Steroid-associated hip joint collapse in bipedal emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients

A total ion chromatogram in full scan mode generated by HPLC/UV/MS/MS.

<p>(A)∼(B) Compared with the blank sera, a peak shown in 38.1 min in the sera from L-EF, M-EF and H-EF group. (C) HPLC profile of standard Icaritin. (D) 391 (<i>m/z</i> [M+Na]⁺) for Icaritin selected for the subsequent selected ion chromatography (SIC), with a peak at 38.1 min. (E) The +MS showed the mass weight by 391 ion (<i>m/z</i> [M+Na]⁺) and the absence of 56 exhibited the existence of prenyl in the +MS² chromatography. (F) <i>Epimedium</i>-derived flavonoids with common stem nuclei intestinally metabolized to Icaritin.</p

Supernate soluble TM in endotoxin induction group (A) and optical density in destained oil red O staining in steroid induction group (B) was significantly higher than the corresponding control group, respectively, whereas Icaritin dose-dependently lowered supernate soluble TM (A) and optical density in destained oil red O staining (B) when compared to the corresponding induction group, respectively.

<p>However, no differences were found between the seven parent flavonoids in EF at the concentration of 10⁻¹⁴ M and corresponding induction group both in supernate soluble TM (C) and optical density in destained oil red O staining (D). Note: * P<0.05 for comparison with the induction group. # P<0.05 for comparison with the low dose group.</p

Hematology/cytology/MRI data analysis.

<p>There was no significant change from baseline in ALT (A) and AST (B) when compared to the CON group. Significantly increased TM (C) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction, and adipocyte positive colonies (D) in the CON group were attenuated in the L-EF group or prevented in both the M-EF and H-EF group after induction. In addition, the significantly decreased PEP (E) from baseline in the CON group was attenuated in the L-EF group or prevented in both the M-EF and H-EF group at week 1 post induction. Note: * P<0.05 for comparison with CON; # P<0.05 for comparison with baseline. • CON group; ⧫ L-EF group; ▪ M-EF group; ▴ H-EF group.</p

Seven major flavonoid compounds are identified in <i>Epimedium</i>-derived Flavonoids (EF).

<p>The common structure is 8-prenylkaempferol. R1 and R2 are substituted by glucose, rhamnose, or xylose, and R3 is replaced by methyl.</p

Key characteristics for histopathological identification and histopathological data analysis.

<p>ON lesion was found with trabecular bone containing considerable empty lacunae and lack of marrow cells (B) when compared to normal bone (A). In ON⁺ rabbits, thrombi were predominantly found in small marrow vessels with lack of angiographic particles (C), and marrow was predominantly occupied by a numerous fat cells (B, C). (D) Incidence of ON in each group: CON (13/14, 93%), L-EF (9/16, 56%), M-EF (2/16, 13%), H-EF (1/16, 6%). (E) There was no significant difference in ON Extent among all the groups. (F) Thrombotic Vessel Counts, and (G) Fat Cell Area Fraction presented similarities in changing patter over time, i.e. either attenuated in the L-EF group or prevented in both the M-EF and H-EF group when compared to that in the CON group. Note: Arrow pointed particle was angiographic substance during microCT-based angiography (data not shown). • CON group; ⧫ L-EF group; ▪ M-EF group; ▴ H-EF group; * P<0.05</p

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

Steroid-associated hip joint collapse in bipedal emus

In this study we established a bipedal animal model of steroid-associated hip joint collapse in emus for testing potential treatment protocols to be developed for prevention of steroid-associated joint collapse in preclinical settings. Five adult male emus were treated with a steroid-associated osteonecrosis (SAON) induction protocol using combination of pulsed lipopolysaccharide (LPS) and methylprednisolone (MPS). Additional three emus were used as normal control. Post-induction, emu gait was observed, magnetic resonance imaging (MRI) was performed, and blood was collected for routine examination, including testing blood coagulation and lipid metabolism. Emus were sacrificed at week 24 post-induction, bilateral femora were collected for micro-computed tomography (micro-CT) and histological analysis. Asymmetric limping gait and abnormal MRI signals were found in steroid-treated emus. SAON was found in all emus with a joint collapse incidence of 70%. The percentage of neutrophils (Neut %) and parameters on lipid metabolism significantly increased after induction. Micro-CT revealed structure deterioration of subchondral trabecular bone. Histomorphometry showed larger fat cell fraction and size, thinning of subchondral plate and cartilage layer, smaller osteoblast perimeter percentage and less blood vessels distributed at collapsed region in SAON group as compared with the normal controls. Scanning electron microscope (SEM) showed poor mineral matrix and more osteo-lacunae outline in the collapsed region in SAON group. The combination of pulsed LPS and MPS developed in the current study was safe and effective to induce SAON and deterioration of subchondral bone in bipedal emus with subsequent femoral head collapse, a typical clinical feature observed in patients under pulsed steroid treatment. In conclusion, bipedal emus could be used as an effective preclinical experimental model to evaluate potential treatment protocols to be developed for prevention of ON-induced hip joint collapse in patients

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

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