Sema 3A as a biomarker of the activated mTOR pathway during hexavalent chromium-induced acute kidney injury.

Semaphorin 3A (sema 3A) is one of a class of secretory proteins belonging to a family of axon-directed factors found in podocytes, distal tubules, and collecting tubes of the kidney. It is considered to be a potential target molecule involved in the mammalian target of the rapamycin (mTOR) pathway in renal injury or renal diseases, but it has an unknown role in the course of hexavalent chromium-Cr(VI) induced nephrotoxicity. In the present study, an acute kidney injury (AKI) model in rats or cultured tubular epithelial HK-2 cells was employed for Cr(VI) exposure alone or in combination with rapamycin (Rap) or N-acetyl-l-cysteine (NAC) or recombinant sema 3A. The methods of histopathology, biochemics, and western blotting were applied to evaluate tubular injury and the role of sema 3A. The results showed that a significant increase of urinary sema 3A indicates an early occurrence of AKI exposed to Cr(VI), accompanied with a significant increase of tubular injury score and phosphorylated mTOR proteins. Further, Cr(VI) treatment, in combination with pretreatment of the mTOR pathway inhibitor, Rap, showed a considerably stronger protective effect of Rap in protecting against Cr(VI)-induced nephrotoxicity than that seen with the free radical scavenger NAC, highlighting the dominant renal protective role of the mTOR pathway in inhibiting toxicity by downregulating the expressed levels of sema 3A in renal tissue. This study has demonstrated that an increased expression of sema 3A occurs in Cr(VI)-induced AKI resulting from activation of the mTOR pathway, and that inhibition of this pathway has been shown to decrease the severity of the toxicity. In conclusion, this study has shown that increased urinary sema 3A is indicative of an activated mTOR pathway and is a valuable biomarker of the early AKI induced by Cr(VI) exposure.

Urinary semaphorin 3A as an early biomarker to predict contrast-induced acute kidney injury in patients undergoing percutaneous coronary intervention.

Contrast-induced acute kidney injury (CI-AKI) is a serious complication of diagnostic coronary angiograph and percutaneous coronary intervention (PCI). However, the exact pathophysiological mechanisms underlying CI-AKI development are largely unknown. The present study examined whether urinary semaphorin 3A levels predict the development of CI-AKI in patients undergoing PCI. This study enrolled 168 patients with stable angina undergoing elective PCI. Serial urine samples, obtained at baseline and 2, 6, 12, 24, 36, and 48 h post-PCI were analyzed by semaphorin 3A and neutrophil gelatinase-associated lipocalin (NGAL) ELISA kit. AKI was defined as an increase in serum creatinine beyond 50% according to the RIFLE classification system. Receiver operator characteristic (ROC) curve analyses identified optimal semaphorin 3A and NGAL values for diagnosing CI-AKI. CI-AKI occurred in 20 of 168 patients. There were no significant differences in the baseline clinical characteristics and angiographic findings between non-AKI patients group and AKI patients group. Both urinary semaphorin 3A and NGAL levels significantly increased at 2 and 6 h post-PCI. ROC analysis showed that the cut-off value of 389.5 pg/mg semaphorin 3A at 2 h post-PCI corresponds to 94% sensitivity and 75% specificity and the cut-off value of 94.4 ng/mg NGAL at 2 h post-PCI corresponds to 74% sensitivity and 82% specificity. Logistic regression showed that semaphorin 3A levels at 2 and 6 h post-PCI were the significant predictors of AKI in our cohort. Urinary semaphorin 3A may be a promising early biomarker for predicting CI-AKI in patients undergoing PCI.

Overexpression of semaphorin 3A in patients with urothelial cancer.

