Remember me
Research ArticleCell biologyNephrology
Open Access | 
10.1172/jci.insight.197643
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Dedhia, C. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Villani, V. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Hou, X. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Neviani, P. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by
Clair, G.
in:
PubMed
|
Google Scholar
|
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Kasravi, M. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Grange, C. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by
Cravedi, P.
in:
PubMed
|
Google Scholar
|
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Aguiari, P. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Alcala, V. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Orlando, G. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Song, X. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Zuckerman, J. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by De Filippo, R. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by
Da Sacco, S.
in:
PubMed
|
Google Scholar
|
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Sedrakyan, S. in: PubMed | Google Scholar
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by
Bussolati, B.
in:
PubMed
|
Google Scholar
|
1The GOFARR Laboratory, The Saban Research Institute, Division of Urology, and
2Extracellular Vesicle Core, The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, California, USA.
3Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
4Department of Medical Sciences, University of Turin, Turin, Italy.
5Department of Medicine, Translational Transplant Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
6Section of Transplantation, Department of Surgery, Wake Forest University School of Medicine, Winston Salem, North Carolina, USA.
7Applied Genomics, Computation, and Translational Core, Cedars-Sinai Medical Center Los Angeles, California, USA.
8Department of Pathology and Lab Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California, USA.
9Department of Urology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Address correspondence to: Laura Perin, Saban Research Institute, Children’s Hospital Los Angeles, 4661 Sunset Boulevard, Los Angeles, California 90027, USA. Phone: 323.361.4584; Email: lperin@chla.usc.edu.
Find articles by Perin, L. in: PubMed | Google Scholar
Published March 23, 2026 - More info
Published in Volume 11, Issue 6 on March 23, 2026
AbstractModulation of miRNA expression in glomerular cells is associated with renal disease. Here, we investigated the role of miR-93-5p in mitigating glomerular damage in Alport syndrome and whether the disease-modifying activity of extracellular vesicles from human amniotic fluid stem cells (hAFSC-EVs) is mediated by their miR-93-5p cargo. We identified downregulation of miR-93-5p specifically in glomerular endothelial cells in Alport syndrome along disease progression. Silencing of miR-93-5p in hAFSC-EVs changed the transcriptomic and proteomic profile, regulating EV disease-modifying activity. Compared with naive hAFSC-EVs, silenced hAFSC-EVs did not rescue glomerular endothelial function in vitro and did not restore kidney function in vivo. We established that hAFSC-EVs regulate VEGFR1 and VEGFR2 signaling by miR-93-5p cargo transfer, highlighting that miR-93-5p can restore glomerular endothelial cell biology. Spatial transcriptomics analysis of hAFSC-EV–injected kidneys showed that these EVs can reverse pathways altered during disease progression by stimulating proregenerative processes, specifically in the glomerulus, by regulating miR-93-5p targets. Alteration of glomerular endothelial cell transcriptomics and miR-93-5p targets was also confirmed in biopsies of patients with Alport syndrome using spatial molecular imaging. We demonstrated the critical role of miR-93-5p in glomerular endothelial cells and the capability of hAFSC-EVs to regulate miR-93-5p and its targets in Alport syndrome.
Graphical Abstract
Introduction
Disturbance of the crosstalk between glomerular cells and changes in their interaction with the glomerular basement membrane (GBM) activate disease processes that lead to kidney failure (1). Alport syndrome (AS) is a progressive glomerular disease caused by mutations in the COL4A3, COL4A4, and COL4A5 genes (2, 3), resulting in the improper assembly of the GBM and leading to changes in cell-matrix interactions and altered glomerular cell crosstalk (4–6). Patients with AS, in addition to kidney disease, also present with auditory and ocular symptoms (2, 7, 8). AS management is centered on angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, and recently on drugs like sparsentan (dual endothelin and angiotensin II receptor antagonist) or sodium-glucose cotransporter 2 inhibitors, which show stronger effects in preserving kidney function (9, 10). Even though these therapies delay the progression to end-stage renal disease, there is an unmet need for new disease-modifying therapies that can directly repair or stabilize glomerular cell biology.
