Renal Primordia Activate Kidney Regenerative Events in a Rat Model of Progressive Renal Disease

<div><p>New intervention tools for severely damaged kidneys are in great demand to provide patients with a valid alternative to whole organ replacement. For repairing or replacing injured tissues, emerging approaches focus on using stem and progenitor cells. Embryonic kidneys represent an interesting option because, when transplanted to sites such as the renal capsule of healthy animals, they originate new renal structures. Here, we studied whether metanephroi possess developmental capacity when transplanted under the kidney capsule of MWF male rats, a model of spontaneous nephropathy. We found that six weeks post-transplantation, renal primordia developed glomeruli and tubuli able to filter blood and to produce urine in cyst-like structures. Newly developed metanephroi were able to initiate a regenerative-like process in host renal tissues adjacent to the graft in MWF male rats as indicated by an increase in cell proliferation and vascular density, accompanied by mRNA and protein upregulation of VEGF, FGF2, HGF, IGF-1 and Pax-2. The expression of SMP30 and NCAM was induced in tubular cells. Oxidative stress and apoptosis markedly decreased. Our study shows that embryonic kidneys generate functional nephrons when transplanted into animals with severe renal disease and at the same time activate events at least partly mimicking those observed in kidney tissues during renal regeneration.</p></div

Vascularization and growth of renal structures in the transplanted metanephroi and intragraft albumin uptake in male MWF rat transplanted with metanephroi.

<p><b>(A)</b> Immunofluorescence detection of WT-1 and rat endothelial cell marker (RECA-1) in metanephroi (MET) developed in male MWF rats. On the left: glomerulus stained for WT1 (red). Scale bar = 10 μm. Center: capillaries of a glomerulus (g) labeled for RECA-1 (red). Scale bar = 20 μm. Right: RECA-1 positive vessels (v, red) and tubuli (t). Scale bar = 20 μm. WGA lectin (green) was used to display renal structures and DAPI (blue) to visualize nuclei. <b>(B)</b> Metanephroi developed in female MWF rats. On the left: glomerulus stained for WT1 (red), FITC-labeled WGA lectin (green) and DAPI (blue). Center: section labeled for rat endothelial cell marker RECA-1 (red), FITC-labeled WGA lectin (green) and DAPI (blue). Right: proximal tubule stained for megalin (green), rhodamine-labeled LCA lectin (red) and DAPI (blue). Scale bars = 10 μm. <b>(C)</b> Localization of FITC-BSA (green) intravenously injected in MET-transplanted male recipient and graft. Host tissues (left), glomerulus (center) and megalin-positive tubule (white, right) of the graft. The tissue was also visualized by labeling with rhodamine-labeled LCA lectin (red) and DAPI (blue). Scale bars = 20 μm (left and center) and 10 μm (right).</p

Glomerulosclerosis, proteinuria and creatinine in male MWF rats pre- and post-metanephros transplantation or saline treatment.

<p>Degree of glomerulosclerosis, proteinuria and creatinine in male MWF rats pre- (25 wks) and post- (31 wks) metanephros (MET) or saline treatment. Creatinine concentration in cysts of MWF animals receiving MET. Urine creatinine is also shown.</p><p>*P < 0.05 vs 25 wks,</p><p>^#P < 0.05 vs urine creatinine of rats receiving MET at 31 wks.</p><p>Glomerulosclerosis, proteinuria and creatinine in male MWF rats pre- and post-metanephros transplantation or saline treatment.</p

Histological appearance of adult male and female MWF rat kidneys and E15 MWF rat metanephros.

<p><b>(A)</b> Representative images of kidney tissues in untreated male MWF rats at 25 (left) and 31 (right) weeks of age. Arrowheads indicate tubular casts. Sections were stained with PAS. Scale bars = 100 μm. <b>(B)</b> Overall appearance of E15 rat metanephros (MET) stained with haematoxylin and eosin. Scale bar = 100 μm. <b>(C)</b> Kidney explanted from male MWF rat, six weeks after MET transplantation, showing metanephroi grown into large structures with fluid-containing cysts. Scale bar = 2.5 mm. Enlargements highlight cyst and vascularization. <b>(D)</b> Macroscopic view of kidney from female MWF rat grafted with MET, six weeks after transplantation. Scale bar = 2.5 mm. <b>(E)</b> Renal histology (haematoxylin and eosin staining) of male MWF rat grafted with MET and sacrificed after 6 weeks from transplant. Scale bars = 100 μm. High magnification of a representative glomerulus developed in the graft is shown in the inset. Scale bar = 50 μm. <b>(F)</b> Histology of renal tissue from female MWF rat transplanted with MET and sacrificed after 6 weeks from transplant. Scale bar = 100 μm. The inset reports a representative glomerulus developed in the graft. Scale bar = 50 μm.</p

Expression of mRNAs and proteins relevant to kidney regeneration in male MWF rats.

