Engineered extracellular vesicles promote the repair of acute kidney injury by modulating regulatory T cells and the immune microenvironment

doi:10.21203/rs.3.rs-5277191/v1

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Research Article

Engineered extracellular vesicles promote the repair of acute kidney injury by modulating regulatory T cells and the immune microenvironment

https://doi.org/10.21203/rs.3.rs-5277191/v1

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Background

Acute kidney injury (AKI) is a common and severe clinical condition. However, the underlying mechanisms of AKI have not been fully elucidated, and effective treatment options remain limited. Studies have shown that immune cells play a critical role in AKI, with regulatory T cells (Tregs) being one of the most important immunosuppressive lymphocytes. Tregs proliferation can attenuate AKI, whereas depletion exacerbates kidney injury. Given that endothelial cells (ECs) are the initial cells that interact with immune cells when they invade the tissue parenchyma, ECs are closely associated with immune reactions.

Methods and Results

In this study, P-selectin binding peptide-extracellular vesicles (PBP-EVs) that target and repair ECs are engineered. Transcriptome sequencing reveals that PBP-EVs reduce the expression of inflammatory genes in AKI mice. Using high-resolution intravital two-photon microscopy (TPM), an increased recruitment of Tregs in the kidneys of AKI Foxp3-EGFP transgenic mice following PBP-EVs treatment is observed, as well as significant Lgr5⁺ renal stem cell proliferation in AKI Lgr5-Cre^ERT2; R26^mTmG mice. Additionally, PBP-EVs treatment result in reduced infiltration of inflammatory cells, pathological damage and fibrosis of AKI mice. Upon depletion of Tregs in Foxp3-DTR transgenic mice, we observe diminished therapeutic effect of PBP-EVs on AKI.

Conclusions

The experimental results indicate that PBP-EVs can promote the repair and regeneration of AKI by mitigating endothelial cell damage and subsequently modulating Tregs and the immune microenvironment. These findings provide novel insights and strategies for the treatment of AKI.

acute kidney injury (AKI)

endothelial cells (ECs)

Tregs

immune microenvironment

Engineered EVs

Damage or functional failure of organs presents a significant challenge to human health, with acute kidney injury (AKI) being particularly notable for its high incidence, elevated mortality rate, and severe impact [1, 2]. AKI is characterized clinically by a sudden loss of kidney function, an increase in serum creatinine levels, and a reduction in urine output typically occurring within the first seven days following an initial insult. One of the primary causes of AKI is renal ischemia/reperfusion injury (IRI), which may result from infections, hypovolaemic shock, sepsis, trauma, dehydration, and other types of injury [1, 3]. Over the past 50 years, the mortality burden of AKI worldwide has surpassed that of cancer, heart failure, or diabetes, becoming an important public health concern affecting the nation's well-being [4]. Recent studies have demonstrated that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (EVs) can improve the function of damaged kidneys, promote the proliferation of renal stem cells, and facilitate renal regeneration and repair. However, the underlying mechanisms of these effects remain unclear [5–7].

The immune microenvironment plays a crucial role in renal regeneration and repair. [8, 9] Research indicates that renal IRI induces the synthesis or activation of pro-inflammatory cytokines and chemokines in the kidney, leading to the recruitment of leukocytes to the post-ischemic kidney and triggering a strong inflammatory response [8, 10]. In the early stages of renal injury, persistent presence of lymphocytes within the kidney may perpetuate damage in the extension phase of IRI [11]. During reperfusion, blood containing innate immune components (such as complement, chemokines, and adhesion molecules) flows through the ischemic tissue, also potentially exacerbating renal injury [8]. Nevertheless, measures that inhibit the inflammatory response, such as blocking various cytokines (IL-1, IL-6, IL-8 et al.), suppressing neutrophil infiltration, and knocking out CD4⁺ T cells, have been demonstrated to alleviate renal injury during ischemic AKI[12]. Regulatory T cells (Tregs), as crucial immunosuppressive cells, play a significant role in modulating the inflammatory response in AKI and promoting renal repair. Studies have shown that Tregs infiltrating the IRI kidney can facilitate renal regeneration and repair by regulating the pro-inflammatory cytokines, such as IFN-γ and TNF-α, produced by other T cell subsets (TCRβ⁺CD4⁺ T cells and TCRβ⁺CD8⁺ T cells) [13]. Simultaneously, Tregs play a significant role in mitigating tissue fibrosis[14]. Single-cell RNA sequencing (scRNA-Seq) indicates that a marked increase tissue-resident IL-33R⁺ and IL-2Ra⁺ Tregs can protect the kidneys from injury and fibrosis[15].

Given that endothelial cells are the first cells with which immune cells interact upon reaching the tissue parenchyma, they play a critical role in immune modulation as a frontline defense system in immune responses[16]. Research indicates that endothelium acts as a regulator of immune cell activation and differentiation; endothelial cells respond actively to lymphocyte adhesion and participate in lymphocyte migration[17]. The self-antigen presentation of damaged endothelial cells can effectively migrate Tregs to inflammatory tissues, thereby inhibiting the regulation of T effector (Teff) cells recruitment and facilitating the establishment of a Teff: Treg ratio optimal for regulation in non-lymphoid tissue[18].

