Hypoxia reoxygenation-increased netrin-1 secretion in TKPTS cells is mediated by ERK MAPK pathway.

<p>Confluent TKPTS cells were subjected to hypoxia followed by 2 hr of reoxygenation and supernatant was then harvested. Netrin-1 was quantified by ELISA. Hypoxia and reoxygenation significantly increased netrin-1 production which was suppressed in the presence of U0126. *, <i>p</i><0.001 vs. control. #, <i>p</i><0.001 vs. cisplatin. n = 6.</p

Pervanadate did not increase netrin-1 mRNA stability.

<p>mRNA degradation was quantified by real time RT-PCR at various times after addition of actinomycin D, actinomycin+pervanadate, pervanadate or cycloheximide. Addition of actinomycin D induced a rapid degradation of netrin-1 mRNA, which was further enhanced in the presence of pervanadate. Similarly, inhibition of translation also increased rapid netrin-1 mRNA degradation. #, <i>p</i><0.05 vs. all other groups.</p

MAPK pathway mediates pervanadate-induced increase in netrin-1 production in TKPTS cells.

<p>A. Pervanadate increases the activation of MAPKs (ERK, p38 and JNK) as seen by increased phosphorylation with increasing dose of pervanadate. B. Pervanadate-induced increase in netrin-1 protein is suppressed in the presence of U0126 and to some extent SB203580 but not with SP2600125 and antioxidant. *, <i>p</i><0.001 vs. control. +, <i>p</i><0.001 vs. pervanadate alone treated group. n = 6. C. Effect of pervanadate and MAPK pathway inhibitors on netrin-1 transcript levels. Pervandate significantly decreased netrin-1 mRNA levels. In the presence of U0126 netrin-1 mRNA levels significantly increased. *, <i>p</i><0.001 vs. pervanadate. #, <i>p</i><0.05 vs. control. +, <i>p</i><0.05 vs. pervanadate+U0126. n = 4.</p

Netrin-1 mRNA and protein levels in kidney in response to ischemia reperfusion of the kidney.

<p>A. Netrin-1 mRNA levels in the kidney at 6 hr after reperfusion in ischemic and sham-operated animals. n = 4, *<i>p</i><0.001 vs. sham-operated. B. Quantification of netrin-1 in urine at 6 hrs after reperfusion. Netrin-1 levels were normalized to per mg of urine creatinine. n = 4. *, <i>p</i><0.001 vs. sham-operated animals.</p

Quantification of serum semaphorin 3A in different forms AKI and diabetes in mouse.

<p>Semaphorin 3A was quantified using ELISA kit as described in Methods. A. Circulating levels of semaphorin 3A before and different time after cisplatin administration. Cisplatin administration was significantly upregulated semaphorin 3A in the blood. *<i>p</i><0.005 vs. 0 hr. B. Circulating levels of semaphorin 3A in sham operated (0 hr) and different time after reperfusion. Ischemia reperfusion rapidly downregulated circulating semaphorin 3A. *<i>p</i><0.005 vs. 0 hr. Values are mean ± SEM. n = 4–6.</p

Regulation of semaphorin 3A expression and excretion after ischemia reperfusion injury of the kidney.

<p>A. Western blot analysis of semaphorin 3A excretion in mice urine before (0 hr) and at different time point after reperfusion. A large increase was detected at 6 hr and 24 hr after reperfusion. B. Serum creatinine levels before and different time after ischemia reperfusion of the kidney. *<i>p</i><0.001 vs. 0 hr. C. Semaphorin 3A expression in kidney tissue was analyzed by Western Blot. A single 95 KDa band was observed and increased expression seen at 24 hr. D. RT-PCR analysis of semaphorin 3A expression in the kidney after ischemia reperfusion. Semaphorin 3A mRNA expression is downregulated at 6 and 24 hr after reperfusion of the kidney. *<i>p</i><0.001 vs. sham operated. E. Graphic representation of known protease (Furin like) cleavage site and expected band of semaphorin 3A. F. Proteolytic cleavage of semaphorin 3A <i>in vitro</i>. 250 ng of recombinant semaphorin 3A-Fc chimera was incubated with 10 µl of human urine for 1 hr at 37°C (lane 1), in the presence of 20 mM EDTA (lane 2), human urine alone (lane 3) and recombinant semaphorin 3A alone. Proteolytic release of smaller fragments of semaphorin 3A was inhibited with addition of EDTA. Values are mean ± SEM. n = 3–5.</p

ROC curve analysis for urinary semaphorin at 2 hours after cardiac surgery.

<p>The values 109.8, 492.1, and 910.0 are urinary semaphorin concentrations (in picograms per milligram urine creatinine) at 2 hours after CPB, which correspond to 96% sensitivity, optimal sensitivity and specificity, and 97% specificity, respectively.</p

Quantification of urine semaphorin 3A in different forms AKI and diabetes in mouse.

<p>Semaphorin 3A was quantified using ELISA kit as described in Methods. A. Semaphorin 3A in urine from animals subjected to sham surgery (0 hr) and different time after reperfusion. Ischemia reperfusion rapidly increased urinary excretion of semaphorin 3A. *<i>p</i><0.005 vs. 0 hr. B. Serum creatinine at different time after reperfusion. *<i>p</i><0.001 vs. 0 hr. C. Semaphorin 3A excretion in urine before and different time after cisplatin administration. Cisplatin administration significantly increased the excretion of semaphorin 3A at 24, 48 and 72 hr. *<i>p</i><0.005 vs. 0 hr. D. Serum creatinine at different time after administration of cisplatin. *<i>p</i><0.05. Values are mean ± SEM. n = 6–8.</p

Spearman correlation coefficients of semaphorin 3A with clinical characteristics.

a<p><i>P</i>≤0.05.</p>b<p><i>P</i>≤0.002.</p

Descriptive statistics of patient characteristics.

<p>Means ± standard deviation (SD) are reported for continuous measures, percentages are reported for categorical variables.</p>a<p>Welch modified two-sample <i>t</i> test.</p>b<p>Fisher exact test.</p