Basic Research

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

Fanny Oliveira Arcolino, Silvia Zia, Katharina Held, Elli Papadimitriou, Koen Theunis, Benedetta Bussolati, Anke Raaijmakers, Karel Allegaert, Thierry Voet, Jan Deprest, Joris Vriens, Jaan Toelen, Lambertus van den Heuvel and Elena Levtchenko
JASN September 2016, 27 (9) 2762-2770; DOI: https://doi.org/10.1681/ASN.2015060664

Abstract

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.

  • stem cell
  • preterm
  • urine

Identification of kidney stem/progenitor cell (KSPC) populations is important for therapeutic potential on kidney injury and also, better understanding nephrogenesis.

During nephrogenesis, after the initial contact of the ureteric bud with the metanephric mesenchyme (MM), a population of kidney progenitor cells is found in the condensed mesenchyme.1 These cells express the transcription factor SIX2, a direct regulator of nephrogenic progenitor self–renewal responsible for maintenance of the MM, suppression of epithelial differentiation,2 and later in development, induction of temporal and spatial nephron differentiation.3

In mice, at embryonic day 11.5, the MM close to the ureteric bud is formed by Six2+ cells2,4 surrounded by Foxd1+ cells that give rise to stromal cells and the mesangial cells of the glomerulus.5 Although these two populations of cells were thought to be distinct and exclusive,3,6,7 single-cell dissection of developing mouse kidney has shown diverse gene expression combinations, including cells transcribing both Six2 and Foxd1, providing evidence that initial organogenesis involves a multilineage priming process.8

Sutherland and Bain9 were the first to culture exfoliated cells from the urine of newborn infants. Currently, it is well established that urinary sediment can serve as a noninvasive source of cells of different parts of the nephron,10 such as renal tubule epithelial cells11,12 and podocytes.13 Recently, it was shown that cells with a renal progenitor phenotype originated from developing kidneys could be isolated from amniotic fluid, mainly composed of fetal urine. These cells presented anti-inflammatory and antiapoptotic effects and inhibited fibrosis in an ischemia-reperfusion model in rats.14

Because stem/progenitor cells from specific adult tissue generally present limited differentiation potential and its existence in the adult kidney is questionable, preterm neonatal urinary cells (born before completion of nephrogenesis) might be a tissue–specific potent source of progenitors with higher potential compared with adult cells.

Here, we introduce preterm urine as novel potent source of KSPCs capable of reducing cisplatin injury and differentiating into functional podocytes and proximal tubule cells (PTECs). Moreover, for the first time to our knowledge, we show that human KSPCs express variable degrees of epithelial and stromal progenitor markers, including coexpression of SIX2 and FOXD1.

Results

Isolation and Culture of Kidney Undifferentiated Cells

It was requisite for collection of amniotic fluid samples and urine of preterm neonates that no renal abnormalities were diagnosed and no nephrotoxic drugs were administered (Supplemental Table 1). Urine was collected from preterm neonates born at median 34 weeks gestational age (GA; range =31–36) 1 day after birth; 51% of the samples gave rise to growing clonal colonies, and bacterial contamination happened in 12.7% of the samples. Adherent cells were detected after 6–7 days of culture and continued growing for at least 21 passages. Amniotic fluid was collected at the median 18th week of GA (range =15–22), and colonies were found in all samples. Adult urine samples at median age 27 years old (range =23–40) were collected. In only three of seven samples from girls, adherent cells appeared in culture, each giving rise to one to three colonies; none of the samples from boys revealed adherent cells.

For further characterization, we selected three clonal subpopulations of each source of cells from different donors: neonatal kidney stem/progenitor cells (nKSPCs) (Figure 1A), amniotic fluid stem cells (AFSCs) (Figure 1F), and adult urine progenitor cells (aUPCs) (Figure 1H).

Figure 1.
Figure 1.

Morphology of KSPCs and KSPC-derived podocytes. (A, F, and H) Representative pictures of preterm nKSPCs, AFSCs, and aUPCs in culture at passage 4. (B, G, and I) nKSPC-podo, AFSC-podo, and aUPC-podo. (C) Scanning electron microscopy of nKSPC-podo. Original magnification, ×370. (D) Detail of the foot process–like structure of nKSPC-podo. (E) ciPodocytes used as control for comparison.