In normal kidneys, the surface of glomerular endothelial cells is covered by glycocalyx, which conceals selectins and cell adhesion molecules. In damaged kidneys, the shedding of the glycocalyx exposes these molecules, facilitating immune cell recruitment [19–21].Among these, P-selectin is an inflammatory cell adhesion molecule. In the early phase of renal IRI, P-selectin is expressed in the glomeruli and interstitial capillaries of the injured kidney, mediating the adhesion of leukocytes to the inner walls of the damaged vessels and their migration to the injured cortex, thereby promoting intrarenal sterile inflammation[22]. Therefore, targeting and repairing damaged endothelial cells is of critical importance for improving the immune microenvironment.

In recent years, based on live fluorescence imaging, the combination of two-photon microscopy (TPM) with lineage-tracing animal models has provided an efficient platform for tracing of the cells in kidney [23–25]. TPM offers advantages such as deep tissue penetration and low phototoxicity, making it suitable for in vivo tracking of Tregs and renal stem cells in this study.

Based on this background, our research group has previously engineered EVs using P-selectin binding peptide (PBP) to create PBP-EVs, which target and repair endothelial cells in IRI kidneys. Additionally, we used transcriptomic sequencing to observe changes in inflammation-related genes in the kidneys of AKI mice before and after treatment. By employing Tregs and Lgr5⁺ stem cell lineage-tracing transgenic mice, combined with TPM, we investigated the role and mechanisms of PBP-EVs in the repair process of AKI mice kidney injury. Ultimately, we found that PBP-EVs can promote the regeneration and repair of AKI by ameliorating endothelial cell damage, thereby regulating Tregs and the immune microenvironment.

Animals

In this study, male C57BL/6 mice, aged 8–10 weeks, weighing 20 to 25 g, were obtained from the Laboratory Animal Center at the Academy of Military Medical Sciences (Beijing, China). Vegfr2-Fluc-KI transgenic mice on a C57BL/6 albino and outbred (Nu/Nu) background were acquired from Xenogen Corporation (Hopkinton, MA, USA). Male C57BL/6 Lgr5-Cre^ERT2; Rosa26^mTmG; Foxp3- DTR/EGFP transgenic mice with expression of a diphtheria toxin receptor-enhanced green fluorescent protein (DTR or GFP) were purchased from Jackson Laboratory. Transgenic mice were identified by genomic PCR analysis or flow cytometry. All animal experimental procedures in this study were approved by the Animal Experiments Ethical Committee of Nankai University, following the guidelines outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (approval no. 20190022). Mice were housed under pathogen-free conditions in a controlled environment with a constant temperature of 21–23°C and humidity levels of 45–50%. The housing facility operated on a 12-hour light-dark cycle (lights on from 07:00 to 19:00), and mice had ad libitum access to food and water throughout the study.

Experimental model and protocol

The renal IRI models were established as previously reported[41]. Mice were anesthetized via intraperitoneal injection of 2.5% avertin (Sigma-Aldrich, Oakville, ON, Canada) at a dosage of 240 mg/kg. The left renal pedicle was clamped for 45 minutes, followed by release to permit blood reperfusion. After 12 h of reperfusion, either PBS, 100 µg of EVs, or PBP-EVs were administered intravenously in a total volume of 200 µL.

Cell culture and EVs

Human placenta-derived mesenchymal stem cells (hP-MSCs) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, Grand Island, NY), supplemented with 10% EV-free fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). All cells were maintained in a humidified incubator (Thermo Scientific, Madison, WI) with 5% CO₂ at 37°C. The hP-MSCs were subcultured every two days. During subculture, the supernatant was collected and sequentially centrifuged at 500 × g for 10 minutes, 2,000 × g for 30 minutes, and 10,000 × g for 30 minutes to remove cell debris and apoptotic bodies. EVs were isolated by ultracentrifugation at 100,000 × g for 70 minutes, washed with phosphate-buffered saline (PBS), and subjected to a second ultracentrifugation at 100,000 × g for 2 hours. The resulting EVs was resuspended in 200 µL PBS and stored at -80°C. All procedures were conducted at 4°C under aseptic conditions. The EVs were quantified by measuring the total protein content using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific).

The endothelial cells (ECs) used in this study were human umbilical vein endothelial cells (HUVECs) sourced from the American Type Culture Collection (ATCC; Manassas, VA). HUVECs were cultured in endothelial cell growth medium-2 (EGM2; Lonza, Walkersville, MD). To simulate the microenvironment of kidneys with IRI, hypoxia/reoxygenation (H/R) was performed on the HUVECs. Cells were incubated in a humidified hypoxia incubator (Thermo Scientific) with 5% CO₂, 1% O₂, and 94% N₂ at 37°C for 6 hours, followed by reoxygenation with 95% air and 5% CO₂ for an additional 12 hours.

Preparation and analysis of PBP-EVs

Dissolve 1 mg of Cy5.5-DPP lyophilized powder in 294.4 µL of DPBS to obtain a 500 µM stock solution. Incubate EVs with 5 µM Cy5.5-DPP at room temperature (25°C) for 30 minutes. Following incubation, wash the EVs more than five times using 5 volumes of PBS and a 100 kDa ultrafiltration centrifuge tube to remove excess DPP, thereby preparing PBP-EVs. To assess the labeling efficiency and stability of PBP-EVs, the PBP-EVs were stored at 4°C for 1, 3, and 7 days, followed by flow cytometry analysis.

