Increase in Angptl4 expression in human MCD patients and associations with desmin, synaptopodin and proteinuria.

<p><b>(A and B)</b> Immunofluorescence of glomerular Angptl4 and desmin in MCD and MsPGN patients with similar nephrotic-range proteinuria (magnification, 200X). <b>(C)</b> Immunofluorescence of glomerular Angptl4 and synaptopodin in MCD patients (magnification, 200X). <b>(D)</b> Immunofluorescence of glomerular Angptl4 and desmin in MCD patients (magnification, 200X). <b>(E and F)</b> Quantifications of the fluorescence staining intensities of glomerular Angptl4 and desmin in MCD and MsPGN patients. <b>(G)</b> Scatter diagram of glomerular Angptl4 and desmin in MCD patients. <b>(H)</b> Scatter diagram of Angptl4 and 24-hour urinary protein in MCD patients. <b>(I)</b> Urine Angptl4 ELISA for MCD and MsPGN patients with similar nephrotic-range proteinuria. <b>(J)</b> Western blot of urinary Angptl4 excretion in MCD, MN, FSGS and MsPGN patients (MCD 1,2,3, MN 1,2,3, FSGS 1 and MsPGN 4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137049#pone.0137049.t001" target="_blank">Table 1</a> are shown in the image) with similar nephrotic-range proteinuria. <b>(K)</b> Quantification of the western blot of urinary Angptl4 excretion in MCD, MN, FSGS and MsPGN patients. *P<0.01 compared with MsPGN patients.</p

Baseline characteristics of the enrolled patients.

<p>M, male; F, female.</p><p>Baseline characteristics of the enrolled patients.</p

The majority of glomerular Angptl4 was secreted by injured podocytes in ADR rats.

<p><b>(A)</b> Immunofluorescence of glomerular Angptl4 and desmin, an injured podocyte marker, in ADR rats on days 14, 21 and 28. <b>(B)</b> Immunofluorescence of glomerular Angptl4 and synaptopodin, a normal podocyte marker, in ADR rats on days 10 and 14. <b>(C)</b> Immunofluorescence of glomerular Angptl4 and laminin, a GBM marker, in ADR rats on day 14. <b>(D)</b> Immunofluorescence of glomerular Angptl4 and RECA-1, an endothelial cell marker, in ADR rats on days 14 and 28. <b>(E)</b> Immunofluorescence of glomerular Angptl4 and OX-7, a mesangial cell marker, in ADR rats on day 14. <b>(F)</b> Immunofluorescence of glomerular Angptl4 and RECA-1 in ADR rats with tacrolimus treatment on day 28. Scale bars: 50 μm. TAC, ADR rats with tacrolimus treatment.</p

Angiopoietin-Like-4, a Potential Target of Tacrolimus, Predicts Earlier Podocyte Injury in Minimal Change Disease

<div><p>Podocyte injury plays central roles in proteinuria and kidney dysfunction, therefore, identifying specific biomarker to evaluate earlier podocyte injury is highly desirable. Podocyte-secreted angiopoietin-like-4 (Angptl4) mediates proteinuria in different types of podocytopathy. In the present study, we established an experimental minimal change disease (MCD) rat model, induced by adriamycin (ADR) and resulted in definite podocyte injury, to identify the dynamic changes in Angptl4 expression. We also investigated the direct effects of tacrolimus on Angptl4 and podocyte repair. We determined that the glomerular Angptl4 expression was rapidly upregulated and reached a peak earlier than desmin, an injured podocyte marker, in the ADR rats. Furthermore, this upregulation occurred prior to heavy proteinuria and was accompanied by increased urinary Angptl4. We observed that the Angptl4 upregulation occurred only when podocyte was mainly damaged since we didn’t observe little Angptl4 upregulation in MsPGN patients. In addition, we observed the glomerular Angptl4 mainly located in injured podocytes rather than normal podocytes. Moreover, we found that tacrolimus treatment significantly promoted podocyte repair and reduced glomerular and urinary Angptl4 expression at an earlier stage with a significant serum Angptl4 upregulation. And similar results were confirmed in MCD patients. In conclusion, this study represents the first investigation to demonstrate that Angptl4 can predict podocyte injury at earlier stages in MCD and the identification of earlier podocyte injury biomarkers could facilitate the prompt diagnosis and treatment of patients with podocytopathy, as well as determination of the prognosis and treatment efficacy in these diseases.</p></div

Tacrolimus promoted podocyte repair in ADR rats.

<p><b>(A)</b> Immunofluorescence of glomerular desmin in normal, tacrolimus-treated and untreated ADR rats. Scale bars = 50 μm. <b>(B)</b> Quantification of the fluorescence staining intensities of glomerular desmin in normal, tacrolimus-treated and untreated ADR rats. <b>(C)</b> Immunofluorescence of glomerular synaptopodin in normal, tacrolimus-treated and untreated ADR rats. <b>(D)</b> Quantification of the fluorescence staining intensities of glomerular synaptopodin in normal, tacrolimus-treated and untreated ADR rats. <b>(E)</b> Transmission electron microscopy of normal, tacrolimus-treated and untreated ADR rats. Foot process effacements are indicated by the black arrows. Scale bars = 2 μm. <b>(F)</b> TUNEL assay of glomeruli from ADR rats. A TUNEL-positive cell is indicated by the black arrow. Scale bars = 50 μm. <b>(G)</b> Quantification of the TUNEL assay of the glomeruli from ADR rats. <b>(H)</b> Western blot of glomerular synaptopodin and desmin expression in ADR rats. <b>(I)</b> Quantification of the western blot of glomerular synaptopodin expression in ADR rats. <b>(J)</b> Quantification of the western blot of glomerular desmin expression in ADR rats. Con, normal rats; Untreated, ADR rats without treatment; TAC, ADR rats with tacrolimus treatment. ##P<0.05 compared with normal rats; #P<0.01, compared with normal rats; *P<0.01 compared with untreated ADR rats.</p

Angptl4 was expressed at an earlier stage of podocyte injury in ADR rats, and tacrolimus diminished glomerular and urinary Angptl4 expression.

