression of the profibrotic markers desmin and vimentin (Figure 3D,E). Podocytes treated with C-EVs showed no alterations in these proteins.

Immunofluorescence analysis revealed alterations in podocyte markers and cytoskeletal structures under EV treatment conditions. Nephrin (Figure 4A), podocin (Figure 4B), and synaptopodin (Figure 4C) expression were markedly reduced in podocytes exposed to HG-EVs. Additionally, vimentin staining (Figure 4C) was observed in podocytes treated with HG-EVs, indicating changes associated with cytoskeletal integrity. Further analysis using phalloidin staining (Figure 4C) demonstrated significant alterations in F-actin organization, suggesting actin cytoskeleton remodeling in HG-EV–treated podocytes. Quantitative analysis of phalloidin fluorescence intensity confirmed these changes, indicating a disruption of podocyte cytoskeletal dynamics in response to HG-EV exposure. These results demonstrate intercellular communication between MCs and podocytes via EVs and suggest that HG stimulation in MCs can modify podocyte function through EVs. This could contribute to glomerular dysfunction in diabetic nephropathy.

2.4. HG-EV-Induced Podocyte Dysfunction

To confirm whether HG-EVs cause podocyte dysfunction, high-glucose-stimulated MCs and normal podocytes were co-cultured using a transwell system in the presence and absence of an exosome release inhibitor, GW4869. Our findings revealed that exosome secretion inhibitor attenuated HG-EVs effects on podocytes’ protein expression of nephrin, synaptopodin, podocin, and vimentin compared to the Control (Figure 5). This confirms that EVs mediated the dysfunction of podocytes. Specifically, the expressions of the critical proteins nephrin, synaptopodin, and podocin were downregulated. Additionally, we observed an upregulation of vimentin, a profibrotic marker associated with podocyte dysfunction and the development of diabetic nephropathy.

A scratch assay was performed to confirm podocyte migratory phenotype after being treated with HG-EVs, Supplemental Data Figure S2. After 24 h, the scraped zone had decreased by 50% in podocytes incubated with HG-EVs, whereas it decreased by 20% in control podocytes. This result supports the migratory phenotype induced by HG-EVs in podocytes (Supplemental Figure S2).

Taken together, these results suggest that the detrimental impact of glucose-stimulated EVs on podocytes is mediated at least partially through the secretion of EVs by MCs and can cause podocyte dysfunction.

3. Discussion

This study demonstrated that the crosstalk between MCs and podocytes via EVs constitutes a mechanism of podocyte lesions in a high-glucose environment. Our previous study showed that HG stimulates human MCs and increases the release of EVs, which may contribute to the pathophysiological signal amplification and transmission for other MCs [27]. Based on this, the current study highlights a similar phenomenon between different cell types, podocytes and MCs. The results showed that MCs produced significantly higher numbers of EVs under HG conditions. Podocytes internalized these “foreign” EVs, which potentially leads to epithelial–mesenchymal transition (EMT) and podocyte dysfunction.

MCs constitutively secrete EVs, and the HG stimulus increases the number of secreted vesicles. According to the International Society of Extracellular Vesicles, the nanoparticle tracking analysis revealed that the average size of the EVs was consistent with the expected size range for EVs [29]. The exosome-enriched EVs were confirmed by the presence of markers CD81 and CD63 and the absence of calnexin, an endoplasmic reticulum protein. This analysis ensures that the observed effects of HG in MCs are related to exosome secretion rather than other EVs.

Here, we showed that EVs secreted by MCs were visualized into the cytoplasm of podocytes exposed to EVs for 1 h. This result indicates that cells can take up and internalize EVs from distinct cell types. Target cells’ incorporation of EVs can be mediated by many mechanisms, including fusion, interaction with receptors, and endocytosis mediated by clathrin or caveolin, phagocytosis, and micropinocytosis [21,30]. This study did not evaluate such mechanisms, and additional studies are required. Previous studies have demonstrated that podocytes are acceptor cells capable of incorporating EVs. One study showed that EVs from glomerular endothelial cells were internalized into podocytes and can mediate podocyte dysfunction [31]. Similarly, another study showed that mesenchymal stem cell-derived EVs were incorporated by podocytes [32]. EVs from stimulated mesangial cells that are internalized by podocytes can induce cytoskeletal remodeling, alter slit diaphragm-associated proteins, and activate profibrotic and EMT-related signaling pathways, thereby amplifying injury signals triggered by HG levels. In this context, cells responsive to HG stimuli can transmit pathological signals to otherwise non-responsive cells, perpetuating podocyte dysfunction and glomerular injury [26,33].