Characterization of Kidney Undifferentiated Cells

Clonal cells from preterm urine and amniotic fluid were positive for kidney progenitor genes SIX2, CITED1, and Vimentin (Figure 2A). Specifically, SIX2 expression was detected in preterm neonatal cells derived from neonates born before 34 weeks GA (Supplemental Figure 1). Adult progenitor cells were negative for SIX2 but expressed CITED1 and Vimentin (Figure 2A) together with CD133 and CD2415 (Figure 2B).

Figure 2.
Figure 2.

Characterization of undifferentiated kidney cells. (A) Quantitative PCR analysis of renal progenitor cell markers SIX2, CITED1, and Vimentin for nKSPCs, AFSCs, and aUPCs normalized to GAPDH. (B) Percentage of cells expressing renal progenitor markers CD133 and CD24 in nKSPCs, AFSCs, and aUPCs in flow cytometry analysis. (C) Representative RT-PCR results of single cells (nKSPCs) from a clonal population of the same passage for early progenitor markers OSR1 and PAX2, nephron progenitor marker SIX2, and stromal progenitor marker FOXD1. Note different combinations of gene expression at the single-cell level. (D) Flow cytometry analysis showing coexpression of SIX2 and FOXD1 (29.9%); the IgG controls are in blue. (E) Immunofluorescence staining of nKSPCs for SIX2/FOXD1 and SIX2/PAX2. (F) Relative expression of caspase 3 in control ciPTECs, ciPTECs damaged with cisplatin (cisplatin), and damaged ciPTECs cocultured with nKSPCs for 48 hours (cisplatin + nKSPC). *P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Undifferentiated cells also showed expression of cell surface antigen markers of mesenchymal stem cells (Supplemental Figure 2) but were negative for hematopoietic cell markers.

For better characterization of nKSPCs, we have performed single–cell RT-PCR. Gene expression profile presented a variety of combinations of nephron and stromal progenitor markers: 17.5% of the cells were SIX2+, 15% were FOXD1+, 10% were OSR1+, and 20% were PAX2+. Surprisingly, 7.5% were SIX2+/FOXD1+, but only one cell was SIX2+/FOXD1+/OSR1+ (Figure 2C). Costaining of SIX2/FOXD1 in nKSPCs using flow cytometry analysis and immunofluorescence confirmed the expression of these markers in single cells at the protein level (Figure 2, D and E).

Protective Effect of Preterm Neonate Urine KSPCs

nKSPCs presented a significant protective effect against cisplatin-induced apoptosis when cocultured with conditionally immortalized proximal tubule cells (ciPTECs) (Figure 2F).

A summary of comparison among nKSPCs, AFSCs, and aUPCs can be found in Table 1. For further experiments, one representative clonal population of each source of cells was used at passages 4–10.

View this table:
Table 1.

Comparison among sources of KSPCs

Differentiation of KSPCs to Podocytes

Podocytes derived from nKSPCs, AFSCs, and aUPCs were named nKSPC-podo, AFSC-podo, and aUPC-podo, respectively. They acquired arborized morphology of the cytoplasm and became bi- or multinucleated, resembling fully differentiated conditionally immortalized podocytes (ciPodocytes), a cell line isolated from human kidney (Figure 1, B–E, G, and I). At the genetic level, there was a significant upregulation of the expression of podocyte-specific genes (Figure 3, A–C). The fold increase was higher in nKSPCs compared with the other cell lines, and only the neonatal- and amniotic fluid–derived podocytes acquired expression of nephrin. The transcription factor LMX1b, essential for the maintenance of differentiated podocytes, presented a 239.2±38.1-fold increase after differentiation in nKSPC-podo and a 138.2±13.6-fold increase in AFSC-podo but only a 1.3±0.4-fold increase in aUPC-podo compared with the respective undifferentiated cells. Because of the fact that ciPodocytes express reduced levels of LMX1b in culture,16 normalization was not suitable.

Figure 3.
Figure 3.

