Renal Expression of FGF23 in Progressive Renal Disease of Diabetes and the Effect of Ace Inhibitor

<div><p>Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone mainly produced by bone that acts in the kidney through FGF receptors and Klotho. Here we investigated whether the kidney was an additional source of FGF23 during renal disease using a model of type 2 diabetic nephropathy. Renal expression of FGF23 and Klotho was assessed in Zucker diabetic fatty (ZDF) and control lean rats at 2, 4, 6, 8 months of age. To evaluate whether the renoprotective effect of angiotensin converting enzyme (ACE) inhibitor in this model was associated with changes in FGF23 and Klotho, ZDF rats received ramipril from 4, when proteinuric, to 8 months of age. FGF23 mRNA was not detectable in the kidney of lean rats, nor of ZDF rats at 2 months of age. FGF23 became measurable in the kidney of diabetic rats at 4 months and significantly increased thereafter. FGF23 protein localized in proximal and distal tubules. Renal Klotho mRNA and protein decreased during time in ZDF rats. As renal disease progressed, serum phosphate levels increased in parallel with decline of fractional phosphorus excretion. Ramipril limited proteinuria and renal injury, attenuated renal FGF23 upregulation and ameliorated Klotho expression. Ramipril normalized serum phosphate levels and tended to increase fractional phosphorus excretion. These data indicate that during progressive renal disease the kidney is a site of FGF23 production which is limited by ACE inhibition. Interfering pharmacologically with the delicate balance of FGF23 and phosphorus in diabetes may have implications in clinics.</p></div

Renal expression of FGF23 and Klotho during the course of the disease of ZDF rats, and modulation by ACE inhibitor therapy.

<p>(<b>a</b>) FGF23 mRNA expression evaluated in kidney tissue from lean and ZDF rats at different ages, and effect of ACE inhibitor treatment (n = 5 rats each group/time). FGF23 mRNA was not detectable in the kidneys of lean rats at all times period nor in ZDF rats at 2 months of age; it was measurable in the kidney of 4-month old ZDF rats. Relative expression values were calculated by the ΔΔCt method using the cDNA expression of ZDF at 4 months as reference. Data are mean ± SEM. *<i>p</i><0.05, **<i>p</i><0.01 vs ZDF rats at 4 months; ^#<i>p</i><0.05, ^##<i>p</i><0.01 vs ZDF+vehicle rats at corresponding age. (<b>b</b>) Representative images of FGF23 staining in the kidney of lean rats and ZDF rats receiving vehicle or ramipril at 8 months of age. FGF23 expression was detected by immunoperoxidase technique using two different anti-FGF23 antibodies (from Santa Cruz Biotechnology, SC, or Abcam, see Methods). Magnification, ×400. (<b>c</b>) Time course of Klotho mRNA expression in the kidney of lean rats and ZDF rats given vehicle or ramipril (n = 5 rats each group/time). Relative expression values in each group were calculated vs lean rats of corresponding age. Data are mean ± SEM. *<i>p</i><0.05 vs lean rats. (<b>d</b>) Representative images of Klotho staining by immunohistochemistry in the kidney of lean rats and ZDF rats treated with vehicle or ramipril, at 8 months of age. Magnification, ×400.</p

Time course of serum FGF23 levels in lean and ZDF rats.

<p>Serum FGF23 was measured in lean rats, and in ZDF rats receiving vehicle or ramipril from 4 to 8 months of age (n = 5 rats each group/time). Values are mean ± SEM. *<i>p</i><0.05 vs lean rats at 8 months.</p

Renal expression of sodium phosphate (NaPi)-2a co-transporter in ZDF rats, and effect of ramipril.

<p>(<b>a</b>) mRNA expression of NaPi-2a co-transporter evaluated in the kidney of lean rats, and ZDF rats treated with vehicle or ramipril from 4 to 8 months of age (n = 5 rats each group/time). Relative expression values were calculated versus lean rats of corresponding age. Data are mean ± SEM. *<i>p</i><0.05 vs age-matched lean rats; ^#<i>p</i><0.01 vs ZDF+vehicle. (<b>b</b>) Western blot analysis for NaPi-2a protein expression, relative to α-tubulin, in lean rats and ZDF rats treated with vehicle or ramipril, at 8 months of age (n = 5 rats each group). Data are mean ± SEM. **<i>p</i><0.01 vs lean rats. (<b>c</b>) Representative images of NaPi-2a staining by immunohistochemistry in the kidney of lean rats, and ZDF rats receiving vehicle or ramipril, at 8 months of age. Magnification, ×400.</p

Systemic and laboratory parameters in lean and ZDF rats at 8 months of age.

<p>Values are mean ± SEM or median (IQR). *<i>p</i><0.05, **<i>p</i><0.01 vs lean; ^#<i>p</i><0.05, ^##<i>p</i><0.01 vs ZDF+vehicle.</p

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

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

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

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