This intercellular signaling between HG-stimulated MCs and podocytes can be analyzed from a pathophysiological point of view through the influence on the expression of nephrin and podocin, which are essential for the structural integrity of the slit diaphragm between podocytes. Disruption in this structure together and thickening of the glomerular basement membrane are the main features of diabetes-induced proteinuria [6]. The effect of HG concentration on the podocytes remains controversial. Some studies suggest that high-glucose conditions in vitro can directly injure podocytes [34,35], but this was not observed by other studies [36,37,38].

Podocytes directly stimulated with high-glucose conditions presented only discrete and non-significant changes in the expression of nephrin and podocin in this study at the studied time points. In addition, the direct exposure of podocytes to high-glucose conditions did not significantly change the expression of desmin, vimentin, and WT1. HG downregulates the protein expressions of podocin and nephrin (Figure 4 and Figure 5). Moreover, HG changed the expression of vimentin significantly (Figure 3 and Figure 5).

Furthermore, TGF-β1 is one of the most critical profibrotic factors in the kidneys and is highly stimulated by a high-glucose environment [39,40]. However, its expression was unchanged in podocytes that were directly stimulated by high-glucose conditions. Another study also evaluated TGF-β1 production in podocytes exposed for 14 days to HG concentration, which did not increase TGF-β1 [36]. In contrast, HG increases TGF-β1 production in mesangial cells and upregulates TGF-β receptor II expression in podocytes, suggesting that mesangial-derived TGF-β1 may act on podocytes through a paracrine mechanism. In this context, extracellular vesicles released from HG-stimulated mesangial cells may serve as carriers of profibrotic cues, amplifying TGFβ signaling in podocytes. Activation of this pathway is known to promote cytoskeletal remodeling and mesenchymal features, including increased vimentin expression. This mechanism provides a plausible explanation for the elevated vimentin levels observed in podocytes in our study (Figure 3D and Figure 5E), linking mesangial–podocyte crosstalk under hyperglycemic conditions to podocyte phenotypic alteration and dysfunction.

The effects of high-glucose conditions on podocyte structure and function are context-dependent and vary between in vitro and in vivo models. In vivo, models have shown that a hyperglycemic environment can induce significant alterations in podocyte phenotype, including the loss of nephrin, perturbations in the synthesis and breakdown of extracellular matrix components, greater signaling of TGF-β1, restructuring of the actin cytoskeleton, and apoptosis [12,16,41,42,43]. The microenvironment within the kidney is not uniform or homogeneous, and it is characterized by a complex microenvironment surrounding specific cells. This environment is influenced by a variety of factors such as neighboring cells, signaling molecules, the extracellular matrix, and intercellular communication in different types of cells by EVs. In this context, the in vitro system imposes a limitation on the effects of these factors, which can interfere with adequate communication among kidney cells of the limited microenvironment and the lack of adequate renal intercellular communication.

The present in vitro study showed that the crosstalk between MCs and podocytes may have a crucial role in the response of podocytes to high-glucose environments in vivo, and this potential mechanism can be triggered through EVs. In contrast to the high-glucose stimulus, our results showed that MC-derived HG-EVs effectively altered the phenotype of health podocytes, indicating their involvement in this process. HG-EVs significantly induced downregulation in the expression of podocin and nephrin genes in podocytes.

Moreover, HG-EVs increased desmin, TGF-β1, and collagen IV levels compared to podocytes directly exposed to HG concentrations. This suggests that HG-EVs transferred information to healthy podocytes, resulting in more significant alterations in their phenotype. The involvement of EVs derived from HG-stimulated MCs on podocyte injury was confirmed through the blockade of exosome generation and release by GW4869. Our findings suggest that mechanisms involving cell–cell communication through EVs can influence different cell types, leading to the potentiation of harmful effects of excess glucose in the kidney through a cascade of signaling to uninjured cells.