AS is considered a podocyte-centric disease, but we (11–13) and others (14) showed a central role of glomerular endothelial cells (GECs) in AS as well as in other renal diseases. The crosstalk between podocytes and GECs is essential for the function of the filtration barrier (1, 15). For example, GECs are particularly vulnerable to changes in VEGF signaling produced by podocytes (1, 15, 16), as VEGF is a crucial regulator of glomerular capillary homeostasis (17) and it is implicated not only in AS (18) but also in various renal pathologies (1, 16).
Extracellular vesicles (EVs) are fundamental modulators of cell-cell communication (19, 20) and, in particular, stem cell–derived EVs play an important role in modulating molecular pathways ranging from fibrosis to immunomodulation to tissue repair, thus slowing down disease progression in many conditions, including renal disease (21–23). Although their organ-protective role is recognized, the mechanisms involved in the EV-mediated glomerular protection are poorly understood, and further investigation into how EVs function as regulators of cell-signaling dynamics is essential to elucidate their contribution to renal protection.
We previously demonstrated (11) that EVs derived from mouse amniotic fluid stem cells (AFSC-EVs) can modulate VEGF signaling in the glomerulus in AS (Col4a5–/–) mice, characterized by a mutation in the α5 chain of collagen IV, the most prominent form of AS in humans (2, 3).
While previous studies using mouse-derived EVs have demonstrated renoprotective effects in experimental models, key questions remain regarding the therapeutic potential of clinically relevant human EV platforms. Therefore, to facilitate the EV clinical translation for chronic kidney disease (CKD), we here studied and characterized human-derived EVs from amniotic fluid.
Among the various components of the EV cargo (proteins, nucleic acids, lipids), microRNAs (miRs), small noncoding RNA molecules (24), are considered key players in exerting EV function; once delivered to the target cells, they modulate gene expression, influencing cell proliferation, differentiation, apoptosis, and other biological processes (25–27).
We previously reported the first evidence that miR-93-5p (hereafter referred to as miR-93), a potent regulator of VEGF signaling (28), plays a key role in GEC biology during AS progression and that miR-93–specific EV cargo transfer is essential for restoring GEC function in AS. Our findings provide insights into the mechanistic role of EVs in regulating GEC biology and emphasize the potential translation of human AFSC-EVs to the clinic as a treatment for patients with AS and other forms of glomerulopathy.
ResultsmiR-93 expression changes in glomerular cells during AS disease progression. To study the role of miR-93 in AS, we first determined the expression of miR-93 within the glomeruli of AS versus WT mice along disease progression, showing that miR-93 is downregulated in glomeruli in AS mice at 5.5 months of age (5.5mAS mice) (Figure 1A), when the level of proteinuria is high (Figure 1B). We detected a significant downregulation of miR-93 expression specifically in GECs in AS, but not in podocytes or mesangial cells (Figure 1C and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.197643DS1). miR-93 exists in 2 mature isoforms (miR-93-5p and miR-93-3p); the 3p form is not highly expressed in WT cells and does not change along disease progression in AS cells (Supplemental Figure 2, A–C); therefore, our study focused on the 5p form.
