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

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CTNSmRNA as a potential treatment for nephropathic cystinosis

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to correct the genetic defect in nephropathic cystinosis using synthetic mRNA. Cystinosis is a prototype disorder of proximal tubular dysfunction caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and most severely affected organs, presenting glomerular and proximal tubular dysfunction. Cysteamine is the current therapeutic standard that reduces cellular cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA is safe and effective to reintroduce functional cystinosin using lipofection in CTNS-/- kidney cells and following direct injection in ctns-/- zebrafish larvae. CTNS mRNA therapy results in prompt lysosomal expression of the functional protein and decreases cellular cystine accumulation for up to 14 days. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. We propose that mRNA-based therapy, if sufficient kidney targeting can be achieved, may be a new approach to treat cystinosis

Gene based therapies for kidney regeneration

In this review we provide an overview of the expanding molecular toolbox that is available for gene based therapies and how these therapies can be used for a large variety of kidney diseases. Gene based therapies range from restoring gene function in genetic kidney diseases to steering complex molecular pathways in chronic kidney disorders, and can provide a treatment or cure for diseases that otherwise may not be targeted. This approach involves the delivery of recombinant DNA sequences harboring therapeutic genes to improve cell function and thereby promote kidney regeneration. Depending on the therapy, the recombinant DNA will express a gene that directly plays a role in the function of the cell (gene addition), that regulates the expression of an endogenous gene (gene regulation), or that even changes the DNA sequence of endogenous genes (gene editing). Some interventions involve permanent changes in the genome whereas others are only temporary and leave no trace. Efficient and safe delivery are important steps for all gene based therapies and also depend on the mode of action of the therapeutic gene. Here we provide examples on how the different methods can be used to treat various diseases, which technologies are now emerging (such as gene repair through CRISPR/Cas9) and what the opportunities, perspectives, potential and the limitations of these therapies are for the treatment of kidney diseases

Urine-Derived Epithelial Cells as Models for Genetic Kidney Diseases

Epithelial cells exfoliated in human urine can include cells anywhere from the urinary tract and kidneys; however, podocytes and proximal tubular epithelial cells (PTECs) are by far the most relevant cell types for the study of genetic kidney diseases. When maintained in vitro, they have been proven extremely valuable for discovering disease mechanisms and for the development of new therapies. Furthermore, cultured patient cells can individually represent their human sources and their specific variants for personalized medicine studies, which are recently gaining much interest. In this review, we summarize the methodology for establishing human podocyte and PTEC cell lines from urine and highlight their importance as kidney disease cell models. We explore the well-established and recent techniques of cell isolation, quantification, immortalization and characterization, and we describe their current and future applications

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..status: publishe

Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction

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A focus on the association of Apol1 with kidney disease in children

Individuals of African origin have an increased risk of developing various progressive chronic kidney diseases (CKD). This risk has been attributed to genetic variants (G1, G2) in apolipoprotein-L1 (APOL1) gene. In the pediatric population, especially in children affected by sickle cell disease (SCD), by human immunodeficiency virus (HIV), or with various glomerular diseases, APOL1 risk variants have been associated with the development of hypertension, albuminuria, and more rapid decline of kidney function. The present review focuses on existing APOL1-related epidemiological data in children with CKD. It also includes data from studies addressing racial disparities in CKD, the APOL1-related innate immunity, and the relationship between APOL1 and CKD and pathogenic pathways mediating APOL1-related kidney injury.status: publishe

Urine-Derived Kidney Progenitor Cells in Cystinosis

Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling

Urine-Derived Kidney Progenitor Cells in Cystinosis

Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling