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

ABSTRACT: 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..

SUBMITTER: Iampietro C

PROVIDER: S-EPMC7589231 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Podocytes are highly specialized cells that play a pivotal role in the blood filtration process in the glomeruli of the kidney, and their dysfunction leads to renal diseases. For this reason, the study and application of this cell type is of great importance in the field of regenerative medicine. Hypertension is mainly regulated by the renin-angiotensin-aldosterone system (RAAS), with its main mediator being angiotensin II (ANG II). Elevated ANG II levels lead to a pro-fibrotic, inflammatory, and hypertrophic milieu that induces apoptosis in podocytes. The activation of RAAS is critical for the pathogenesis of podocyte injury; as such, to prevent podocyte damage, patients with hypertension are administered drugs that modulate RAAS signaling. A prime example is the orally active, non-peptide, selective angiotensin-II-type I receptor (AGTR1) blocker losartan. Here, we demonstrate that SIX2-positive urine-derived renal progenitor cells (UdRPCs) and their immortalized counterpart (UM51-hTERT) can be directly differentiated into mature podocytes. These podocytes show activation of RAAS after stimulation with ANG II, resulting in ANG II-dependent upregulation of the expression of the angiotensin-II-type I receptor, AGTR1, and the downregulated expression of the angiotensin-II-type II receptor 2 (AGTR2). The stimulation of podocytes with losartan counteracts ANG II-dependent changes, resulting in a dependent favoring of the specific receptor from AGTR1 to AGTR2. Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced the exclusive expression of 106 genes associated with DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, and calcium ion transmembrane transport. These findings highlight the usefulness of UdRPC-derived podocytes in studying the RAAS pathway and nephrotoxicity in various kidney diseases.

Project description:Alport syndrome (AS) is a rare disease characterized by defective glomerular basement membranes, caused by mutations in COL4A3, COL4A4, and COL4A5, which synthesize collagen type IV. Patients present with progressive proteinuria, hematuria and podocyte loss. There is currently no cure for Alport syndrome, and this is mainly due to its complex and variable pathogenesis, as well as the lack of models that can faithfully mimic the human phenotype. Here we have developed a novel human culture model of Alport syndrome and used it to study the effects of different mutations on podocyte development and biology. First, we established a differentiation protocol that allowed us to generate podocyte spheroids from patient-derived human induced pluripotent stem cells (hiPSCs). We have then carried out discovery proteomics and demonstrated that a total of 178 proteins were differentially expressed between Alport (AS1 and AS3) and control (LT) podocytes. GO analysis indicated alterations in several metabolic pathways, such as oxidative phosphorylation, RNA maturation, chromatin condensation, and proliferation. Although functional assays showed no changes in lactate production and mitochondrial potential compared to healthy controls, immunofluorescence and electron microscopy analysis showed key morphological changes related to the phenotypical maturation of Alport podocytes. Moreover, the studied mutations led to persistent proliferation, increased reactive oxygen species (ROS) production and the concomitant expression of peroxisome proliferator-activated receptors α and γ (PPARα and PPARγ) in podocytes. These data on patient-derived podocytes provide evidence that collagen mutations, in addition to playing a central role in the defective development of the glomerular filtration barrier, cause significant alterations in podocyte development and metabolism very early in development, even before the formation of the filtering apparatus. In conclusion, our study provides a new methodological platform for the differentiation of podocytes and to study human podocytopathies in a personalized manner, and reveals new insights into the etiopathogenesis and pathobiology of Alport syndrome.

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

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