Figure 1miR-93 expression decreases along disease progression in mice and humans. (A) Graph showing miR-93 expression in glomeruli isolated from WT mice (C57BL/6J) at 4 months old (4m) and AS (Col4a5–/–; B6.Cg-Col4a5tm1Yseg/J) mice at 2m, 3.5m, and 5.5m. (B) Graph showing proteinuria levels, measured as albumin-to-creatinine ratio in WT mice and AS mice at 2m, 3.5m, and 5.5m. (C) Graph showing miR-93 expression in GECs, podocytes, and mesangial cells isolated from WT (4m), and AS mice at 2m, 3.5m, and 5.5m. No significant difference in miR-93 expression was noted in podocytes and mesangial cells. (D) Graph showing miR-93-5p (left) and miR-93-3p (right) expression in the kidney cortex of a human biopsy of AS patient (no. 2; male, 17 years old) compared to healthy tissue of a partial nephrectomy (used as reference; reference no. 1; male, 66 years old), showing that miR-93-5p is less expressed in AS and that miR-93-3p is much less expressed compared with miR-93-5p. (E) Representative images of human glomeruli, healthy tissue (reference no. 3) of a partial nephrectomy (male, 7 years old) showing miR-93 expression by in situ hybridization. Upper left corner: DAPI in blue. Upper right corner: EHD3 (green), identifying GECs. Lower left corner: miR-93 probe (red). Lower right corner: merged panels. White arrows: GECs showing coexpression of the miR-93 probe and EHD3. Yellow arrows: RBCs (autofluorescence in red). Scale bars: 20 μm. For miR-93 expression, small nuclear RNA U6 was used as a reference to calculate relative expression. All experiments were run in triplicate. All values are reported as mean ± SEM. Statistical significance was assessed using 1-way ANOVA with post hoc uncorrected Fisher’s LSD test. *P < 0.05; **P < 0.01; ****P < 0.0001.
We confirmed that miR-93 expression is downregulated in biopsies of patients with AS and confirmed the biological relevance of the 5p form versus the 3p isoform also in human samples (Figure 1D). Since the mouse data showed that miR-93 is expressed in GECs, we confirmed by in situ hybridization that miR-93 is mainly expressed in GECs also in human glomeruli (Figure 1E and Supplemental Figure 2D). To characterize the glomerular gene signature in AS and to determine whether miR-93 targets are altered in AS glomeruli, we performed bulk RNA-seq analysis on glomeruli isolated from AS mice and age-matched WT mice (Supplemental Figure 3, Supplemental Dataset 1, and NCBI Gene Expression Omnibus [GEO] dataset GSE318476). Principal component analysis (PCA) revealed a clear separation between AS and WT glomeruli (Supplemental Figure 3A), and transcriptional differences were also confirmed by hierarchical clustering (Supplemental Figure 3B). A volcano plot (Supplemental Figure 3C) revealed a significant shift in gene expression in AS versus WT. Specifically, Tnfrsf21 and Tgfbr2, predicted miR-93 targets, were altered in AS glomeruli (Supplemental Figure 3D), thus correlating transcriptional changes in AS with changes in miR-93 targets. Our studies were performed in male AS mice because of the X-linked nature of the disease, and even though heterozygous females were not the focus of our analysis, alteration of miR-93 targets was also present in glomeruli of female AS mice compared with age-matched WT female mice (Supplemental Figure 4, A–D, and Supplemental Dataset 1). Importantly, even if heterozygous females were to present with milder disease with no evident changes in proteinuria during our analysis timeline (Supplemental Figure 4E), they were analyzed to document that their glomerular transcriptome is different versus WT and that miR-93 targets are also modified in the milder form of AS disease.
Loss of miR-93 alters the EV cargo. miR-93 is one of the most highly expressed miRs in human EVs (Supplemental Dataset 2), with comparable expression in mouse and human EVs (Supplemental Figure 5A). For data reproducibility, we have characterized (and used in all experiments) EVs derived from our established human AFSC (hAFSC) clonal line, as published previously (11, 29–31). The data indicate that in 24 hours, 1 × 106 hAFSCs produced 2.8 × 1010 EVs, with a mode size of approximately 108 nm (Figure 2A) and had an intact membrane, as confirmed by TEM (Figure 2B). Super-resolution microscopy confirmed the presence of the tetraspanins CD9, CD63, and CD81, known to be commonly expressed in EVs (19). The majority of EVs were CD63+ or CD63+CD81+, with smaller fractions of CD81+, CD63+CD9+, and CD63+CD81+CD9+ (Figure 2, C and D). We also determine that hAFSC-EVs are CD63+VEGFR2+, CD63+CD81+VEGFR2+, CD81+VEGFR1+, CD63+VEGFR1+, and CD81+CD62+VEGFR1+ (Supplemental Figure 5, B–E).