OBJECTIVE: A highly sensitive and specific urine marker for the detection of recurrent urothelial cancer and for screening healthy population or people at risk for urothelial cancer has not been found yet. As urine cytology is not sensitive enough, patients with non-muscle-invasive bladder cancer need lifelong follow-up involving multiple invasive cystoscopies. Our aims of study were to examine the expression of semaphorin 3A in urothelial cancer patients and to evaluate semaphorin 3A as a potential marker for urothelial cancer. MATERIALS AND METHODS: Urine samples were taken from patients with known bladder tumor, hospitalized for transurethral resection of lesions, from patients with history of urothelial cancer admitted for endoscopic follow up, from patients with other nonmalignant urological conditions such as prostatic hyperplasia, stress incontinence, urethral stricture, ureteral and kidney stones, and from healthy volunteers with no history of urothelial malignancy and no urological symptoms. Semaphorin 3A (sema3A) protein level was measured using enzyme-linked immunosorbent assay in every sample and levels were correlated with endoscopic and pathological findings. In addition, we performed immunohistochemically staining with semaphorin 3A of 15 tissue samples (various tumors and normal bladder tissues). RESULTS: A total of 183 urine samples were tested. Out of them, 116 patients (mean age 70.7; 94 males and 22 females) had positive cystoscopy, and 67 (mean age 64.7; 51 males and 16 females) had negative cystoscopy. Higher sema3A values were significantly correlated (P = 0.006) with presence of urothelial cancer, as determined by positive cystoscopy or urethroscopy and pathological biopsy. Sema3A levels also showed positive correlation with the number of tumors. Sema3A levels combined with urine cytology showed much higher sensitivity compared with cytology alone (66% vs. 33%), with smaller reduction of specificity (77% vs. 90%). Immunohistochemical staining showed intense staining in high stage and grade tumors, and almost no staining in normal tissue. CONCLUSIONS: Semaphorin 3A is overexpressed in urothelial cancer patients, as evidenced both in its presence in urine and in bladder tissue. Semaphorin 3A in urine is a promising potential urothelial cancer biomarker either independently or in conjunction with cytology. Further tests are needed to elucidate the sex difference in the expression of Sema3A in the urine of bladder cancer patients.

Semaphorin-3A and Netrin-1 predict the development of kidney injury in children with congenital hydronephrosis.

Congenital obstructive nephropathy is amongst the main causes of chronic renal failure in children. Early diagnosis and initiation of the treatment will delay progressive renal tubular atrophy and interstitial fibrosis with the loss of nephrons. The aim of this study was to evaluate whether urinary (u) semaphorin-3A (SEMA-3A) and Netrin-1 may be potential biomarkers in children with congenital hydronephrosis due to ureteropelvic junction obstruction (UPJO). The study consisted of 42 children with severe hydronephrosis who needed surgery and two control groups (Control One: 42 children with mild, non-obstructive hydronephrosis; Control Two: 44 healthy children). All children had normal renal function. Urinary semaphorin-3A and Netrin-1 levels were measured in different groups using immunoenzymatic ELISA commercial kits. Compared with Control One and Control Two groups, the preoperative median uSEMA-3A/creatinine (cr.) and uNetrin-1/cr. levels increased significantly in the children with severe hydronephrosis (p < .01). One month after surgery, uSEMA-3A/cr. and uNetrin-1/cr. levels had decreased significantly in the children with severe hydronephrosis (p < .01), but were still higher than those in both control groups (p < .05). Receiver operator characteristic (ROC) analyses revealed a good diagnostic profile for uSEMA-3A and uNetrin-1 in terms of identifying children with a differential renal function of <40% [area under the curve (AUC) 0.825 and 0.745, respectively]. Our results indicate that increased concentrations of uSEMA-3A and uNetrin-1 are found in urine from children with severe hydronephrosis and that their concentrations are related to the degree of obstruction.

Urinary semaphorin 3A as an early biomarker to predict contrast-induced acute kidney injury in patients undergoing percutaneous coronary intervention

Contrast-induced acute kidney injury (CI-AKI) is a serious complication of diagnostic coronary angiograph and percutaneous coronary intervention (PCI). However, the exact pathophysiological mechanisms underlying CI-AKI development are largely unknown. The present study examined whether urinary semaphorin 3A levels predict the development of CI-AKI in patients undergoing PCI. This study enrolled 168 patients with stable angina undergoing elective PCI. Serial urine samples, obtained at baseline and 2, 6, 12, 24, 36, and 48 h post-PCI were analyzed by semaphorin 3A and neutrophil gelatinase-associated lipocalin (NGAL) ELISA kit. AKI was defined as an increase in serum creatinine beyond 50% according to the RIFLE classification system. Receiver operator characteristic (ROC) curve analyses identified optimal semaphorin 3A and NGAL values for diagnosing CI-AKI. CI-AKI occurred in 20 of 168 patients. There were no significant differences in the baseline clinical characteristics and angiographic findings between non-AKI patients group and AKI patients group. Both urinary semaphorin 3A and NGAL levels significantly increased at 2 and 6 h post-PCI. ROC analysis showed that the cut-off value of 389.5 pg/mg semaphorin 3A at 2 h post-PCI corresponds to 94% sensitivity and 75% specificity and the cut-off value of 94.4 ng/mg NGAL at 2 h post-PCI corresponds to 74% sensitivity and 82% specificity. Logistic regression showed that semaphorin 3A levels at 2 and 6 h post-PCI were the significant predictors of AKI in our cohort. Urinary semaphorin 3A may be a promising early biomarker for predicting CI-AKI in patients undergoing PCI.