EVs and PBP-EVs were analyzed using a FACSCalibur flow cytometer (FCM; BD Biosciences, San Jose, CA)[42]. PBS and DPP solutions were initially tested to evaluate background noise. Unmodified EVs were utilized to set appropriate voltages and thresholds, gating the EV population in the forward-scatter and side-scatter channels for measurements.

The size distribution and zeta potential of EVs and PBP-EVs were assessed using a Malvern Particle Size Analyzer (Zeta sizer Nano ZS, Malvern Panalytical, Malvern, UK). The morphological characteristics of EVs and PBP-EVs were then examined via transmission electron microscopy (TEM; Talos L120C, Hillsboro, OR). Additionally, the expression of three EVs marker proteins (Alix, TSG101, and CD63) was analyzed through Western blotting.

To determine the Cy5.5 radiance of EVs or PBP-EVs in cells and renal sections, laser scanning confocal microscopy (LSCM; excitation at 647 nm; FV1000, Olympus, Lake Success, NY) was employed. Fluorescence intensity was quantified from specified regions of interest.

Preparation and analysis of Gluc-labeled EVs

A gene fragment encoding the Gluc-lactadherin fusion protein was transduced into human placenta-derived mesenchymal stem cells (hP-MSCs), resulting in the production of Gluc-labeled EVs. The hP-MSCs were infected with lentivirus particles (MOI = 10) carrying the Gluc and human lactadherin fusion protein genes and were subsequently selected using puromycin (2 µg/mL, Sigma-Aldrich). Gluc-labeled EVs were then isolated from the supernatant of the infected hP-MSCs by ultracentrifugation.

For in vitro experiments, the detection of Gluc-labeled EVs or PBP-EVs was performed in 24-well plates using 0.1 µg/mL water-soluble coelenterazine (CTZ; Nanolight Technology, Pinetop, AZ) as the substrate. In vivo experiments involved the intravenous injection of 100 µg of Gluc-labeled EVs or PBP-EVs via the caudal vein. At designated time points, mice were imaged immediately following the intraperitoneal injection of water-soluble CTZ (5 mg/kg) for bioluminescence imaging. The bioluminescence radiance of EVs or PBP-EVs was monitored using an IVIS Lumina Imaging System (Xenogen Corporation), with all measurements taken as the average radiance from regions of interest.

Lymphocyte transmigration assays

As previously described, hypoxia-induced injured ECs were seeded at a density of 3 × 10⁴ cells per well and cultured on gelatin-coated Transwell inserts (Corning) with a diameter of 6.5 mm and polycarbonate membranes featuring 3 µm pores[18]. After 16 hours, this process resulted in the formation of a confluent monolayer. Subsequently, T cells (5 × 10⁵) suspended in RPMI 1640 medium supplemented with 2% FBS (Gibco) were added to each insert and allowed to migrate through the EC monolayer. The number of transmigrated T cells was quantified by counting the cells present in the well media. The results are presented as the number of cells that successfully transmigrated.

Intravital two-photon imaging in mice

An abdominal imaging window (AIW) was surgically implanted in the mouse, secured with an adapter, and the mouse was maintained under anesthesia using avertin. Imaging was conducted using a 25× objective lens immersed in water, focused on the AIW. Two-photon excitation was performed at 835 nm wavelength with 10% laser power, and emission signals were collected at wavelengths of 495–540 nm (EGFP) and 575–630 nm (tdTomato). Imaging involved scanning with 5 µm Z-steps and using a zoom factor under the 25× objective lens, covering a single scanning area of 580 µm × 580 µm with an image resolution of 800 × 800 pixels.

Bioluminescence imaging of renal angiogenesis

The angiogenesis in the injured kidney was evaluated in Vegfr2-Fluc KI transgenic mice using bioluminescence imaging. Twelve hours after the induction of severe ischemia-reperfusion injury (IRI), mice were intravenously injected with 100 µg of Gluc-labeled EVs or PBP-EVs. Control mice received an equal volume of PBS. At specified time points, the mice were administered D-luciferin intraperitoneally (150 mg/kg; Biosynth International, Naperville, IL) and subsequently imaged using the IVIS Lumina Imaging System. The Fluc signals were quantified as the average radiance from regions of interest, analyzed with Living Image software.

Flow cytometry

As previously described, flow cytometry was employed to assess the ratio of inflammatory cells in the kidneys of each group of mice[43]. Following perfusion with phosphate-buffered saline, mouse kidneys were processed using a Stromacher 80 Biomaster. Renal tissue was homogenized and filtered through a 70-µm cell strainer to obtain single-cell suspensions. The cells were then resuspended in 35% Percoll and subjected to gradient centrifugation at 400 × g for 30 minutes. Flow cytometry analyses were conducted to assess both the absolute number and proportion of leukocytes present in the kidneys of mice.

Renal function analysis

On days 3 and 7 AKI, serum samples were collected to assess renal function markers, specifically blood urea nitrogen (BUN) and serum creatinine (SCr). The concentrations of BUN and SCr were determined using a BUN assay kit and an SCr assay kit, both from Nanjing Jiancheng Bioengineering Institute.

Histopathology and immunostaining

Mice were euthanized for tissue collection at specified time points. The harvested tissues were fixed in 4% paraformaldehyde, followed by dehydration through a graded ethanol series, clearing with xylene, embedding in paraffin, and sectioning into 5 µm thick paraffin slices. Hematoxylin and eosin (H&E) staining, along with Masson staining, were conducted on the paraffin sections according to established protocols.

