Upregulation of miR145 and miR126 in EVs from Renal Cells Undergoing EMT and Urine of Diabetic Nephropathy Patients.

Diabetic nephropathy (DN) is a severe kidney-related complication of type 1 and type 2 diabetes and the most frequent cause of end-stage kidney disease. Extracellular vesicles (EVs) present in the urine mainly derive from the cells of the nephron, thus representing an interesting tool mirroring the kidney's physiological state. In search of the biomarkers of disease progression, we here assessed a panel of urinary EV miRNAs previously related to DN in type 2 diabetic patients stratified based on proteinuria levels. We found that during DN progression, miR145 and miR126 specifically increased in urinary EVs from diabetic patients together with albuminuria. In vitro, miRNA modulation was assessed in a model of TGF-ß1-induced glomerular damage within a three-dimensional perfusion system, as well as in a model of tubular damage induced by albumin and glucose overload. Both renal tubular cells and podocytes undergoing epithelial to mesenchymal transition released EVs containing increased miR145 and miR126 levels. At the same time, miR126 levels were reduced in EVs released by glomerular endothelial cells. This work highlights a modulation of miR126 and miR145 during the progression of kidney damage in diabetes as biomarkers of epithelial to mesenchymal transition.

Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

Urinary Extracellular Vesicles: Uncovering the Basis of the Pathological Processes in Kidney-Related Diseases.

Intercellular communication governs multicellular interactions in complex organisms. A variety of mechanisms exist through which cells can communicate, e.g., cell-cell contact, the release of paracrine/autocrine soluble molecules, or the transfer of extracellular vesicles (EVs). EVs are membrane-surrounded structures released by almost all cell types, acting both nearby and distant from their tissue/organ of origin. In the kidney, EVs are potent intercellular messengers released by all urinary system cells and are involved in cell crosstalk, contributing to physiology and pathogenesis. Moreover, urine is a reservoir of EVs coming from the circulation after crossing the glomerular filtration barrier-or originating in the kidney. Thus, urine represents an alternative source for biomarkers in kidney-related diseases, potentially replacing standard diagnostic techniques, including kidney biopsy. This review will present an overview of EV biogenesis and classification and the leading procedures for isolating EVs from body fluids. Furthermore, their role in intra-nephron communication and their use as a diagnostic tool for precision medicine in kidney-related disorders will be discussed.

Modeling a biofluid-derived extracellular vesicle surface signature to differentiate pediatric idiopathic nephrotic syndrome clinical subgroups.

Idiopathic Nephrotic Syndrome (INS) is a common childhood glomerular disease requiring intense immunosuppressive drug treatments. Prediction of treatment response and the occurrence of relapses remains challenging. Biofluid-derived extracellular vesicles (EVs) may serve as novel liquid biopsies for INS classification and monitoring. Our cohort was composed of 105 INS children at different clinical time points (onset, relapse, and persistent proteinuria, remission, respectively), and 19 healthy controls. The expression of 37 surface EV surface markers was evaluated by flow cytometry in serum (n = 83) and urine (n = 74) from INS children (mean age = 10.1, 58% males) at different time points. Urine EVs (n = 7) and serum EVs (n = 11) from age-matched healthy children (mean age = 7.8, 94% males) were also analyzed. Tetraspanin expression in urine EVs was enhanced during active disease phase in respect to the remission group and positively correlates with proteinuria levels. Unsupervised clustering analysis identified an INS signature of 8 markers related to immunity and angiogenesis/adhesion processes. The CD41b, CD29, and CD105 showed the best diagnostic scores separating the INS active phase from the healthy condition. Interestingly, combining urinary and serum EV markers from the same patient improved the precision of clinical staging separation. Three urinary biomarkers (CD19, CD44, and CD8) were able to classify INS based on steroid sensitivity. Biofluid EVs offer a non-invasive tool for INS clinical subclassification and "personalized" interventions.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Pass through the Filtration Barrier and Protect Podocytes in a 3D Glomerular Model under Continuous Perfusion.

BACKGROUND: Dynamic cultures, characterized by continuous fluid reperfusion, elicit physiological responses from cultured cells. Mesenchymal stem cell-derived EVs (MSC-EVs) has been proposed as a novel approach in treating several renal diseases, including acute glomerular damage, by using traditional two-dimensional cell cultures and in vivo models. We here aimed to use a fluidic three-dimensional (3D) glomerular model to study the EV dynamics within the glomerular structure under perfusion. METHODS: To this end, we set up a 3D glomerular model culturing human glomerular endothelial cells and podocytes inside a bioreactor on the opposite sides of a porous membrane coated with type IV collagen. The bioreactor was connected to a circuit that allowed fluid passage at the rate of 80 µl/min. To mimic glomerular damage, the system was subjected to doxorubicin administration in the presence of therapeutic MSC-EVs. RESULTS: The integrity of the glomerular basal membrane in the 3D glomerulus was assessed by a permeability assay, demonstrating that the co-culture could limit the passage of albumin through the filtration barrier. In dynamic conditions, serum EVs engineered with cel-miR-39 passed through the glomerular barrier and transferred the exogenous microRNA to podocyte cell lines. Doxorubicin treatment increased podocyte apoptosis, whereas MSC-EV within the endothelial circuit protected podocytes from damage, decreasing cell death and albumin permeability. CONCLUSION: Using an innovative millifluidic model, able to mimic the human glomerular barrier, we were able to trace the EV passage and therapeutic effect in dynamic conditions.