Semaphorin3a promotes advanced diabetic nephropathy.

The onset of diabetic nephropathy (DN) is highlighted by glomerular filtration barrier abnormalities. Identifying pathogenic factors and targetable pathways driving DN is crucial to developing novel therapies and improving the disease outcome. Semaphorin3a (sema3a) is a guidance protein secreted by podocytes. Excess sema3a disrupts the glomerular filtration barrier. Here, using immunohistochemistry, we show increased podocyte SEMA3A in renal biopsies from patients with advanced DN. Using inducible, podocyte-specific Sema3a gain-of-function (Sema3a(+)) mice made diabetic with streptozotocin, we demonstrate that sema3a is pathogenic in DN. Diabetic Sema3a(+) mice develop massive proteinuria, renal insufficiency, and extensive nodular glomerulosclerosis, mimicking advanced DN in humans. In diabetic mice, Sema3a(+) exacerbates laminin and collagen IV accumulation in Kimmelstiel-Wilson-like glomerular nodules and causes diffuse podocyte foot process effacement and F-actin collapse via nephrin, αvß3 integrin, and MICAL1 interactions with plexinA1. MICAL1 knockdown and sema3a inhibition render podocytes not susceptible to sema3a-induced shape changes, indicating that MICAL1 mediates sema3a-induced podocyte F-actin collapse. Moreover, sema3a binding inhibition or podocyte-specific plexinA1 deletion markedly ameliorates albuminuria and abrogates renal insufficiency and the diabetic nodular glomerulosclerosis phenotype of diabetic Sema3a(+) mice. Collectively, these findings indicate that excess sema3a promotes severe diabetic nephropathy and identifies novel potential therapeutic targets for DN.

Increased urine semaphorin-3A is associated with renal damage in hypertensive patients with chronic kidney disease: a nested case-control study.

BACKGROUND: Semaphorins are guidance proteins implicated in several processes such as angiogenesis, organogenesis, cell migration, and cytokine release. Experimental studies showed that semaphorin-3a (SEMA3A) administration induces transient massive proteinuria, podocyte foot process effacement and endothelial cell damage in healthy animals. While SEMA3A signaling has been demonstrated to be mechanistically involved in experimental diabetic glomerulopathy and in acute kidney injury, to date its role in human chronic kidney disease (CKD) has not been investigated. METHODS: To test the hypothesis that SEMA3A may play a role in human CKD, we performed a cross-sectional, nested, case-control study on 151 matched hypertensive patients with and without CKD. SEMA3A was quantified in the urine (USEMA) by ELISA. Glomerular filtration rate was estimated (eGFR) by the CKD-EPI formula and albuminuria was measured as albumin-to-creatinine ratio (ACR). RESULTS: USEMA levels were positively correlated with urine ACR (p = 0.001) and serum creatinine (p < 0.001). USEMA was higher in patients with both components of renal damage as compared to those with only one and those with normal renal function (p < 0.007 and <0.001, respectively). The presence of increased USEMA levels (i.e. top quartile) entailed a fourfold higher risk of combined renal damage (p < 0.001) and an almost twofold higher risk of macroalbuminuria (p = 0.005) or of reduced eGFR, even adjusting for confounding factors (p = 0.002). CONCLUSIONS: USEMA is independently associated with CKD in both diabetic and non diabetic hypertensive patients. Further studies may help clarify the mechanisms underlying this association and possibly the pathogenic changes leading to the development of CKD.

Netrin-1 and semaphorin 3A predict the development of acute kidney injury in liver transplant patients.