For cryosectioning, tissues were similarly fixed in 4% paraformaldehyde, dehydrated in a 30% sucrose solution, embedded in Optimal Cutting Temperature (OCT) compound, and then sectioned to a thickness of 5 µm.

Statistical analysis

Each experiment included a minimum of three independent evaluations per condition to ensure reproducibility. Data are presented as scatter plots with mean values and standard deviations (mean ± s.d.). Statistical significance between different groups was assessed using two-tailed unpaired Student’s t-tests for comparisons between two groups, and one- or two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple group comparisons. Statistical analyses were conducted using GraphPad Prism software, with significance defined as P < 0.05.

Preparation and characterization of the PBP-EVs

Based on the foundation of previous laboratory research, we designed PBP-EVs that can target the P-selectin which on surface of damaged endothelial cells (Fig. 1A). The peptide fragment (DAEWVDVS) serves as a P-selectin binding peptide (PBP), enabling specific targeting and binding to P-selectin (Fig. S1a). We synthesized DMPE-PEG-PBP (DPP) by conjugating PBP onto an amphiphilic substance, DMPE-PEG5000-maleimide (DMPE-PEG-MAL), to anchor the EV membrane (Fig. S1a). Subsequently, we utilized nuclear magnetic resonance to conduct one-dimensional hydrogen spectrum detection on the synthesized DPP product. The presence of characteristic peaks corresponding to the indole ring of PBP (7.0-7.6 ppm region) in the DPP product was observed, confirming successful conjugation of PBP onto DMPE-PEG (Fig. S1b). For subsequent visualization detection, the Cy5.5 fluorescent dye was conjugated to the amino groups on the side chains of PBP to obtain Cy5.5-labeled DPP. At room temperature, 5 µM of Cy5.5-labeled DPP with EVs were incubated for 30 minutes to obtain PBP-EVs (Fig. 1A). We characterized and compared the prepared PBP-EVs and EVs. The particle size detection results reveal that both EVs and PBP-EVs predominantly exhibit a size distribution around 100nm, with PBP-EVs displaying a slightly increased size after DPP modification compared to EVs (Fig. 1B). Membrane potential analysis indicated that both EVs and PBP-EVs carried a negative charge (Fig. 1C). Western blotting revealed that PBP-EVs retained the characteristic protein markers of EVs (Fig. 1D). Transmission electron microscopy (TEM) demonstrated that both EVs and PBP-EVs exhibited vesicular structures (Fig. 1E). Subsequently, flow cytometry was utilized to assess EVs and labeling efficiency (Fig. 1F), revealing a labeling efficiency of over 90% for PBP-EVs (Fig. 1G). Simultaneously, PBP-EVs can be stably preserved for over one week at 4℃ (Fig. 1H and S1c, d). These findings collectively indicate that we successfully prepared and preserved PBP-EVs for subsequent experiments without compromising the physicochemical properties of EVs.

PBP-EVs can target and repair damaged renal endothelial cells

P-selectin is an inflammatory cell adhesion molecule expressed on ECs, typically at low levels under normal conditions. Upon endothelial cell injury, P-selectin can recruit leukocytes and platelets, and its expression is positively correlated with the pathophysiological progression of various injuries[26, 27]. These characteristics suggest that P-selectin may be a reliable target for the targeted delivery of exosomes to injured sites. Given the critical association between ECs and the immune microenvironment, and the role of P-selectin in pathological injury targeting, we successfully prepared PBP-EVs, which target injured ECs. Our findings indicate that PBP-EVs can target and ameliorate ECs injury, promote the recruitment of Tregs, and restore the immune microenvironment.

To further visualize PBP-EVs in subsequent experiments, we employed the bioluminescent Gaussia luciferase (Gluc) lactadherin fusion proteins report system to obtain Gluc/Cy5.5-labeled EVs and PBP-EVs (Fig. S2a, b, c). Mice underwent 12 h of renal IRI, followed by tail vein injection of 100µg of Gluc/Cy5.5-labeled EVs or PBP-EVs. At 2, 6, 12, and 24 h post-injection, Gluc imaging demonstrated significantly higher signal intensity in the renal region for PBP-EVs compared to EVs, indicating the strong targeting capability of PBP-EVs to injured kidneys in vivo (Fig. S2d, e). Concurrently, Cy5.5 fluorescence imaging revealed higher Cy5.5 fluorescence signals in the kidneys of the PBP-EVs group than in the EVs group, suggesting that PBP-EVs specifically target injured renal tissues (Fig. 2A-C). Kidney tissues injected with Cy5.5-labeled EVs and PBP-EVs were cryosectioned for immunofluorescence analysis. The results showed a significant increase in the accumulation of PBP-EVs in the injured kidneys compared to the EVs group (Fig. 2D), with a predominant distribution in renal endothelial cells (Fig. 2E). Subsequent in vitro experiments further confirmed these findings, demonstrating greater adhesion of PBP-EVs to H/R injured endothelial cells when co-cultured with Cy5.5/Gluc-labeled EVs and PBP-EVs (Fig. 2F and S2f, g, h). These results indicate that PBP-EVs can specifically target damaged renal endothelial cells both in vivo and in vitro, compared to EVs.

We further investigated the reparative effects of EVs and PBP-EVs on endothelial cells. Using vascular endothelial growth factor receptor 2 (Vegfr2)-Fluc mice, we established a renal IRI model to dynamically track angiogenesis in injured kidneys in real-time via

bioluminescence imaging. Mice subjected to 12 hours of IRI were injected via the tail vein with either EVs or PBP-EVs. The results showed that bioluminescence signals (Fluc) were consistently higher in the PBP-EVs group compared to the EVs group at 3, 7, 10, and 14 days post-injection, indicating enhanced angiogenesis in the injured kidneys mediated by PBP-EVs (Fig. 2G, H). Additionally, real-time qPCR revealed significantly elevated expression levels of angiogenesis-related genes Vegfα, Vegfr2, Ang1, and Ang2 in the PBP-EVs group compared to the EVs group, suggesting that PBP-EVs possess a strong capacity to promote angiogenesis (Fig. 2I). The above research findings demonstrated that PBP-EVs exhibits effective targeting and repair capabilities for renal endothelial cells.

Transcriptomic sequencing indicated that PBP-EVs reduced the expression levels of inflammatory genes which associated with renal regeneration

ECs are crucial components of the circulatory system. Interestingly, ECs and immune cells share a common ancestor, directly supporting the important role of ECs in immune responses[16, 28, 29]. Research indicates that vascular ECs can regulate inflammation by modulating the trafficking, activation status, and function of immune cells[30, 31].

Given the close relationship between endothelial cells and the immune microenvironment, we utilized transcriptomic sequencing to observe changes in inflammatory genes in the kidneys across different groups. The quality assessment of sequencing data and the Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) density distribution for each group are as follows (Fig. S3a-c). Principal Component Analysis (PCA) revealed significant clustering overlap between the PBP-EVs group and the Sham group, suggesting fewer differentially expressed genes between these two groups. In contrast, the PBP-EVs group and the PBS group are likely to have a greater number of differentially expressed genes (Fig. 3A). The Pearson correlation coefficient heatmap represents the degree of correlation in gene expression levels between samples. A correlation coefficient closer to 1 indicates a higher similarity in expression patterns between samples. The heatmap shows that the PBP-EVs group has a higher similarity in gene expression patterns with the Sham group. In contrast, the PBP-EVs group exhibited lower similarity with the PBS group (Fig. S3d). Furthermore, trend clustering analysis demonstrated consistent gene expression trends between the PBP-EVs and Sham groups, with trends opposite to those observed in the PBS group (Fig. 3B).

Then we compared the number of differentially expressed genes (DEGs) among the groups. A bar chart depicting the number of DEGs showed that, compared to the PBS group, there were 2911 DEGs in the PBP-EVs group, with 1768 upregulated and 1143 downregulated genes (Fig. 3C). In the EVs group compared to the PBS group, there were 479 DEGs, with 352 upregulated and 127 downregulated genes (Fig. 3C). Compared to the Sham group, the PBS group had 4042 DEGs, with 1864 upregulated and 2178 downregulated genes (Fig. 3C). UpSet and Venn diagrams further illustrated the common DEGs among the different comparison groups (Fig. S3e, f). Clustering of DEGs was performed to assess the variations in gene expression between different groups. The hierarchical clustering heatmap of differentially expressed genes between comparison groups shows that the PBP-EVs group clusters more closely with the Sham group and less closely with the PBS group (Fig. 3D).

Volcano plots visually displayed the distribution and expression changes of DEGs between two groups. Compared to the PBS group, genes such as Anxa2, Anxa3, Ankrd1, Akr1b8, and Krt18 were downregulated in the PBP-EVs group, while Hpd, Rdh16f2, Atp4a, Slc4a1, and Car3 were upregulated (Fig. 3E). Compared to the Sham group, the PBS group had upregulated genes including Havcr1, Tnfrsf12a, Fgb, Clu, and Lgals3, and downregulated genes such as Acmsd, Egf, Al314278, Slc34a3, and Pvalb (S4a). Gene Set Enrichment Analysis (GSEA) indicated that DEGs between the PBP-EVs and PBS groups were enriched in inflammation-related pathways such as the NOD-like receptor signaling pathway (hsa04621) and the fibrin complement receptor 3 signaling pathway (WP4136) (Fig. 3F). Additionally, compared to the PBS group, PBP-EVs suppressed these inflammation-related pathways. Compared to the Sham group, the PBS group upregulated inflammation-related pathways such as overview of proinflammatory and profibrotic mediators (WP5095) and nod like receptor signaling pathway (hsa04621) (Fig. S4b).

KEGG pathway analysis showed that, compared to PBS, the top 20 downregulated pathways in the PBP-EVs group included numerous inflammation-related pathways such as TNF signaling, NF-kappa B signaling, IL-17 signaling, cytokine-cytokine receptor interaction, and apoptosis and cellular senescence pathways (Fig. 3G). Additionally, compared to the Sham group, the PBS group upregulated inflammation-related pathways (Fig. S4c). Conversely, the top 20 upregulated pathways in the PBP-EVs group included pathways related to cell proliferation and renal regeneration and repair, such as the PPAR signaling pathway and the cAMP signaling pathway (Fig. S5a). Heatmap analysis of genes related to these pathways revealed that, compared to the PBS group, the PBP-EVs group exhibited decreased expression of inflammation, apoptosis and senescence-related genes and increased expression of genes related to cell proliferation and renal regeneration and repair (Fig. 3H and S5b). Using the STRING protein-protein interaction database, the analysis of differential gene protein interaction network revealed that inflammation is closely related to cell proliferation, apoptosis, and organ regeneration (Fig. 3I and S5c).