Reduced Thiamine Availability and Hyperglycemia Impair Thiamine Transport in Renal Glomerular Cells through Modulation of Thiamine Transporter 2.

Thiamine helps transketolase in removing toxic metabolites, counteracting high glucose-induced damage in microvascular cells, and progression of diabetic retinopathy/nephropathy in diabetic animals. Diabetic subjects show reduced thiamine levels. Hyperglycemia and reduced thiamine availability concur in impairing thiamine transport inside the blood-retinal barrier, with thiamine transporter-2 (THTR2) primarily involved. Here, we examined the behavior of thiamine transporter-1 (THTR1), THTR2, and their transcription factor Sp1 in response to high glucose and altered thiamine availability in renal cells involved in diabetic nephropathy. Human proximal tubule epithelial cells, podocytes, glomerular endothelial, and mesangial cells were exposed to high glucose and/or thiamine deficiency/oversupplementation. Localization and modulation of THTR1, THTR2, and Sp1; intracellular thiamine; transketolase activity; and permeability to thiamine were examined. Reduced thiamine availability and hyperglycemia impaired thiamine transport and THTR2/Sp1 expression. Intracellular thiamine, transketolase activity, and permeability were strongly dependent on thiamine concentrations and, partly, excess glucose. Glomerular endothelial cells were the most affected by the microenvironmental conditions. Our results confirmed the primary role of THTR2 in altered thiamine transport in cells involved in diabetic microvascular complications. Lack of thiamine concurs with hyperglycemia in impairing thiamine transport. Thiamine supplementation could represent a therapeutic option to prevent or slow the progression of these complications.

Novel Human Podocyte Cell Model Carrying G2/G2 APOL1 High-Risk Genotype.

Apolipoprotein L1 (APOL1) high-risk genotypes (HRG), G1 and G2, increase the risk of various non-diabetic kidney diseases in the African population. To date, the precise mechanisms by which APOL1 risk variants induce injury on podocytes and other kidney cells remain unclear. Trying to unravel these mechanisms, most studies have used animal or cell models created by gene editing. We developed and characterised conditionally immortalised human podocyte cell lines derived from urine of a donor carrying APOL1 HRG G2/G2. Following induction of APOL1 expression by polyinosinic-polycytidylic acid (poly(I:C)), we assessed functional features of APOL1-induced podocyte dysfunction. As control, APOL1 wild type (G0/G0) podocyte cell line previously generated from a Caucasian donor was used. Upon exposure to poly(I:C), G2/G2 and G0/G0 podocytes upregulated APOL1 expression resulting in podocytes detachment, decreased cells viability and increased apoptosis rate in a genotype-independent manner. Nevertheless, G2/G2 podocyte cell lines exhibited altered features, including upregulation of CD2AP, alteration of cytoskeleton, reduction of autophagic flux and increased permeability in an in vitro model under continuous perfusion. The human APOL1 G2/G2 podocyte cell model is a useful tool for unravelling the mechanisms of APOL1-induced podocyte injury and the cellular functions of APOL1.

Potential Applications of Extracellular Vesicles in Solid Organ Transplantation.

Extracellular vesicles (EVs) play an important role in cell-to-cell communication by delivering coding and non-coding RNA species and proteins to target cells. Recently, the therapeutic potential of EVs has been shown to extend to the field of solid organ transplantations. Mesenchymal stromal cell-derived EVs (MSC-EVs) in particular have been proposed as a new tool to improve graft survival, thanks to the modulation of tolerance toward the graft, and to their anti-fibrotic and pro-angiogenic effects. Moreover, MSC-EVs may reduce ischemia reperfusion injury, improving the recovery from acute damage. In addition, EVs currently considered helpful tools for preserving donor organs when administered before transplant in the context of hypothermic or normothermic perfusion machines. The addition of EVs to the perfusion solution, recently proposed for kidney, lung, and liver grafts, resulted in the amelioration of donor organ viability and functionality. EVs may therefore be of therapeutic interest in different aspects of the transplantation process for increasing the number of available organs and improving their long-term survival.