Human Urine as a Noninvasive Source of Kidney Cells

Urine represents an unlimited source of patient-specific kidney cells that can be harvested noninvasively. Urine derived podocytes and proximal tubule cells have been used to study disease mechanisms and to screen for novel drug therapies in a variety of human kidney disorders. The urinary kidney stem/progenitor cells and extracellular vesicles, instead, might be promising for therapeutic treatments of kidney injury. The greatest advantages of urine as a source of viable cells are the easy collection and less complicated ethical issues. However, extensive characterization and in vivo studies still have to be performed before the clinical use of urinederived kidney progenitors

APOL1 C-Terminal Variants May Trigger Kidney Disease through Interference with APOL3 Control of Actomyosin

The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.info:eu-repo/semantics/publishe

Human urine as a mirror of kidney development, disease and regeneration

Urine represents an unlimited source of kidney specific cells that can be harvested non-invasively. Urine derived podocytes and proximal tubule cells have been used for diagnosis of kidney diseases but also to study disease mechanisms and to screen for novel drug therapies in a variety of human kidney disorders. The urinary kidney stem/progenitor cells and extracellular vesicles, instead, might be promising for treatments of kidney disease. The greatest advantages of urine as a source of viable cells are the easy collection and less complicated ethical issues. However, extensive characterization and in vivo studies still have to be performed before the clinical use of urine-derived kidney cells. In the first part of this PhD thesis, we introduced a novel source of potent cells derived from the urine of preterm neonates. These kidney stem/progenitor cells (nKSPC) presented characteristics of stromal and nephron progenitors simultaneously, meaning that nKSPCs had capacity to prevent apoptosis of mature proximal tubule epithelial cells via a paracrine action, which is a feature of mesenchymal stem cells; and also, they had capacity to differentiate into functional podocytes and proximal tubule epithelial cells in vitro, which is a feature of nephron progenitor cells. Besides that, nKSPCs might be a unique source of kidney progenitors due to the easiness of non-invasive sample collection raising nearly no ethical concerns, and because they show proper equilibrium between unlimited self-renewal and commitment to kidney tissue, representing an ideal source tool for renal cell therapies. In the second chapter, we have shown that urinary podocyte cell lines are an important tool to model cystinosis, a genetic lysosomal disorder that often leads to renal failure. We investigated whether there was an excessive loss of podocytes in urine (podocyturia) of cystinosis patients in comparison to controls and the possible explanations for that, evaluating the structural and molecular abnormalities of urinary podocytes. Our results have shown that cystinosis patients void off in urine a significantly higher amount of podocytes compared to healthy individuals. We observed that conditionally immortalized cystinotic podocyte cell lines had cystine accumulation compatible with cystinosin deficiency and also had altered cytoskeleton, impaired cell adhesion sites and increased individual cell motility. These observations, together with the fact that cystinotic cells showed enhanced phosphorylation of Akt1 and Akt2 isoforms, gave us new insights for the explanation of excessive proteinuria and podocyturia in cystinosis patients. Podocyturia might influence the severity of cystinosis and since podocytes have very low or even no capacity to proliferate, novel therapies are needed for glomerular regeneration. In the third chapter of this thesis, we have described a peculiar source of undifferentiated kidney cells isolated from urine of cystinosis patients. These cells presented progenitor cell phenotype with long-term growth capacity and potential to differentiate into functional podocytes. Once we are able to correct the cystinosin mutation by means of gene therapy in the cystinosis progenitor cells, they might signify a novel tool for personalized cell therapy in cystinosis and the developed methodology will be useful for other genetic kidney diseases.status: publishe

Gene based therapies for kidney regeneration

n 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.status: publishe

Human Urine as a Noninvasive Source of Kidney Cells

Urine represents an unlimited source of patient-specific kidney cells that can be harvested noninvasively. Urine derived podocytes and proximal tubule cells have been used to study disease mechanisms and to screen for novel drug therapies in a variety of human kidney disorders. The urinary kidney stem/progenitor cells and extracellular vesicles, instead, might be promising for therapeutic treatments of kidney injury. The greatest advantages of urine as a source of viable cells are the easy collection and less complicated ethical issues. However, extensive characterization and in vivo studies still have to be performed before the clinical use of urine-derived kidney progenitors