These findings suggested that PBP-EVs reduced the expression of renal inflammatory genes which related to promote renal regeneration and cell proliferation in AKI.

PBP-EVs reduce the generation of renal pro-inflammatory immune cells and cytokines

We subsequently validated the transcriptomic sequencing results by examining the levels of pro-inflammatory immune cells and cytokines in the kidney. Real-time qPCR results indicated that, compared to the PBS group, the PBP-EVs group and the EVs group exhibited reduced levels of pro-inflammatory genes TNF-α, IL-6, and TGF-β in the kidney, while the anti-inflammatory gene IL-10 was elevated (Fig. 4A). Moreover, the anti-inflammatory capability of the PBP-EVs group was superior to that of the EVs group. Immunofluorescence staining of renal CD45⁺ cells and myeloperoxidase (MPO) assays revealed that PBP-EVs reduced leukocyte infiltration in the injured kidneys (Fig. 4B, C). Further flow cytometry analysis identified the types and proportions of pro-inflammatory immune cells in the kidney. The results showed that, compared to the PBS group, the PBP-EVs group had a lower proportion of neutrophils in the kidneys of mice (Fig. 4D, E and S6a). PBP-EVs reduced the proportion of iNOS⁺ pro-inflammatory M1 macrophages and increased the proportion of CD206⁺ anti-inflammatory M2 macrophages in the injured kidneys (Fig. 4F-H and S6b, c). Additionally, PBP-EVs reduced the proportion of monocytes in the injured kidneys, particularly the proportion of intermediate monocytes and non-classical monocyte subtypes (Fig. 4I, J and S6d).

In summary, PBP-EVs decreases the levels of inflammatory cells and cytokines in injured kidneys, exhibiting superior anti-inflammatory capabilities compared to EVs.

PBP-EVs promote the accumulation of Tregs in the kidney following IRI

Tregs, characterized by the expression of the X chromosome-encoded transcription factor Foxp3, represent a distinct lineage of T lymphocytes [32]. Their critical function is to suppress T cell responses to self-antigens, commensal microbiota, dietary, and environmental antigens [33]. In addition to mitigating tissue damage by suppressing post-infection inflammatory responses, Tregs can facilitate tissue repair through mechanisms such as attenuating the pro-inflammatory responses of cells in the innate and adaptive immune systems, and reducing endothelial cell activation[34]. The blunted recruitment and responsiveness of inflammatory cells reduce the positive feedback loop mediated by activated T cells and promote the transition from inflammation to tissue repair processes. Studies have also shown that Tregs can directly contribute to tissue repair by producing amphiregulin[35]. Consequently, when tissues and organs are damaged, circulating or resident Tregs are rapidly recruited or expanded, thereby promoting tissue repair[36]. So, as crucial immunosuppressive cells within the body, Tregs play a significant role in regulating the immune microenvironment and tissue repair.

Although Tregs constitute a small proportion of lymphocytes and are at a numerical disadvantage, they can migrate rapidly and efficiently to target tissues. The levels of adhesion molecules and chemokines are insufficient to support their robust migratory capacity [37]. Studies have shown that the expression of antigens by ECs is an essential condition for the recruitment of Tregs [18].

We employed Foxp3-GFP transgenic mice to trace Tregs and subsequently used TPM for in vivo observation of the effects of PBP-EVs on renal Tregs (Fig. S7a). Flow cytometry was utilized to comparatively analyze Tregs in the kidneys of wild-type and Foxp3-GFP transgenic mice, revealing a strong GFP signal in the kidneys of Foxp3-GFP transgenic mice, which was undetectable in wild-type mice (Fig. S7b).

As depicted in the experimental flowchart, we established a renal IRI model in Foxp3-GFP transgenic mice, followed by the administration of PBP-EVs, EVs, or PBS (Fig. 5A). On the third day, TPM was employed to observe the distribution of Tregs in the kidneys of each group. We observed that PBP-EVs promoted the recruitment of Tregs to the injured kidneys, with a higher recruitment rate compared to the EVs group (Fig. 5B and Videos S1, S2, S3, S4). Immunofluorescence staining of renal sections from each group also demonstrated that PBP-EVs facilitated the accumulation of Tregs in the kidneys (Fig. 5C). Flow cytometry analysis further indicated that the proportion and number of Tregs in the kidneys were increased in the PBP-EVs group compared to the PBS group (Fig. 5D, E). In vitro experiment, we isolated Tregs from Foxp3-GFP mice and co-cultured them with hypoxia-induced injured endothelial cells on transwells. The experimental results indicated that, compared to the PBS group, PBP-EVs increased the migration of Tregs (Fig. S7c, d).

In summary, PBP-EVs enhanced the recruitment of Tregs to the kidneys following IRI, exhibiting superior recruitment capabilities compared to the PBS and EVs group.