<p><b>(A)</b> Real Time RT-PCR analysis of VEGF, FGF2, HGF, IGF-1 and Pax-2 in whole host renal tissues of male MWF rats receiving saline or metanephroi (MET) (in adjacent area), 6 weeks after transplantation. Data are expressed as mean ± SE; *P < 0.05, **P < 0.01 <i>vs</i> saline (n = 4 rats/group, VEGF n = 6 rats/group). (<b>B)</b> Immunohistochemical staining of VEGF (arrows) in male animals receiving saline or MET transplant, after 6 weeks. Scale bars = 50 μm. Representative immunofluorescence stainings (red) of FGF2, HGF, IGF-1 and Pax-2 in rats receiving saline or MET, on renal tissues labeled with WGA-lectin (green) and DAPI (blue). Scale bars = 50 μm, = 25 μm (for FGF2 and IGF-1). Semiquantitative analysis of immunohistochemical staining of VEGF, FGF2, HGF, IGF-1 and Pax-2 in male MWF rats receiving saline or MET. Data are expressed as mean ± SE; *P < 0.05, **P < 0.01 <i>vs</i> saline (n = 3/group).</p

Metanephros effect on oxidative damage, apoptosis and proliferation of recipient renal tissues.

<p>(<b>A)</b> Oxidative damage was quantified in renal tissues of male animals receiving saline or methanephroi (MET), after 6 weeks, as percentage of tubuli positive for nitrotyrosine (NT), in fields distant from or adjacent to the grafts. Data are expressed as mean ± SE. *P < 0.05 <i>vs</i> areas distant from the graft and <i>vs</i> saline (n = 3 rats/group). Pictures show renal tissues of male MWF rats receiving saline or MET stained for nitrotyrosine (red), FITC-labeled lectin WGA (green) and DAPI (blue). Scale bars = 50 μm. (<b>B)</b> Quantification of oxidative damage in female animals receiving saline or MET, in fields distant from or adjacent to the grafts. Data are expressed as mean ± SE (n = 3 rats/group). (<b>C</b>) Saline or MET-treated male animals were evaluated for the presence of apoptotic cells by counting the number of cells positive for TUNEL in fields adjacent to or distant from the graft. Data are expressed as mean ± SE. *<i>P</i> < 0.05 <i>vs</i> areas distant from the graft and <i>vs</i> saline (n = 3 rats/group). Immunofluorescence images show host renal tissues stained with TUNEL (green), rodhamine-LCA lectin (red) and DAPI (blue), obtained from saline or MET-treated animals. Arrows indicate apoptotic nuclei. Scale bars = 50 μm. (<b>D)</b> Quantification of apoptotic cells in female animals receiving saline or MET, in fields distant from or adjacent to the grafts. Data are expressed as mean ± SE (n = 3 rats/group). (<b>E)</b> Quantification of Ki-67 positive cells in renal tissues of male animals treated with saline or MET after 6 weeks, in fields distant or adjacent to the grafts. Data are expressed as mean ± SE. *P < 0.05 <i>vs</i> areas distant from the graft and <i>vs</i> saline (n = 5 rats/group). Pictures show representative fields of host renal tissues of male MWF rats receiving saline or MET showing Ki-67 positive nuclei (green). Tissues are also stained with rodhamine-LCA lectin (red) and DAPI (blue). Scale bars = 50 μm. (<b>F)</b> Quantification of proliferating cells in female animals receiving saline or MET, in fields distant from or adjacent to the grafts. Data are expressed as mean ± SE (n = 3 rats/group).</p

Effect of fibroblasts transplanted under the kidney capsule of male MWF rats.

<p>(<b>A</b>) Histological appearance of renal tissue from male MWF rat transplanted with fibroblasts in adjacent (left, arrow) or distant (right) area. Scale bars = 200 μm. (<b>B</b>) Quantification of endothelium volume density (Vv), peritubular capillary length density (Jv), nitrotyrosine (NT)-positive tubuli, TUNEL-positive cells and Ki-67-positive cells, in renal tissues of MWF rats receiving fibroblasts or saline. Data are expressed as mean ± SE (n = 3 rats/group). (<b>C</b>) Representative immunohistochemical images of SMP30 (green), NCAM (red), VEGF (brown signal), FGF2 (red), HGF (red), IGF-1 (red) and Pax-2 (red) in male rats with fibroblast transplant. Sections are co-stained with rhodamine-LCA (in SMP30 panel, red) or with FITC-WGA lectin (in panels showing NCAM, FGF2, HGF, IGF-1, Pax2, green) and DAPI (blue). Scale bars = 50 μm.</p

Metanephros effect on endothelium volume density and peritubular capillary length density.

<p><b>(A)</b> Representative images of renal tissue of male MWF rats receiving saline or methanephroi (MET), 6 weeks after transplantation, stained for RECA-1 (red), FITC-labeled WGA lectin (green) and DAPI (blue); Scale bars = 20 μm. <b>(B-C)</b> Endothelial volume density (Vv, on the left), and length density of peritubular capillaries (Jv, on the right), evaluated in renal tissues of male <b>(B)</b> and female <b>(C)</b> animals receiving saline or MET distant from or adjacent to the graft. The results are expressed as mean ± SE. *P < 0.01 <i>vs</i> respective adjacent tissues and <i>vs</i> saline (n = 4 rats/group).</p

LpX plasma clearance and glomerular upake <i>in vivo.</i>

<p>LCAT deficiency markedly decreases LpX plasma clearance. WT and <i>Lcat</i>^-/- mice were injected with lissamine rhodamine B PE-tagged LpX and plasma samples were taken at the indicated times. (A) Plasma-associated fluorescence. Each data point represents the total fluorescence of pooled mouse plasma samples (mean ± S.D.; n = 3). (B) Agarose gel electrophoresis of pooled mouse plasma lipoprotein PE fluorescence (same samples as in (A)). LpX cleared from WT plasma by 240 min, whereas <i>Lcat</i>^-/- LpX remained elevated at all times. HDL-associated fluorescence was increased in WT plasma. W<i>hite line</i> indicates origin. (C) Fluorescent LpX retention in renal glomeruli is markedly increased in <i>Lcat</i>^-/- mice. Representative confocal maximum projection images of 10 μm fixed frozen kidney sections 4 hrs after injection of fluorescent-PE tagged LpX in mice chronically treated with 3 mg/wk synthetic LpX. Note the markedly increased retention of LpX in <i>Lcat</i>^-/- mice glomeruli. (D) Electron microscopic analysis of LpX in renal glomerular capillaries. Representative TEM of renal glomerular capillaries in WT (<i>left panels</i>) and <i>Lcat</i>^-/- mice (<i>right panels</i>). Endogenous multilamellar structures with features of LpX particles were occasionally present in the capillaries of (-) LpX <i>Lcat</i>^-/-, but not (-) LpX WT mice. Synthetic LpX particles resembling endogenous LpX were frequently observed in renal capillaries of both (+) LpX WT and (+) LpX <i>Lcat</i>^-/- mice. Both endogenous and exogenous synthetic LpX were often seen to be engulfed by endothelial cell processes (<i>insets</i>). Exogenous LpX in the capillary lumen bound to red blood cells in LpX-treated WT and <i>Lcat</i>^-/- mice. GBM: Glomerular Basement Membrane; PFP: Podocyte Foot Process. Scale bars = 500nm. Inset scale bars = 250 nm (WT+LpX); 100 nm (<i>Lcat</i>^-/- ± LpX).</p

Electron microscopic analysis of LpX movement through renal glomerular compartments.

<p>Circulating LpX particles (small arrows in (A, B)) bind to endothelial cell lamellipodia in (A) WT and (B) <i>Lcat</i>^-/- mouse glomerular capillaries (arrowheads), are internalized (long arrows in (A), and degraded (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>). LpX bound to the cell surface (B1), is partially (B2), small arrows in inset) and then completely engulfed (B3). LpX penetrates the glomerular basement membrane (GBM) in WT (C) and <i>Lcat</i>^-/- mice ((D) and, inset in (D), arrowheads), markedly disrupting its structure (C, D; asterisks). The typical intramembranous lesion as found in the peripheral GBM of human FLD is seen in the inset in D, displaying a characteristic lamellar structure within a lucent lacuna in <i>Lcat</i>^-/- mice. In (D), several lamellipodia (arrows) engulf an LpX particle in the GBM. LpX penetrates the glomerular urinary space of both WT (E, G) and <i>Lcat</i>^-/- (F, H) mice. LpX binds to podocyte cell bodies (PCBs) and foot processes (PFPs) at multiple sites (E, F: small arrows; H: arrowheads), and was internalized into PCBs (F; large arrow). Large vacuoles (G, H; large arrows) containing partially degraded LpX particles (G, H; small arrows) as well as numerous small unilamellar vesicles are often observed, consistent with cell-mediated LpX degradation. (I) In WT mice, LpX did not accumulate in the mesangial matrix and occasional foamy mesangial cells were observed. (J) Mesangial cells near the sites of LpX deposition engulf LpX particles. (K) Marked retention of LpX in <i>Lcat</i>^-/- mouse mesangial matrix. The regions near large arrows 1 & 2 in (K) are shown enlarged in K1&2. LpX binds to the mesangial cell prior to engulfment. Scale bars: A, B1, F, H, J = 200 nm; B2, D (inset), K1, K2 = 250 nm; B–E, G, I, K = 500 nm. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s003" target="_blank">S3 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150083#pone.0150083.s004" target="_blank">S4 Fig</a>, for additional examples.</p