Cystinosin deficiency causes podocyte damage and loss associated with increased cell motility

The involvement of the glomerulus in the pathogenesis of cystinosis, caused by loss-of-function mutations in cystinosin (CTNS, 17p13), is a matter of controversy. Although patients with cystinosis demonstrate glomerular lesions and high-molecular-weight proteinuria starting from an early age, a mouse model of cystinosis develops only signs of proximal tubular dysfunction. Here we studied podocyte damage in patients with cystinosis by analyzing urinary podocyte excretion and by in vitro studies of podocytes deficient in cystinosin. Urine from patients with cystinosis presented a significantly higher amount of podocytes compared with controls. In culture, cystinotic podocytes accumulated cystine compatible with cystinosin deficiency. The expression of podocyte specific genes CD2AP, podocalyxin, and synaptopodin and of the WT1 protein was evident in all cell lines. Conditionally immortalized podocyte lines of 2 patients with different CTNS mutations had altered cytoskeleton, impaired cell adhesion sites, and increased individual cell motility. Moreover, these cells showed enhanced phosphorylation of both Akt1 and Akt2 (isoforms of protein kinase B). Inhibition of Akt by a specific inhibitor (Akti inhibitor 1/2) resulted in normalization of the hypermotile phenotype. Thus, our study extends the list of genetic disorders causing podocyte damage and provides the evidence of altered cell signaling cascades resulting in impaired cell adhesion and enhanced cell motility in cystinosis.publisher: Elsevier articletitle: Cystinosin deficiency causes podocyte damage and loss associated with increased cell motility journaltitle: Kidney International articlelink: http://dx.doi.org/10.1016/j.kint.2016.01.013 content_type: article copyright: Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.status: publishe

Connective tissue growth factor (CTGF) from basics to clinics

Connective tissue growth factor, also known as CCN2, is a cysteine-rich matricellular protein involved in the control of biological processes, such as cell proliferation, differentiation, adhesion and angiogenesis, as well as multiple pathologies, such as tumor development and tissue fibrosis. Here, we describe the molecular and biological characteristics of CTGF, its regulation and various functions in the spectrum of development and regeneration to fibrosis. We further outline the preclinical and clinical studies concerning compounds targeting CTGF in various pathologies with the focus on heart, lung, liver, kidney and solid organ transplantation. Finally, we address the advances and pitfalls of translational fibrosis research and provide suggestions to move towards a better management of fibrosis

A Human Proximal Tubular Epithelial Cell Model to Explore a Knowledge Gap on Neonatal Drug Disposition

Finding the right drug-dosage for neonates is still a challenge. Until now, neonatal doses are extrapolated from adults and children doses. However, there are differences between neonatal and adult kidney physiology that should be considered, especially when it comes to drug metabolism and/or transport. Studying renal drug disposition in neonates is limited by the lack of reliable human cell models.status: publishe

De novo SIX2 activation in human kidneys treated with neonatal kidney stem/progenitor cells.

During development, nephron structures are derived from a SIX2+ stem cell population. After 36 weeks of gestation, these cells are exhausted, and no new nephrons are formed. We have previously described a non-invasive strategy to isolate and expand the native SIX2+ kidney stem cells from the urine of preterm neonates, named neonatal kidney stem/progenitor cells (nKSPC). Here, we investigated the safety and feasibility of administering nKSPC into human kidneys discarded for transplantation during normothermic machine perfusion (NMP) and evaluated the regenerative and immunomodulatory potential of nKSPC treatment. We found that nKSPC administration during NMP is safe and feasible. Interestingly, nKSPC induced the de novo expression of SIX2 in proximal tubular cells of the donor kidneys and upregulated regenerative markers such as SOX9 and VEGF. This is the first time that SIX2 re-expression is observed in adult human kidneys. Moreover, nKSPC administration significantly lowered levels of kidney injury biomarkers and reduced inflammatory cytokine levels via the tryptophan-IDO-kynurenine pathway. In conclusion, nKSPC is a novel cell type to be applied in kidney-targeted cell therapy, with the potential to induce an endogenous regenerative process and immunomodulation