PBP-EVs promote the proliferation of renal Lgr5⁺ stem cells

In adult mammals, the regenerative capacity of tissues and organs is limited. Tissue regeneration involves multiple complex steps and the participation of various cell sources, among which the proliferation of endogenous stem cells is considered the most promising strategy for tissue regeneration[5]. Increasing evidence suggests that endogenous stem cells can interact with the extracellular matrix, soluble cytokines, and cellular signals to promote tissue damage repair[38]. Lgr5, also known as leucine-rich repeat-containing G-protein coupled receptor 5, is an important membrane protein and a crucial regulator of intracellular signal transduction. Recent studies have identified Lgr5⁺ cells as homeostatic stem cells in various tissues, including the kidney, hair follicles, and intestine [39]. In neonatal and young mice, Lgr5 contributes to kidney development, is activated in response to ischemic kidney injury, and aids in vascular repair following acute kidney injury [40].Currently, Lgr5⁺ stem cells are believed to represent a type of endogenous stem cell within kidney tissue capable of participating in tissue damage repair. In this study, we found that PBP-EVs not only ameliorated pathological damage to the kidneys in AKI and reduced renal fibrosis but also promoted the proliferation of Lgr5⁺ stem cells.

Given the close relationship between the immune microenvironment and renal regeneration, we next investigated the effects of PBP-EVs on renal regeneration and repair. We used Lgr5-Cre^ERT2; R26^mTmG mice to label renal stem cells, administering tamoxifen (100 mg/kg daily, 3 times a week) a week prior to the experiment (Fig. S8a, b). Subsequently, an IRI model was established, and the mice were treated with PBP-EVs, EVs, or PBS. Fourteen days later, TPM was used to observe the activation and proliferation of renal stem cells in each group. The experimental workflow is shown in the schematic (Fig. S8b).

In vivo observations using TPM revealed that PBP-EVs increased the number of Lgr5⁺ renal stem cells in the injured kidneys (Fig. 6A and Videos S5-1, S5-2, S6-1, S6-2, S7-1, S7-2, S8-1, S8-2). Immunofluorescence staining of the kidneys also indicated that PBP-EVs promoted the proliferation of Lgr5⁺ renal stem cells, with a more pronounced effect than the EVs group (Fig. 6B, C).

In conclusion, we found that PBP-EVs can enhance the activation and proliferation of Lgr5⁺ renal stem cells, thereby promoting the regeneration of injured kidneys.

PBP-EVs alleviate mice renal IRI

We further investigated the effect of PBP-EVs on renal repair following injury. The results showed that compared to the PBS group, both PBP-EVs and EVs reduced serum creatinine (SCr) and blood urea nitrogen (BUN) in IRI mice, with PBP-EVs exhibiting superior efficacy over EVs (Fig. 7A, B). Hematoxylin and eosin (H&E) staining and kidney injury molecule 1 (Kim1) immunofluorescence staining indicated that PBP-EVs alleviated the severity of IRI kidney and restored renal pathological structure (Fig. 7C, D). Masson staining and α-SMA immunofluorescence staining demonstrated that, compared to the PBS group, PBP-EVs reduced the degree of fibrosis in the kidneys of IRI mice (Fig. 7E, F). Additionally, caspase3 immunofluorescence staining showed that PBP-EVs decreased apoptosis in damaged renal cells (Fig. S9a). Real-time qPCR results revealed that PBP-EVs significantly reduced the expression of apoptosis-related genes (Bax, Bad, Fasl, and Fas) as well as renal injury markers (Kim1, Ngal, Nphs1, and Nphs2) (Fig. 7G, H and S9b).

In conclusion, PBP-EVs can promote the repair of renal injury, reduce fibrosis and cell apoptosis in the kidneys, and is more effective than EVs.

The knockdown of Tregs reduces the protective effects of PBP-EVs on AKI

Studies have shown that Tregs depletion exacerbates histological damage and delays tubular regeneration in kidney of IRI mice; conversely, the infusion of Tregs into IRI mice can effectively suppress the expression of pro-inflammatory cytokines such as IFN-γ and promote renal repair [13]. In this study, we depleted Tregs in Foxp3-GFP transgenic mice with diphtheria toxin and found that the PBP-EVs + DT group exhibited exacerbated renal pathological damage and fibrosis, as well as diminished renal regeneration and repair. These findings are consistent with previous research results.

To further elucidate that PBP-EVs enhance renal regeneration by ameliorating endothelial cell damage and consequently modulating Tregs, we utilized Foxp3-DTR transgenic mice to knockdown Tregs and observed the effects of PBP-EVs on injured kidneys. The schematic and experimental workflow are as shown (Fig. 8A and S10a). We found that following Tregs knockdown, the expression levels of pro-inflammatory genes TGF-β, IL-6, and TNF-α were elevated, while the anti-inflammatory gene IL-10 expression level was reduced in both the PBS + DT and PBP-EVs + DT groups, indicating an exacerbation of the inflammatory response (Fig. 8B). Compared to the PBS and PBP-EVs groups, the SCr and BUN levels were elevated in the PBS + DT and PBP-EVs + DT groups, suggesting worsened renal injury upon Tregs knockdown (Fig. 8C). Furthermore, H&E staining revealed increased pathological damage in the PBP-EVs + DT group compared to the PBP-EVs group, indicating that the renal reparative effect of PBP-EVs was diminished after Tregs knockdown (Fig. 8D). α-SMA immunofluorescence staining and Masson staining demonstrated elevated renal fibrosis levels in the PBS + DT and PBP-EVs + DT groups compared to the PBS and PBP-EVs groups after Tregs knockdown (Fig. 8E). Concurrently, after Tregs depletion, the expression levels of apoptosis and kidney injury-related genes increased, while the regeneration of Lgr5⁺ renal stem cells decreased in the PBS + DT and PBP-EVs + DT groups, suggesting a reduction in the renal repair effect of PBP-EVs (Fig. S10b-d).