<p><b>(A)</b> Immunofluorescence of glomerular Angptl4 in normal, tacrolimus-treated and untreated ADR rats. Scale bars: 50 μm. <b>(B)</b> Quantifications of the fluorescence staining intensities of glomerular Angptl4 in normal, tacrolimus-treated and untreated ADR rats. <b>(C)</b> Western blot of glomerular Angptl4 expression in ADR rats. <b>(D)</b> Western blot of urine Angptl4 excretion in ADR rats. <b>(E)</b> Quantification of the western blot of glomerular Angptl4 expression in ADR rats. <b>(F)</b> Quantification of the western blot of urine Angptl4 excretion. <b>(G)</b> Urine Angptl4 ELISA for ADR rats. <b>(H)</b> Serum Angptl4 ELISA for ADR rats. <b>(I)</b> Quantification of the real-time PCR of glomerular Angptl4 in ADR rats. <b>(J)</b> Relationship between glomerular Angptl4 and glomerular desmin in ADR rats. <b>(K)</b> Relationship between glomerular Angptl4 and 24-hour urinary protein excretion in ADR rats. <b>(L)</b> Relationship between glomerular Angptl4 and glomerular synaptopodin in ADR rats. Con, normal rats; Untreated, ADR rats without treatment; TAC, ADR rats with tacrolimus treatment. ##P<0.05 compared with normal rats; #P<0.01 compared with normal rats; *P<0.01 compared with untreated ADR rats. The arrow indicates that tacrolimus treatment was initiated on day 14.</p

Tacrolimus-treated rats exhibited ameliorated ADR-induced proteinuria and lipid metabolism disorders.

<p><b>(A)</b> Twenty-four hour urinary protein excretion in ADR rats. <b>(B)</b> Serum albumin levels in ADR rats. <b>(C)</b> Serum triglyceride levels in ADR rats. <b>(D)</b> Serum cholesterol levels in ADR rats. Con, normal rats; Untreated, ADR rats without treatment; TAC, ADR rats with tacrolimus treatment. ##P<0.05 compared with normal rats; #P<0.01 compared with normal rats; *P<0.01 compared with untreated ADR rats. The arrow indicates that tacrolimus treatment was initiated on day 14.</p

HO-1 overexpression reduced peripheral capillary loss and inhibited the activation and proliferation of renal interstitial myofibroblasts after UUO.

<p><b>(A)</b> Representative immunofluorescence images of CD31-labeled PTCs (red) from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(B)</b> Graph showing the morphometric quantification of CD31-positive staining. <b>(C)</b> Representative immunofluorescence images of kidney sections co-labeled with PDGFRβ (green) and α-SMA (red) in the Sham and UUO groups on days 7 and 10. PDGFRβ is a specific marker of pericytes, fibroblasts and myofibroblasts, and α-SMA labels activated fibroblasts and myofibroblasts (magnification, ×400; bars = 100 μm). <b>(D)</b> Graph displaying the morphometric quantification of the PDGFRβ-positive area per high-power field. <b>(E)</b> Histogram showing the quantification of the α-SMA -positive area per high-power field. <b>(F)</b> Graph showing the morphometric quantification ratios of the double-positive/PDGFR-β-positive areas. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 decreased tubulointerstitial inflammation in the UUO model.

<p><b>(A)</b> Representative immunofluorescence images of F4/80-labeled macrophages (green) from the Sham and UUO groups on days 3, 7, 10 and 14. DAPI labels the nucleus (blue). (Magnification, ×200; bars = 250 μm) <b>(B)</b> Histogram indicating the morphometric quantification of the number of F4/80-positive cells per high-power field. <b>(C-D)</b> Graph showing the mRNA expression of inflammation-related cytokines at day 7 after UUO in the TG and WT groups. <b>(E)</b> Representative ELISA for inflammatory markers (TNF-α, IL-1β, IL-6, IL-4 and IL-10) in kidney tissue homogenates of HO-1 TG mice on the 7^th day after UUO. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 overexpression regulated cell proliferation and apoptosis after renal fibrosis induced by UUO.

<p><b>(A)</b> Representative images of TUNEL staining (brown) of kidney sections from the Sham and UUO groups on days 7, 10, and 14. (magnification, ×200; bars = 250 μm) <b>(B)</b> Graph indicating the number of apoptotic cells in mice after UUO. <b>(C)</b> Representative Ki67 immunofluorescence staining of kidneys with UUO at days 7 (magnification, ×200; bars = 250 μm). Red arrows indicate Ki67+ epithelial cells, and white arrows indicate Ki67+ tubulointerstitial cells. <b>(D)</b> Graph showing the number of proliferative cells in renal tubule and interstitial compartments. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p