Genetic and protein expression analyses of podocytes derived from undifferentiated kidney cells. (A–C) Quantitative PCR analysis of podocyte–specific genes Podocalyxin, Synaptopodin, CD2AP, and Nephrin in (A) nKSPC and nKSPC-podo, (B) AFSC and AFSC-podo, and (C) aUPC and aUPC-podo normalized to the gene expression of ciPodocytes and normalized to glyceraldehyde-3-phosphate dehydrogenase. *P<0.01. (D–F) Immunofluorescence staining for podocyte–specific proteins podocin (red) and WT-1 (green) and nuclear staining DAPI (blue). (D) nKSPC and nKSPC-podo, (E) AFSC and AFSC-podo, and (F) aUPC and aUPC-podo. It is of note that cells derived from preterm urine and amniotic fluid acquired this expression on differentiation to podocytes, whereas cells isolated from adult urine already expressed those proteins (although at lower levels), indicating a more differentiated state. Original magnification, ×200. DAPI, 4′,6-diamidino-2-phenylindole.

Podocyte–specific proteins podocalyxin, synaptopodin, podocin, and WT-1 were expressed in all derived podocytes (Figure 3, D–F, Supplemental Figure 3).

Derived podocytes presented arrangement and distribution of actin filaments in long bundles similar to ciPodocytes, but they had more distantly distributed focal adhesion molecules (Supplemental Figure 4).

Functionality of Podocytes Derived from Undifferentiated KSPCs

Albumin Endocytosis

The temperature–dependent endocytic uptake of 555-labeled albumin by nKSPC-podo was significantly higher than that by ciPodocytes, showing high activity of the cells (Figure 4).

Figure 4.
Figure 4.

Albumin endocytosis in podocytes derived from undifferentiated kidney cells. Functional assay of albumin-555 (red) endocytosis at 37°C by (A) nKSPC-podo, (B) AFSC-podo, and (C) aUPC-podo compared with control; (D) ciPodocytes incubated at 37°C; and (E) ciPodocytes incubated at 4°C (inhibition of endocytosis). Nuclear staining 4′,6-diamidino-2-phenylindole (blue). Original magnification, ×400. (F) Quantification of total fluorescence corrected to background. *P<0.05.

Transient Receptor Potential Cation Channel, Subfamily C, Member 6 Activity

We have also tested the functionality of podocyte–specific transient receptor potential cation channel, subfamily C, member 6 (TRPC6)17 as a marker for successful podocyte differentiation. On stimulation with the TRPC6 agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG), cells showed a robust increase in [Ca2+]i in 79% (335 of 424 cells) of the ciPodocytes, 70.5% (41 of 58 cells) of the nKSPC-podo, 97.4% (40 of 42 cells) of the AFSC-podo, and 44.2% (11 of 25 cells) of the aUPC-podo in the presence of extracellular Ca2+ (Figure 5, A and B). In contrast, a significantly lower amount of undifferentiated cells responded to OAG stimulation (15.8% of the nKSPCs, 70.2% of the AFSCs, and 11.8% of the aUPCs). In the absence of extracellular Ca2+, stimulation with OAG did not induce increase in [Ca2+]i (Supplemental Figure 5). The increased expression of the TRPC6 protein was shown in nKSPC-podo and compared with nKSPCs and ciPodocytes by immunofluorescence staining (Supplemental Figure 3, D–G).

Figure 5.
Figure 5.

Calcium influx in podocytes derived from kidney undifferentiated cells and whole–cell patch clamp. (A) TRPC6 activity is shown by cell change in fluorescence ratio because of stimulation with OAG over time. (B) Quantification of OAG–responder progenitor cells and podocytes derived from progenitor cells. (C) Time course of currents obtained in ciPodocytes at ±80 mV in the whole–cell patch clamp configuration. At indicated time points, OAG (100 µM) was applied, or all cations were exchanged for NMDG+ in the extracellular solution. (D) Current-voltage curves corresponding to the indicated time points in C. (E) Time course of currents obtained in nKSPC-podo at ±80 mV in the whole–cell patch–clamp configuration. (F) Current-voltage curves corresponding to the indicated time points in E. (G) Time course of currents obtained in nKSPC-Podo at ±80 mV in the whole–cell patch–clamp configuration. (H) Mean current increases at +80 mV (black bars) and −80 mV (red bars) after treatment with OAG in ciPodocytes, nKSPC, and nKSPC-Podo. Note that currents are presented as currents related to the membrane surface (current density). Iono, ionomycin; pA/pF, picoamperes per picofarad (current density). *P<0.05 (statistically significant).

Patch-clamp experiments confirmed the specific TRPC6 activity in nKSPC-podo and ciPodocytes (Figure 5, C and D). Replacing all cations in the external solution by NMDG+ resulted in a block of the inward current, indicating that the OAG-induced current was carried by cations. Similar to ciPodocytes, nKSPC-podo showed an increase in in- and outward currents on stimulation with OAG (Figure 5, E–G).

Differentiation of KSPCs to PTECs

By culturing nKSPCs in PTEC-specific medium, we observed a significant upregulation of PTEC-specific genes, including significant increased expression of p-glycoprotein (Pgp) (Figure 6, A–C). Pgp activity of derived PTECs was evaluated by calcein-AM efflux assay and compared with ciPTECs isolated from human kidneys.18 Cell lysates showed a significant accumulation of calcein inside the cells when incubated with Pgp inhibitor compared with cells with noninhibited transporters, suggesting the successful differentiation of nKSPCs toward PTECs.

Figure 6.
Figure 6.

Differentiation and functionality of nKSPCs into PTECs. (A) Representative image of preterm nKSPCs and differentiated nKSPC-PTECs. (B) Quantitative PCR analysis of PTEC markers for ciPTECs, nKSPCs, and nKSPC-PTECs normalized to GAPDH. (C) Western blot analysis of Pgp and the endogenous control GAPDH for nKSPCs and nKSPC-PTEC and respective quantification. (D) Fluorescence measurements after incubation of cells with calcein-AM or calcein-AM plus Pgp inhibitor. An increase in fluorescence after incubation with inhibitor indicates specific Pgp activity. *P<0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Discussion

Our study shows that renal stem/progenitor cells can be isolated from urine of preterm neonates. We have not only characterized nKSPCs in detail but also, proven their potential to differentiate into functional podocytes and PTECs. At the single-cell level, nKSPCs expressed different combinations of nephron and stromal progenitor markers, even sometimes being detected in the same cells. The latter observation was unexpected, because SIX2+ and FOXD1+ cells were assumed to be distinct populations.3,7 Self-renewing SIX2+ cells retain the potential to differentiate into mature nephron structures, whereas FOXD1+ cells show no epithelial potential and develop instead into interstitial, perivascular, and possibly, endothelial elements of the kidney.19 Although our finding is novel in humans, the existence of SIX2/FOXD1 double–positive cells was previously reported in FOXD1-CRE transgenic mice8 by both immunofluorescence staining and single–cell mRNA analysis.

These results support the idea that the cap mesenchyme is composed of a heterogeneous population of cells that changes with time rather than restricted lineages. Therefore, it seems that the concept of lineage restriction in two distinct populations of stromal and epithelial progenitors in the cap mesenchyme should be re-evaluated in human tissue. It also reinforces the fact that, although mouse and human embryogeneses share similarities, the dynamics of nephrogenesis can be very different.20

The expression of OSR1 was not expected in our cultured cells, because it is a very early expressed gene in the intermediate mesoderm.21 However, because it is a common precursor marker of SIX2+ and FOXD1+ cells, nKSPC might have undifferentiated when put in culture and re-expressed OSR1. Possibly, the double-positive cells represent an earlier state of differentiation compared with the more committed epithelial (only SIX2+) or stromal cells (only FOXD1+). Apparently, there is an age-dependent balance between cell self-renewal and differentiation.22,23 Because our cells were isolated during an active state of nephrogenesis, these two processes might have continued in culture, with cells either maintaining progenitors features or following their fate and spontaneously committing to more mature stages. This might also explain why some of cells did not express any of the early markers. Nevertheless, the population of nKSPCs guaranteed their potential to differentiate into mature nephron cells and also, presented stromal features as shown by their paracrine effect.

We have compared the feasibility of isolation and potency of KSPCs derived from preterm urine with AFSCs and aUPCs. Stem cells derived from fetal tissue have the advantage of being more stable and plastic, offering a valid alternative to their adult counterpart.24 Because fetal urine contributes to the contents of amniotic fluid, one can assume that subpopulations of cells cultured from amniotic fluid derive from the developing kidneys.9 However, amniotic fluid represents a heterogeneous source of fetal cells,25 containing lung progenitors and fibroblasts, and therefore, extensive characterization has to be performed to select the kidney-derived clones. In this regard, using preterm urine might be more straightforward, because it contains cells already committed to the renal fate. In addition, urine is a more advantageous source of cells compared with amniotic fluid because of the noninvasiveness; moreover, repeated samples can be collected from the same donor.

