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

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.

Keywords: preterm; stem cell; urine.

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.

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.

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.

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.

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).

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.