Fanny Oliveira Arcolino

Amniotic Fluid Derived Stem Cells with a Renal Progenitor Phenotype Inhibit Interstitial Fibrosis in Renal Ischemia and Reperfusion Injury in Rats

Objectives Mesenchymal stem cells derived from human amniotic fluid (hAFSCs) are a promising source for cellular therapy, especially for renal disorders, as a subpopulation is derived from the fetal urinary tract. The purpose of this study was to evaluate if hAFSCs with a renal progenitor phenotype demonstrate a nephroprotective effect in acute ischemia reperfusion (I/R) model and prevent late stage fibrosis. Methods A total of 45 male 12-wk-old Wistar rats were divided into three equal groups;: rats subjected to I/R injury and treated with Chang Medium, rats subjected to I/R injury and treated with hAFSCs and sham-operated animals. In the first part of this study, hAFSCs that highly expressed CD24, CD117, SIX2 and PAX2 were isolated and characterized. In the second part, renal I/R injury was induced in male rats and cellular treatment was performed 6 hours later via arterial injection. Functional and histological analyses were performed 24 hours, 48 hours and 2 months after treatment using serum creatinine, urine protein to creatinine ratio, inflammatory and regeneration markers and histomorphometric analysis of the kidney. Statistical analysis was performed by analysis of variance followed by the Tukey’s test for multiple comparisons or by nonparametric Kruskal-Wallis followed by Dunn. Statistical significance level was defined as p <0.05. Results hAFSCs treatment resulted in significantly reduced serum creatinine level at 24 hours, less tubular necrosis, less hyaline cast formation, higher proliferation index, less inflammatory cell infiltration and less myofibroblasts at 48h. The treated group had less fibrosis and proteinuria at 2 months after injury. Conclusion hAFSCs contain a renal progenitor cell subpopulation that has a nephroprotective effect when delivered intra-arterially in rats with renal I/R injury, and reduces interstitial fibrosis on long term follow-up.

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.

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.

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.

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

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.

Urine of Preterm Neonates as a Novel Source of Kidney Progenitor Cells

In humans, nephrogenesis is completed prenatally, with nephrons formed until 34 weeks of gestational age. We hypothesized that urine of preterm neonates born before the completion of nephrogenesis is a noninvasive source of highly potent stem/progenitor cells. To test this hypothesis, we collected freshly voided urine at day 1 after birth from neonates born at 31-36 weeks of gestational age and characterized isolated cells using a single-cell RT-PCR strategy for gene expression analysis and flow cytometry and immunofluorescence for protein expression analysis. Neonatal stem/progenitor cells expressed markers of nephron progenitors but also, stromal progenitors, with many single cells coexpressing these markers. Furthermore, these cells presented mesenchymal stem cell features and protected cocultured tubule cells from cisplatin-induced apoptosis. Podocytes differentiated from the neonatal stem/progenitor cells showed upregulation of podocyte-specific genes and proteins, albumin endocytosis, and calcium influx via podocyte-specific transient receptor potential cation channel, subfamily C, member 6. Differentiated proximal tubule cells showed upregulation of specific genes and significantly elevated p-glycoprotein activity. We conclude that urine of preterm neonates is a novel noninvasive source of kidney progenitors that are capable of differentiation into mature kidney cells and have high potential for regenerative kidney repair.

Nephropathic cystinosis: an update

PURPOSE OF REVIEW Over the past few decades, cystinosis, a rare lysosomal storage disorder, has evolved into a treatable metabolic disease. The increasing understanding of its pathophysiology has made cystinosis a prototype disease, delivering new insights into several fundamental biochemical and cellular processes. RECENT FINDINGS In this review, we aim to provide an overview of the latest advances in the pathogenetic, clinical, and therapeutic aspects of cystinosis. SUMMARY The development of alternative therapeutic monitoring strategies and new systemic and ocular cysteamine formulations might improve outcome of cystinosis patients in the near future. With the dawn of stem cell based therapy and new emerging gene-editing technologies, novel tools have become available in the search for a cure for cystinosis.

Vascular Endothelial Growth Factor Up-regulation in Human Amniotic Fluid Stem Cell Enhances Nephroprotection After Ischemia-Reperfusion Injury in the Rat

Objective: To evaluate if the up-regulation of vascular endothelial growth factor strengthens the protective effect of amniotic fluid stem cells in a renal ischemia-reperfusion injury model. Design: Randomized animal study. Settings: University research laboratory. Subjects: A total of 40 males 12-week-old Wistar rats were subjected to ischemia-reperfusion and assigned to four groups: amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, and vehicle. Ten animals were used as sham-controls. Intervention: Six hours after induction of renal ischemia-reperfusion injury, amniotic fluid stem cells, vascular endothelial growth factor-amniotic fluid stem cells in two different doses, or vehicle were injected intraarterially. Measurements and Main Results: Analyses were performed at 24 hours, 48 hours, and 2 months after treatment. Outcome measures included serum creatinine, urine microprotenuira, and immunohistomorphometric analyses. Vascular endothelial growth factor-amniotic fluid stem cells induced a significantly higher nephroprotection than amniotic fluid stem cells. This effect was mediated mainly by immunomodulation, which led to lower macrophage infiltration and higher presence of regulatory T cell after ischemia-reperfusion injury. At medium term, it inhibited the progression toward chronic kidney disease. Vascular endothelial growth factor-amniotic fluid stem cells can worsen the ischemia-reperfusion injury when delivered in a high dose. Conclusions: Up-regulation of vascular endothelial growth factor enhances the therapeutic effect of human amniotic fluid stem cells in rats with renal ischemia-reperfusion injury, mainly by mitogenic, angiogenic, and anti-inflammatory mechanisms.

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.

Allogeneic HSCT transfers wild-type cystinosin to nonhematological epithelial cells in cystinosis: First human report

Cystinosis is an autosomal recessive lysosomal storage disorder characterized by the defective transport of the amino acid cystine out of the lysosome due to a deficiency of cystinosin, the lysosomal cystine transporter. Patients have lysosomal cystine accumulation in various tissues, leading to cellular stress and damage, particularly in the kidney, cornea, and other extrarenal tissues. Cysteamine, a cystine‐depleting agent, improves survival and delays the progression of disease, but it does not prevent the development of either renal failure or extrarenal complications. Furthermore, the drug has severe adverse effects that significantly reduce patient compliance. Allogeneic hematopoietic stem cell transplantation (HSCT) is currently established as a therapeutic option for many inborn errors of metabolism, where the main pathologic driving factor is an enzyme deficiency. Recent studies in the cystinosis mouse‐model suggested that HSCT could be a curative treatment alternative to cysteamine therapy. We treated a 16‐year‐old boy who had infantile cystinosis and side effects of cysteamine therapy with HSCT. We were able to demonstrate successful transfer of the wild‐type cystinosin protein and CTNS mRNA to nonhematological epithelial cells in the recipient, as well as a decrease in the tissue cystine‐crystal burden. This is the first report of allogeneic HSCT in a patient with cystinosis, the prototype of lysosomal membrane‐transporter disorders.