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

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

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

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

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

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

Baseline characteristics of the enrolled patients.

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