In summary, the knockdown of Tregs in IRI mice led to an exacerbated inflammatory response, disruption of the immune microenvironment, reduced regeneration of Lgr5⁺ renal stem cells, and weakened therapeutic effects of PBP-EVs on IRI.

In this study, we developed PBP-EVs targeted to and capable of repairing damaged endothelial cells, demonstrating significant therapeutic effects on AKI. Transcriptome sequencing also revealed that PBP-EVs inhibited the expression of inflammation-related genes and pathways in renal tissue after IRI. Compared to the PBS group, PBP-EVs reduced the expression levels of IL-6, TNF-α, and TGF-β, and decreased the numbers of neutrophils, M1 macrophages and monocytes in AKI mice. In contrast, PBP-EVs promoted the recruitment of M2 macrophages and the expression of IL-10. In addition, we observed via intravital TPM that PBP-EVs treatment promoted the recruitment of Tregs and the proliferation of Lgr5⁺ renal stem cells.in AKI mice. Immunofluorescence staining and flow cytometry further confirmed these findings. So, PBP-EVs can regulate the immune microenvironment in the kidney, facilitating post-injury renal repair. (Fig. 9).

In summary, our study indicates that PBP-EVs can promote AKI regeneration and repair by mitigating endothelial cells damage, subsequently modulating Tregs and the immune microenvironment. We innovatively utilized the interaction mechanisms between ECs and the immune microenvironment to develop PBP-EVs which target and repair ECs, providing a potential therapeutic strategy for renal IRI.

Supplementary Information

Supporting Information is available.

Author contributions

Z.L. and L.X conceptualized the project. L.X., K.Z., P.K., and L.R. complete the experiment. X.S., X.Z., H.P., E.F., and Y.X. analyzed the data. Z.L. provided funding support. L.X. wrote the manuscript.

Funding

This study was financially supported by the National Key R&D Program of China (2017YFA0103200), the National Natural Science Foundation of China (Nos. U2004126, 82330066, 82270565, and 81925021), the Tianjin Natural Science Foundation (Nos. 22JCZXJC00170 and 21JCZDJC00070), and Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-043A).

Ethics approval and consent to participate

All animal experimental procedures in this study were approved by the Animal Experiments Ethical Committee of Nankai University, following the guidelines outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (approval no. 20190022).

Consent for publication

All authors approved the final manuscript and the submission to this journal.

Conflict of Interest

The authors declare no competing interests.

Data availability Statement

The main data supporting the results in this study are available within the paper and its Supplementary Information.

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Engineered extracellular vesicles promote the repair of acute kidney injury by modulating regulatory T cells and the immune microenvironment

Status:

Version 1

Abstract

Background

Methods and Results

Conclusions

Figures

Introduction

Methods

Animals

Experimental model and protocol

Cell culture and EVs

Preparation and analysis of PBP-EVs

Preparation and analysis of Gluc-labeled EVs

Lymphocyte transmigration assays

Intravital two-photon imaging in mice

Bioluminescence imaging of renal angiogenesis

Flow cytometry

Renal function analysis

Histopathology and immunostaining

Statistical analysis

Results and Discussion

Preparation and characterization of the PBP-EVs

PBP-EVs can target and repair damaged renal endothelial cells

PBP-EVs reduce the generation of renal pro-inflammatory immune cells and cytokines

PBP-EVs promote the accumulation of Tregs in the kidney following IRI

PBP-EVs promote the proliferation of renal Lgr5⁺ stem cells

PBP-EVs alleviate mice renal IRI

The knockdown of Tregs reduces the protective effects of PBP-EVs on AKI

Conclusion

Declarations

References

Supplementary Material

Additional Declarations

Supplementary Files

Status:

Version 1

Engineered extracellular vesicles promote the repair of acute kidney injury by modulating regulatory T cells and the immune microenvironment

Status:

Version 1

Abstract

Background

Methods and Results

Conclusions

Figures

Introduction

Methods

Animals

Experimental model and protocol

Cell culture and EVs

Preparation and analysis of PBP-EVs

Preparation and analysis of Gluc-labeled EVs

Lymphocyte transmigration assays

Intravital two-photon imaging in mice

Bioluminescence imaging of renal angiogenesis

Flow cytometry

Renal function analysis

Histopathology and immunostaining

Statistical analysis

Results and Discussion

Preparation and characterization of the PBP-EVs

PBP-EVs can target and repair damaged renal endothelial cells

PBP-EVs reduce the generation of renal pro-inflammatory immune cells and cytokines

PBP-EVs promote the accumulation of Tregs in the kidney following IRI

PBP-EVs promote the proliferation of renal Lgr5+ stem cells

PBP-EVs alleviate mice renal IRI

The knockdown of Tregs reduces the protective effects of PBP-EVs on AKI

Conclusion

Declarations

References

Supplementary Material

Additional Declarations

Supplementary Files

Status:

Version 1

PBP-EVs promote the proliferation of renal Lgr5⁺ stem cells