Adult stem cells from different organs were able to differentiate into any cell type of the organ of origin. These cells generally present very low risk of tumorigenesis, better integration into the related tissue, and better acquisition of relevant functions.26 In the mature kidney, the source and even the existence of endogenous stem cells are still uncertain. It has been suggested that a population of glomerular parietal epithelial cells can function as stem cells, displaying self-renewal properties and potential to differentiate into podocytes and PTECs. These cells are CD133+CD24+ and widely present during kidney development, becoming more restricted to the urinary pole of Bowman’s capsule after kidney maturation and possibly, persisting in adulthood as kidney progenitor cells.27,28

Recently, cells isolated from adult urine with characteristics of stem cells, such as clonogenicity, cell surface marker expression, multipotent differentiation, and immune-modulatory properties, were described.29–32 It is likely that they originate from the kidneys because of the expression of specific renal cells markers.30 In our study, we collected samples of healthy adults; we were not able to detect proliferating cells in any urine from men, and only five clonal cell lines grew out of three of seven samples from girls and still presented low proliferation rate after four passages. Our findings were in line with those in the work by Lazzeri et al.,33 which showed that adult kidney progenitors could only be isolated from urine of proteinuric kidneys. Another limitation in the isolation of adult kidney progenitor cells is the lack of specific multipotent markers. Although cells expressed CD24 and CD133, they were also positive for podocyte-specific proteins, indicating an existing commitment.

Instead, the isolation of preterm neonate progenitors was highly successful. We only collected samples from boy neonates because of the easiness of attaching a plastic bag with a sticky strip to the penis and avoiding infections of samples during collection by cleaning the genital region; however, sample collection from girls should also be possible.

Although regenerative potential via paracrine effect has been mostly attributed to mesenchymal stem cells,34 renal progenitors have also shown protective effect in AKI.35 Adult renal progenitors protected PTECs from cisplatin toxicity, preventing apoptosis and enhancing proliferation of survived cells, probably because of secretion of chemokines and specific microvesicle mRNA through the activation of Toll-like receptor 2.36 Also, human–induced pluripotent stem cell–derived renal progenitors showed significant amelioration of AKI in mice without integration or differentiation of the cells, suggesting a paracrine effect.37 In analogy, we investigated this protective effect using nKSPCs in cisplatin-damaged ciPTECs and confirmed their capacity to prevent apoptosis via a paracrine action using a coculture system.

After expansion and characterization of the KSPCs, we evaluated their potential to differentiate into podocytes and PTECs. Retinoic acid is known to contribute to renal morphogenesis and differentiation.38 The use of all trans retinoic acid maintains expression of nephrin for extended periods39 but also, induces differentiation of kidney progenitors stimulating transcription of target genes to modulate expression of nephrin and podocin in vitro and in vivo.40,41 On differentiation, we observed clear difference in size (preterm neonatal cells grew from 14 to 22 μm) and morphology of the cells. Differentiated nKSPCs and AFSCs were associated with higher upregulation of podocyte genes compared with aUPCs, and these were the only cells expressing nephrin.

At the functional level, nKSPC-podo presented high levels of albumin endocytosis. Recent studies have shown albumin endocytosis in podocytes in culture42 and human and animal models under albuminuric conditions.43 In addition, we studied the activity of the podocyte–specific TRPC6 channel, which is expressed in podocyte foot processes and along the slit diaphragm, where it colocalizes with nephrin and podocin.17 In cultured podocytes, OAG is described to induce a TRPC6–dependent calcium influx.44 In whole–cell patch clamp, direct application of OAG induced the activation of an in- and outwardly rectifying current, similar to currents previously described for TRPC6.45 So far, we are the first to show the activity of TRPC6 in podocytes derived of progenitor cells and suggest the implementation of this technique for validation of podocyte functionality.

Differentiation of nKSPCs into PTECs was successful after 4 days in PTEC medium. We evaluated the functionality of the derived cells via activity of Pgp, a membrane transporter that mediates efflux of cationic drugs.46 Fluorescent calcein is actively removed by Pgp, and this specific transport can be inhibited using PSC833. nKSPC-PTECs incubated with inhibitor showed a significant increased accumulation of calcein in the cytoplasm compared with nKSPCs cells, indicating full differentiation into PTEC cells.