The experimental outcomes elucidated the paracrine communication between MCs and podocytes via EVs. The results suggested that HG stimulation in MCs can modify podocyte function, contributing to diabetic nephropathy. In summary, high-glucose conditions increased the number of EVs secreted by MCs but not their size, and HG alone induced non-significant trends toward changes in podocyte phenotype. In contrast, EVs from HG-stimulated MCs induced significant changes in mRNA expression levels in healthy podocytes with increased synthesis of profibrotic markers, collagen IV, desmin, vimentin, and TGF-β1 and decreased nephrin, podocin, alpha-actinin-4, p-cadherin, and synaptopodin. HG-EV-induced changes in podocyte protein levels were attenuated in the presence of the extracellular vesicle secretion inhibitor GW4869.

Podocyte migration represents an early functional response that can occur before detectable changes in podocyte marker expression [44]. High-glucose conditions are known to rapidly affect cytoskeletal dynamics and cell motility, whereas downregulation of slit diaphragm and differentiation markers typically requires longer exposure times [45,46,47]. In contrast, HG-EVs were sufficient to induce both early migratory behavior and changes in podocyte marker expression within the same timeframe.

These data suggest that high-glucose EVs can cause dysfunction in healthy podocytes and mediate intercellular communication between MCs and podocytes. This has important implications as mesangial cell secreted EVs can serve as potential therapeutic targets in diseases like DN.

4. Materials and Methods

4.1. Cell Culture

4.1.1. Mesangial Cells

Murine mesangial cells were obtained from the American Type Culture Collection (reference CRL-1927) and cultured in 6-well plates at a temperature of 37 °C. The culture medium was a mixture of Dulbecco’s Modified Eagle’s medium and Ham’s F12 medium (DMEM/F12) at a 3:1 ratio. This medium was supplemented with 10% fetal bovine serum (FBS) and penicillin at 50 U/mL. To maintain conditions for cell growth, the plates were placed in an incubator with an atmosphere containing 95% air and 5% carbon dioxide. Once the MCs reached confluence, they were incubated for 24 h in DMEM/F12 medium without FBS.

MCs were cultured in DMEM/F12 medium containing either standard 5 mM D-glucose or high 30 mM D-glucose for 24 h. MCs were also incubated in media containing 30 mM mannitol as an osmotic control. The culture media were collected, and EVs from the control group (C-EVs), high-glucose group (HG-EVs), and mannitol group (M-EVs) were isolated and incubated with healthy podocytes.

4.1.2. Podocytes

Healthy podocytes are immortalized murine podocytes provided by Dr. Niels Olsen Saraiva Camara (Institute of Biomedical Science, University of São Paulo, São Paulo, São Paulo, Brazil). Podocytes (Cell Lines Service GmbH, Baden-Württemberg, Germany) carrying temperature-sensitive variant SV40 T antigen under the control of IFN-γ-inducible H-2Kb promoter were maintained in RPMI 1640 medium, which was supplemented with 10% fetal bovine serum and 1% streptomycin/penicillin solution. Cells were grown and expanded in Type I collagen-coated flasks (Corning™, New York, NY, USA) under permissive conditions at 33 °C with 50 IU/mL IFN-γ (Cranbury, NJ, USA). Upon reaching 70–80% confluence, they were switched to non-permissive conditions (37 °C without IFN-γ) for 14 days for complete differentiation.

The control group of podocytes had no EV treatment, and the high-glucose group was incubated with RPMI and 30 mM glucose. The HG-EV group of podocytes was set with HG-treated MC-derived EVs, and the control-EV group was incubated with normal MC-derived EVs. To analyze protein expression, the mannitol group was incubated with 30 mM mannitol-treated MC-derived EVs. Podocytes were then exposed to EVs for 24 h, and the cells were analyzed.

