Overview of the study of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> Time-line of Chr I/R procedure and <b>(b)</b> the study schema was showed.</p

Renal pathology scoring at 20 weeks post-nephrectomy of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> The representative figures of renal cortical staining by Periodic Acid-Schiff (PAS), and Masson’s trichrome among sham-operated, placebo (PB), and cilostazol-treated (CZ) mice were demonstrated. <b>(b)</b> Glomerulosclerosis, the percentage of the glomerular area that was sclerotic determined from PAS-stained sections, (left-side panel) and relative interstitial volume, the percentage of total surface area of the sampled cortical area in Masson’s trichrome stained sections that is occupied by interstitial space (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187872#sec002" target="_blank">method</a>), (right-side panel) from Chr I/R in sham-operated (n = 6), placebo (PB) (n = 10), and cilostazol-treated mice (CZ) (n = 10) were demonstrated. *<i>p</i>< 0.05, #<i>p</i>< 0.0001. Scale bar = 200 μm.</p

Aortic pathology at 20 weeks post-nephrectomy of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> The representative figures of aorta by H&E staining of sham-operated, placebo (PB) and cilostazol-treated mice (CZ) at 20 weeks post-nephrectomy were demonstrated. The semi-quantitative analysis regarding: <b>(b)</b> the average area of the IH area, <b>(c)</b> the ratio of tunica intima area/ tunica media area, <b>(d)</b> the ratio of tunica intima area/ (tunica intima area + tunica media area) and <b>(e)</b> aortic diameter of sham-operated (n = 6), placebo (PB) (n = 10), and cilostazol-treated mice (CZ) (n = 10) were demonstrated. *<i>p</i>< 0.01, #<i>p</i>< 0.0001.</p

Serum concentration of several mediators at 20 weeks post-nephrectomy of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> asymmetrical dimethylarginine (ADMA), <b>(b)</b> high sensitivity C-reactive protein (hs-CRP), <b>(c)</b> platelet-derived growth factor (PDGF), <b>(d)</b> vascular endothelium growth factor (VEGF), <b>(e)</b> interleukin (IL)-6 and <b>(f)</b> tumor necrosis factor alpha (TNF-α) in sham-operated (n = 6), placebo (PB) (n = 10), and cilostazol-treated mice (CZ) (n = 10) at 20 weeks post-nephrectomy were demonstrated. *<i>p</i>< 0.05, **<i>p</i>< 0.01, #<i>p</i>< 0.001, ##<i>p</i><0.0001.</p

The correlation analysis between microRNAs (miRs) in aortic tissue and other serum parameters.

<p>The correlation between asymmetrical dimethylarginine <b>(</b>ADMA) and miR-143 <b>(a)</b>, miR-145, <b>(b)</b> miR-221 <b>(c)</b>; between vascular endothelium growth factor (VEGF) and these miRs <b>(d-f)</b> and between platelet-derived growth factor (PDGF) and these miRs <b>(g-i)</b> were demonstrated.</p

Effect of various concentrations of cilostazol on microRNAs (miRs) expression of human umbilical vein endothelial cells (HUVECs).

<p>HUVECs were incubated with 3, 10, 30 uM of cilostazol for 24 hours. The number of expression is presented as the percentage of expression determined by ΔΔCT and expression fold change relative to control. Data are represented as mean±standard error. *<i>p</i>< 0.05, **<i>p</i>< 0.01, and #<i>p</i>< 0.001 indicate significance relative to control. Sample size (n) = 3 for each group from 3 independent replicates. Ctrl, control (DMSO in EBM-2-treated) group; CZ, cilostazol-treated group.</p

An analysis of microRNAs in aortic tissue at 20 weeks post-nephrectomy of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> MicroRNA in aortic tissue from sham-operated (n = 6), placebo (PB) (n = 10), and cilostazol-treated mice (CZ) (n = 10) at 20 weeks post-nephrectomy were showed. RNU48 was used for the normalization (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187872#sec002" target="_blank">method</a>). <b>(b)</b> A graphical heat-map presentation was demonstrated. Higher and lower ΔCt values were colored in red and blue, respectively; *<i>p</i>< 0.05, **<i>p</i>< 0.01.</p

Clinical and laboratory manifestations of the ischemic-reperfusion injury with unilateral nephrectomy (Chr I/R) mouse model.

<p><b>(a)</b> Body weight (BW), <b>(b)</b> systolic blood pressure (SBP), <b>(c)</b> serum creatinine (Cr), and <b>(d)</b> hematocrit (Hct) alteration of sham-operated (n = 6), placebo (PB) (n = 10), and cilostazol-treated mice (CZ) (n = 10/group) during the period of study were demonstrated in longitudinal assessment. <b>(e)</b> Quantitative proteinuria determined with urine protein creatinine ratio (UPCR) of Chr I/R mice at 1 week (randomization time) and at 20 weeks post-nephrectomy was showed. *<i>p</i>< 0.05 <i>vs</i> baseline in the same group; # <i>p</i>< 0.05, ## <i>p</i>< 0.01 <i>vs</i> baseline in sham-operated mice at wk-1; $ <i>p</i>< 0.05.</p