Figure 1.
Generation of EpoGFP/Δ3′E mouse.
A Diagram shows structures of the wild type (wt, upper), EpoGFP (middle) and EpoΔ3′E (lower), respectively. IRES, internal ribosomal entry site. B,C An EpoGFP/wt heterozygote and an EpoΔ3′E/Δ3′E homozygote were bred to obtain the compound EpoGFP/Δ3′E genotype. EpoGFP/Δ3′E mice exhibited transient anemia during the perinatal stage: B at P5 looked pale; C presented a decreased Hct value even lower than that of EpoΔ3′E/Δ3′E at P4–P6 stage. The EpoΔ3′E/wt littermates were used as a normal control. *p<0.05.
Figure 2.
GFP expression in the anemic kidney of EpoGFP/Δ3′E mouse.
Representative photos of P5 neonates: kidney slices A from a nonanemic EpoΔ3′E/wt littermate as a negative control; B–D from EpoGFP/Δ3′E newborns. Light and confocal microscopy revealing the GFP signal concentrated in the deep-cortex∼outer-medulla region (B), where the Epo-producing peritubular interstitial cells are localized (C,D). Anti-GFP signals: green (Alexa 488), and dark-brown (diaminobenzidine). Scale bars: 25 µm. The sketch at the bottom illustrates peritubular localization of the GFP-expressing cells in the kidney.
Figure 3.
Cellular marker characterization of the GFP+ kidney cells of EpoGFP/Δ3′E mouse.
Dual immunostaining combined with confocal microscopy indicating GFP-expressing cells are not endothelial (CD31, A), dendritic (MHCII, B), and tubular cells (E-cadherin, Movie S1). GFP-positive interstitial cells express fibroblast-like markers (PDGFRβ, C), but not myofibroblast marker (α-SMA, D). Kidney slices from P5 EpoGFP/Δ3′E mice were stained with GFP (green) and the indicated antibodies directly labeled with phycoerythrin (PE, red) or indirectly labeled with Alexa 594 (red) respectively. Merged images of the same kidney section are shown. DAPI: blue/nucleus; Scale bars: 25 µm.
Figure 4.
Co-localization of GFP and CD73 in the peritubular interstitial cells of the EpoGFP/Δ3′E kidneys.
A–C Representitive images of confocal microscopy of kidney sections from P5 EpoGFP/Δ3′E mice. B A high-power view, demonstrated that GFP and CD73 were mostly co-localized in peritubular fibroblast cells (merged in yellow). Global merged view of the kidney sections from P5 EpoGFP/Δ3′E neonates (C), and EpoΔ3′E/wt littermates (D). D Under normal conditions CD73 showed a wide expression spectrum in a variety of cellular types including a few interstitial fibroblasts in the kidney. C During anemia note that CD73+ cells appeared to be increased mainly in the peritubular fibroblast population. GFP: green; CD73: red; DAPI: blue/nucleus; merged: yellow. Scale bars: 20 µm.
Table 1.
Expression of cellular markers in REPs by multiple immunostaining methods.
Figure 5.
Isolation of REPs from the P4–6 EpoGFP/Δ3′E anemic kidneys.
A Cell purification protocol for a rare population of REPs from kidney. B, upper panel Representative FACS scatter plot of kidney cells from pooled P4–6 EpoGFP/Δ3′E neonates demonstrating GFP+ cells above the gate (set using EpoΔ3′E/wt control cells). B, lower panel Assessment by FACS of percentage of GFP+ and CD73+ fractions in the kidney cells from P4–6 anemic EpoGFP/Δ3′E neonates. Pooled results were from three independent experiments. Note CD73 expression divides GFP+ cells into two parts: CD73+ and CD73−. C Representative confocal microscopy of the FACS-sorted GFP+ cells (REPs) with anti-GFP immunostaining. Scale bar: 5 µm. D Analysis of relative Epo mRNA levels by qRT-PCR (Hprt as a loading control) in FACS-purified GFP+ or the remaining GFP− kidney cells from P4–6 EpoGFP/Δ3′E mice; CD73+ vs. CD73− fractionated cells were also evaluated. The data shown are from four experiments, each performed in duplicate. nd: not detectable; *p<0.05.
Figure 6.
Expression profile of cellular oxygen-sensing and hypoxia-inducible molecules in the isolated REPs.
qRT-PCR analysis of the genes related to hypoxia response using RNA extracted from the sorted GFP− and GFP+ renal cells from P4–6 EpoGFP/Δ3′E mice. Hprt as a loading control *p<0.05. Data are for three experiments performed in duplicate. A Four genes related to cellular oxygen-sensing; B three Hifα isoforms. Also note that HIF1α targets: Pgk1, Phd2 are not enriched in the GFP+ fraction (REPs). C Three transcript variants of Hif3α in the purified GFP+ fraction (REPs), using a method for exponential PCR amplification at a fixed threshold (see also Materials and Methods).
Table 2.
Oligo-nucleotide sequences of primers used in this study.