4.2. Extracellular Vesicles Isolation

As described previously, the differential ultracentrifugation method was used to isolate EVs enriched with EVs from the supernatants of serum-free MC cultures [27]. In brief, supernatants of MC cultures were collected and sequentially centrifuged at 300× g for 10 min, 2000× g for 20 min, and 10,000× g for 30 min. This was done to remove lifted cells, cellular debris, and large vesicles. The cleared samples were then subjected to ultracentrifugation twice at 100,000× g for 120 min at 4 °C to obtain a pellet of EVs enriched with exosomes. The EVs were then resuspended in a small amount of phosphate-buffered saline (PBS) for direct use. EV characterization was performed as recommended by the International Society of Extracellular Vesicles (MISEV2023) [29].

4.3. Quantification of Particles

The size and concentration of extracellular vesicles from MCs were determined by nanoparticle tracking analysis (NTA) to measure the ratio of Brownian motion to particle size. This was done using a Malvern NanoSight NS300 system (Malvern, Worcestershire, UK) to track individual particles and the Stokes–Einstein equation to calculate their diameters. Three replicates of diluted aliquots of vesicle fractions (1 mL in PBS) were injected into the system’s specimen chamber. The vesicles were tracked and measured three times for each sample at a constant flow rate for 30 seconds each time.

4.4. Incorporation of EVs

EVs isolated from the culture supernatants of high-glucose MCs were labeled with a fluorescent dye, CellTracker Red CMTPX (20 µM) (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), at room temperature for 45 min [48]. The dyed vesicles were washed, ultracentrifuged again to remove the remaining dye, and filtered with a 0.22 µM filter to remove aggregates. The labeled EVs were incubated with healthy podocytes for one hour and observed in a confocal microscope to verify incorporation. Images were acquired using a Zeiss LSM 780 confocal microscope (Jena, Thuringia, Germany) (20× objective).

4.5. Western Blot Analysis

EV pellets or Total protein was extracted from podocytes using RIPA buffer supplemented with protease and phosphatase inhibitors. Total protein was purified from podocytes or EV pellets were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. The cell suspensions were homogenized in ice-cold lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 2.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 44 mM o-phenanthroline (all reagents were from Sigma-Aldrich Chemical Co., Burlington, MA, USA). The Lowry method determined the protein concentration (DC Protein Assay; Bio-Rad Laboratories Inc., Richmond, CA, USA). Equal amounts of total extracted proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Blots of cell protein were subjected to immunoblot analyses with primary polyclonal antibodies against nephrin (Abcam Cambridge, Cambridgeshire, UK), podocin (Abcam), WT1, vimentin, desmin, and alpha-tubulin (1:1000; Sigma-Aldrich, Saint Louis, MO, USA). Antibodies verified the exosomal protein expression against CD63 (1:100, Abcam), CD81 (1:1000 Abcam), and calnexin 1:1000 (Sigma-Aldrich, USA). Immunodetection was achieved by incubating the blots in horseradish peroxidase-conjugated (HRP) anti-rabbit or anti-mouse secondary antibody (1:30,000 dilution). The protein bands were visualized using the Immobilon Western HRP substrate (Millipore, Saint Louis, MO, USA). Densitometric analysis of bands was done using UVIband analysis software v12.14 (Uvitec Limited, Cambridge, UK).

4.6. mRNA Expression Analysis

According to the manufacturer’s instructions, the mRNA expression levels were estimated by quantitative RT-PCR. The total RNA was purified from podocytes using a commercial TRIzol (Gibco BRL, Rockland, MD, USA). The RNA quantity and purity were determined using the NanoVue spectrophotometer (GE Healthcare Life Sciences, Chicago, IL, USA). To avoid genomic DNA contamination, 2 μg of total RNA was treated with DNase (RQ1 RNase-free DNase; Promega, Madison, WI, USA). The RNA pellet was resuspended in RNase-free water and reverse transcribed into cDNA by the addition of a mixture containing 0.5 mg/mL oligo-deoxy thymidylate (oligo-d(T)), 10 mM dithiothreitol (DTT), 0.5 mM deoxynucleoside triphosphates (Amersham Pharmacia Biotech, Uppsala, Sweden), and 200 U reverse transcriptase enzyme (SuperScript RT; Gibco BRL).