In preterm neonates, nephrogenesis is still ongoing at the time of birth and continues in the ex utero environment.47 The presence of progenitor cells in urine of preterm neonates is of great interest for autologous therapy. They can be noninvasively collected, expanded, and stored for future usage, because low birth weight is associated with increased risk of developing renal insufficiency later in life.48 Additional studies should evaluate the potential of nKSPCs to ameliorate outcomes in renal injury models.

We concluded that KSPCs can be efficiently isolated from preterm neonatal urine, representing a potent noninvasive source of kidney progenitor cells, with potential to reduce toxic kidney injury and differentiate into functional podocytes and PTECs. These cells may be a promising tool for regenerative medicine aimed at kidney repair.

Concise Methods

Isolation and Culture of Undifferentiated Cells from Urine and Amniotic Fluid

Isolation and culture of cells from urine of preterm neonates, adults, and amniotic fluid is described in detail in Supplemental Material.

Characterization of Kidney-Undifferentiated Cells Derived from Urine and Amniotic Fluid

Clonal cell lines were characterized using flow cytometry analysis, quantitative PCR, immunofluorescence staining, and Western blotting as described in Supplemental Material.

Single–Cell cDNA Synthesis

Single-cell cDNA was prepared adapting the protocol in the work by Picelli et al.49 and is detailed in Supplemental Material.

Paracrine Protective Effect of Preterm Neonate Urine KSPCs

We investigated the potential of nKSPCs to protect against cisplatin-induced apoptosis. Urinary ciPTECs18 were seeded in six–well culture dishes in a density of 150,000 cells per well. The control condition was cultured in 5% FBS medium. To induce caspase 3 cascade activation, cells were damaged for 24 hours with cisplatin (5 μg/ml) in 5% FBS medium; 150,000 nKSPCs were seeded in 1-μm-pore size BD Falcon Cell Culture Inserts (353102; BD Biosciences, San Jose, CA) and cocultured with the damaged ciPTECS for 48 hours. Activation of caspase 3 cascade in ciPTECs was analyzed by quantitative PCR in three independent experiments.

Differentiation of KSPCs to Podocytes and PTECs

The detailed protocol of KSPCs differentiation into podocytes and PTECs as well as functional analyses are described in Supplemental Material.

Ethics

Parents of neonates and adult donors signed an informed consent for the collection of urine or amniotic fluid, and the Ethics Committee of the Universitaire Ziekenhuizen Leuven approved the research protocol (S53345) under the Belgian registration number B322201317777.

Disclosures

None.

Acknowledgments

The authors acknowledge Dr. M. Saleem for offering conditionally immortalized podocyte cell lines to be used as a control in the study. The intellectual support of Prof. Dr. Laura Perin and the technical support of Harm Kwak and Flore Lesage are also acknowledged.

F.O.A. and E.P. are supported by Seventh Framework Programme 7 NephroTools Marie Curie Initial Training Networks grant 289754. K.H. is funded by the Fonds Wetenschappelijk Onderzoek (FWO) Belgium. K.A. is supported by Research Foundation–Flanders (FWO Vlaanderen) grant 1800214N. J.D. is supported by Fonds voor Wetenschappelijk Onderzoek Vlaanderen grant FWO 1.801207. J.V. is supported by Research Council of the Katholieke Universiteit Leuven grant OT/13/113 and Research Foundation–Flanders grant G.0856.13N. J.T. is a beneficiary of a fundamental clinical research grant from the Klinische Opleidings en Onderzoeks Raad of the University Hospitals Leuven. E.L. is supported by Research Foundation–Flanders (FWO Vlaanderen) grant 1801110N and EURenOmics Study grant 305608.

Abstracts regarding this study have been presented at the following conferences: 17th Annual Meeting of the American Society of Gene and Cell Therapy, May 20–24, 2014, in Washington, DC and the European Society of Pediatric Nephrology Conference, September 18–20, 2014, in Porto, Portugal. This work was selected for an oral presentation at Kidney Week 2015 of the American Society of Nephrology, November 3–8, 2015, in San Diego, California.

Footnotes

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