Figure 2Loss of miR-93 alters EV cargo. (A) Nanoparticle tracking analysis representing the average mode of hAFSC-EVs (~108 nm). (B) TEM representing typical EV morphology (scale bar: 200 nm). (C and D) Super-resolution microscopy of the surface tetraspanins CD9, CD63, and CD81 (C), with a graph representing the single-, double-, and triple-positive fractions (D). Scale bars: 50 nm. (E) miR-93 expression in EVs derived from hAFSCs and KD hAFSCs. SC EVs: EVs treated with scramble control for miR-93. (F) Principal component analysis (PCA) of the miR sequencing of EVs (blue, n = 3) and KD-EVs (orange, n = 3) showing the distribution of samples along PC1 (49% variance) and PC2 (19% variance). (G) Hierarchical clustering heatmap of miRs expression in EVs (green, left) and KD_EVs (orange, right), showing a shift following miR-93 KD. (H) Volcano plot showing the differentially expressed (DE) miRs, upregulated (red) or downregulated (blue), in EVs versus KD_EVs (FC > 1.5 or FC < −1.5; P < 0.05). (I) Venn diagram representing DE miRs in EVs (blue, left) and KD_EVs (orange, right). Enriched pathways are shown below arrows. (J) PCA from the proteomic analysis of EVs (blue, n = 3) and KD_EVs (orange, n = 3) showing separation along PC1 (59.9% variance) and PC2 (18.85% variance). (K) Hierarchical clustering representing expression of proteins in EVs (green group, right) and KD_EVs (orange, left), showing shifts following KD_EVs. (L) Volcano plot of DE proteins in KD_EVs vs. EVs (log2FC > 0 or log2FC < 0; adjusted P < 0.05) upregulated (red) and downregulated (blue). (M) Venn diagram of DE proteins in EVs (blue, left) and KD_EVs (orange, right). The enriched pathways of 32 EV and 183 KD_EV proteins are indicated. miR-93 expression was normalized to small nuclear RNA U6 and measured in triplicate. Data are reported as mean ± SEM. ****P < 0.0001 by 1-way ANOVA with post hoc uncorrected Fisher’s LSD test (D and E).
To understand how miR-93 expression affects EV cargo, we generated miR-93–knockdown EVs (hAFSC-EVsmiR-93–/–, hereafter KD_EVs) using a transient antagomiR method and confirmed silencing in the cells (Supplemental Figure 5F) and EVs (Figure 2E). We performed miR sequencing on naive EVs and KD_EVs (Supplemental Dataset 2). Analysis showed a clear separation between KD_EVs and EVs (Figure 2F), and an overall shift in miR expression between the 2 groups (Figure 2G). A volcano plot indicates that miR-93 KD resulted in significant changes in specific miRs (Figure 2H); the most significantly upregulated and downregulated miRs in KD_EVs are shown. KD of miR-93 resulted in upregulation of miR-1973 and miR-29b-2-5p (only in KD_EVs) and downregulation of miR-4325p, miR-151a-3p, and miR-10b-5p expression in the KD_EVs (Supplemental Dataset 2). Prediction targets of the above-cited miRs include genes that play a key role in CKD progression, like PDGRB, WNK1, TGFb1, VEGF, FBN1, FN, and CADM2 (32–39), with miR-432-5p having the highest predicted target gene, COL4A5. We identified (Figure 2I) the exclusively upregulated miRs in EVs (19 miRs) and KD_EVs (6 miRs). Enrichment analysis of these miRs’ predicted genes in EVs is associated with pathways related to blood vessel morphogenesis, angiogenesis, cell cycle, and the insulin signaling pathway, whereas the analysis of the miR predicted genes in KD_EVs identified pathways such as IL-1 signaling, IL-17 signaling, VEGF and WNT signaling, and apoptosis.
We performed proteomics on the same EVs (Supplemental Dataset 3). Analysis showed that KD_EVs differ from the naive EVs (
Comments (0)