The RNA pellet was resuspended in RNase-free water and reverse transcripted into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR amplification was performed using SYBR Green (Applied Biosystems) in the QuantStudio^TM^ 7 Flex System (Applied Biosystems), with specific primers for each molecule as follows. Primers were purchased from Sigma-Aldrich and validated by melt curve analysis, demonstrating a single amplification peak for each target gene. The mRNA expression levels were normalized to HPRT, and the results were expressed as arbitrary units with the control cells as a reference sample. Primers used for RT-PCR are listed in Supplemental Data Table S1.

4.7. Immunofluorescence and Phalloidin Staining

Podocytes were grown on Lab-Tek^®^ chamber slides (Thermo Fisher Scientific) and treated with HG (HG), HG-EVs, or C-EVs for 24 h. Cells were fixed with 3.5% formaldehyde for 15 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min, and blocked in PBS containing 0.5% bovine serum albumin (BSA) for 1h.

Cells were incubated overnight at 4 °C with primary antibodies against nephrin (Abcam, 1:100), podocin (Abcam, 1:100), synaptopodin (Santa Cruz Biotechnology, 1:100), or vimentin (Thermo Fisher Scientific 1:100), followed by Alexa Fluor–conjugated secondary antibodies (488 or 594; Thermo Fisher Scientific, 1:1000) for 60 min at room temperature in the dark.

Actin cytoskeleton organization was assessed using Alexa Fluor Plus 647-conjugated phalloidin (Invitrogen), following the manufacturer’s instructions. Nuclei were counterstained with DAPI (Invitrogen). Coverslips were mounted in glycerol buffer (pH 9.0).

Nephrin and podocin images were acquired using a Leica TCS SP8 confocal microscope, whereas phalloidin, vimentin, and synaptopodin images were obtained using a Nikon Eclipse i90 fluorescence microscope. Phalloidin fluorescence intensity was quantified using Fiji/ImageJ 1.54p. Individual podocytes were outlined as regions of interest, and fluorescence intensity was measured as mean gray value, corresponding to the sum of pixel intensities divided by the total number of pixels. Mean values were calculated per image (5–15 cells per image) and normalized to control conditions to correct for staining variability [49]. Data were analyzed in GraphPad Prism 10 using one-way ANOVA followed by Tukey’s post hoc test.

4.8. Co-Culture of MCs and Podocytes

The effect of HG-EVs on normal podocytes was analyzed in the presence and absence of an exosome release inhibitor, GW4869. MCs were cultured at DMEM/F12 medium (3:1 ratio) at the upper compartment of a transwell insert in a culture medium containing HG (30 mM) or mannitol (30 mM) in the presence or absence of 20 μM GW4869 for 24h. GW4869 (Sigma-Aldrich) was purchased and used as an EV release inhibitor at a concentration selected based on previously published studies [50,51]. A 0.4 μM microporous membrane separated the upper and lower compartments to mimic direct contact between MCs and podocytes. To allow cells sufficient time for the secretion of EVs, MCs were initially stimulated with HG or mannitol for 24 h, and then the upper compartment with stimulated MCs was inserted into the lower coated chamber of the well-containing podocytes, cultured in RPMI. The medium of MCs was not changed. The upper and lower compartments were in contact for 24 h, and then the podocyte protein expression was analyzed by Western blot.

4.9. In Vitro Scratch Assay

Mouse podocytes (5 × 10^5^ cells per well) were plated into 24-well plates. A scratch was introduced in each well using a sterile 200 μL pipette tip. The wells were then washed with phosphate-buffered saline (PBS) to remove debris and replenished with fresh culture medium. The control condition consisted of a medium containing 0% FBS, while the positive control included a medium supplemented with 25 ng/mL of Epidermal growth factor (EGF). For experimental conditions, podocytes were treated with HG, C-EVs, or HG-EVs in a medium containing 0% FBS. Images were captured by microscopy under an x10 objective for 48 h, every hour after scratching. The wound rate was quantified with measurements of the gap size over time. Three different areas in each assay were chosen to measure the distance of migrating cells. The experiment was carried out in triplicate.

4.10. Statistical Analysis

The results are expressed as the means ± standard error of the mean (SEM). Statistical significance was determined through one-way analysis of variance (ANOVA), followed by Tukey’s test. p-values less than 0.05 were considered statistically significant. Statistical analysis and graph construction were performed using GraphPad Prism version 10.00 for macOS (GraphPad Software, San Diego, CA, USA).

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.