Intracellular Kinases Mediate Increased Translation and Secretion of Netrin-1 from Renal Tubular Epithelial Cells

Background: Netrin-1 is a laminin-related secreted protein, is highly induced after tissue injury, and may serve as a marker of injury. However, the regulation of netrin-1 production is not unknown. Current study was carried out in mouse and mouse kidney cell line (TKPTS) to determine the signaling pathways that regulate netrin-1 production in response to injury. Methods and Principal Findings: Ischemia reperfusion injury of the kidney was induced in mice by clamping renal pedicle for 30 minutes. Cellular stress was induced in mouse proximal tubular epithelial cell line by treating with pervanadate, cisplatin, lipopolysaccharide, glucose or hypoxia followed by reoxygenation. Netrin-1 expression was quantified by real time RT-PCR and protein production was quantified using an ELISA kit. Cellular stress induced a large increase in netrin-1 production without increase in transcription of netrin-1 gene. Mitogen activated protein kinase, ERK mediates the drug induced netrin-1 mRNA translation increase without altering mRNA stability. Conclusion: Our results suggest that netrin-1 expression is suppressed at the translational level and MAPK activation leads to rapid translation of netrin-1 mRNA in the kidney tubular epithelial cells

Guidance Cue Netrin-1 and the Regulation of Inflammation in Acute and Chronic Kidney Disease

Acute kidney injury (AKI) is a common problem in the hospital setting and intensive care unit. Despite improved understanding, there are no effective therapies available to treat AKI. A large body of evidence strongly suggests that ischemia reperfusion injury is an inflammatory disease mediated by both adaptive and innate immune systems. Cell migration also plays an important role in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5B receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Recent identification of netrin-1 as regulators of immune cell migration has led to a large number of studies looking into how netrin-1 controls inflammation and inflammatory cell migration. This review will focus on recent advances in understanding netrin-1 mediated regulation of inflammation during acute and chronic kidney disease and whether netrin-1 and its receptor activation can be used to treat acute and chronic kidney disease

Semaphorin 3A is a new early diagnostic biomarker of experimental and pediatric acute kidney injury.

BACKGROUND: Semaphorin 3A is a secreted protein that regulates cell motility and attachment in axon guidance, vascular growth, immune cell regulation and tumor progression. However, nothing is known about its role in kidney pathophysiology. Here, we determined whether semaphorin3A is induced after acute kidney injury (AKI) and whether urinary semaphorin 3A can predict AKI in humans undergoing cardiopulmonary bypass (CPB). METHODS AND PRINCIPAL FINDINGS: In animals, semaphorin 3A is localized in distal tubules of the kidney and excretion increased within 3 hr after reperfusion of the kidney whereas serum creatinine was significantly raised at 24 hr. In humans, using serum creatinine, AKI was detected on average only 48 hours after CPB. In contrast, urine semaphorin increased at 2 hours after CPB, peaked at 6 hours (2596±591 pg/mg creatinine), and was no longer significantly elevated 12 hours after CPB. The predictive power of semaphorin 3A as demonstrated by area under the receiver-operating characteristic curve for diagnosis of AKI at 2, 6, and 12 hours after CPB was 0.88, 0.81, and 0.74, respectively. The 2-hour urine semaphorin measurement strongly correlated with duration and severity of AKI, as well as length of hospital stay. Adjusting for CPB time and gender, the 2-hour semaphorin remained an independent predictor of AKI, with an odds ratio of 2.19. CONCLUSION: Our results suggest that semaphorin 3A is an early, predictive biomarker in experimental and pediatric AKI, and may allow for the reliable early diagnosis and prognosis of AKI after CPB, much before the rise in serum creatinine

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

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

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

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