Acute kidney injury (AKI) is a serious complication after liver transplantation. Currently there are no validated biomarkers available for early diagnosis of AKI. The current study was carried out to determine the usefulness of the recently identified biomarkers netrin-1 and semaphorin 3A in predicting AKI in liver transplant patients. A total of 63 patients' samples were collected and analyzed. AKI was detected at 48 hours after liver transplantation using serum creatinine as a marker. In contrast, urine netrin-1 (897.8 ± 112.4 pg/mg creatinine), semaphorin 3A (847.9 ± 93.3 pg/mg creatinine) and NGAL (2172.2 ± 378.1 ng/mg creatinine) levels were increased significantly and peaked at 2 hours after liver transplantation but were no longer significantly elevated at 6 hours after transplantation. The predictive power of netrin-1, as demonstrated by the area under the receiver-operating characteristic curve for diagnosis of AKI at 2, 6, and 24 hours after liver transplantation was 0.66, 0.57 and 0.59, respectively. The area under the curve for diagnosis of AKI was 0.63 and 0.65 for semaphorin 3A and NGAL at 2 hr respectively. Combined analysis of two or more biomarkers for simultaneous occurrence in urine did not improve the AUC for the prediction of AKI whereas the AUC was improved significantly (0.732) only when at least 1 of the 3 biomarkers in urine was positive for predicting AKI. Adjusting for BMI, all three biomarkers at 2 hours remained independent predictors of AKI with an odds ratio of 1.003 (95% confidence interval: 1.000 to 1.006; P = 0.0364). These studies demonstrate that semaphorin 3A and netrin-1 can be useful early diagnostic biomarkers of AKI after liver transplantation.

Urinary semaphorin 3A correlates with diabetic proteinuria and mediates diabetic nephropathy and associated inflammation in mice.

Semaphorin 3A (sema3A) was recently identified as an early diagnostic biomarker of acute kidney injury. However, its role as a biomarker and/or mediator of chronic kidney disease (CKD) related to diabetic nephropathy is unknown. We examined the expression of sema3A in diabetic animal models and in humans and tested whether sema3A plays a pathogenic role in the development of diabetic nephropathy. The expression of sema3A was localized to podocytes and epithelial cells in distal tubules and collecting ducts in control animals, and its expression was increased following the induction of diabetes. Quantification of sema3A urinary excretion in three different diabetic mouse models showed that excretion was increased as early as 2 weeks after the induction of diabetes and increased over time, in conjunction with the development of nephropathy. Consistent with the mouse data, increased sema3A urinary excretion was detected in diabetic patients with albuminuria, particularly in those with macroalbuminuria. Genetic ablation of sema3A or pharmacological inhibition with a novel sema3A inhibitory peptide was protected against diabetes-induced albuminuria, kidney fibrosis, inflammation, oxidative stress, and renal dysfunction. We conclude that sema3A is both a biomarker and a mediator of diabetic kidney disease and could be a promising therapeutic target in diabetic nephropathy. Key messages Diabetes induced sema3A excretion in urine. Increased semaphorin 3A was associated with severity of albuminuria. Seme3A-mediated diabetes induced glomerulosclerosis. Peptide-based inhibition of semaphorin3A suppressed diabetic nephropathy.

Repulsive guidance cue semaphorin 3A in urine predicts the progression of acute kidney injury in adult patients from a mixed intensive care unit.

BACKGROUNDS: Predicting the development of acute kidney injury (AKI) in the critical care setting is challenging. Although several biomarkers showed somewhat satisfactory performance for detecting established AKI even in a heterogeneous disease-oriented population, identification of new biomarkers that predict the development of AKI accurately is urgently required. METHODS: A single-center prospective observational cohort study was undertaken to evaluate for the first time the reliability of the newly identified biomarker semaphorin 3A for AKI diagnosis in heterogeneous intensive care unit populations. In addition to five urinary biomarkers of L-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), IL-18, albumin and N-acetyl-ß-d-glucosaminidase (NAG), urinary semaphorin 3A was measured at intensive care unit (ICU) admission. RESULTS AND CONCLUSION: Three hundred thirty-nine critically ill adult patients were recruited for this study. Among them, 131 patients (39%) were diagnosed with AKI by the RIFLE criteria and 66 patients were diagnosed as AKI at post-ICU admission (later-onset AKI). Eighty-four AKI patients showed worsening severity during 1 week observation (AKI progression). Although L-FABP, NGAL and IL-18 showed significantly higher area under the curve (AUC)-receiver operating characteristic (ROC) values than semaphorin 3A in detecting established AKI, semaphorin 3A was able to detect later-onset AKI and AKI progression with similar AUC-ROC values compared with the other five biomarkers [AUC-ROC (95% CI) for established AKI 0.64 (0.56-0.71), later-onset AKI 0.71 (0.64-0.78), AKI progression 0.71 (0.64-0.77)]. Urinary semaphorin 3A was not increased in non-progressive established AKI, while the other biomarkers were elevated regardless of further progression. Finally, sepsis did not have any impact on semaphorin 3A while the other urinary biomarkers were increased with sepsis. Semaphorin 3A is a new biomarker of AKI which may have a distinct predictive use for AKI progression when compared with other AKI biomarkers.

Tubular injury marker netrin-1 is elevated early in experimental diabetes.

BACKGROUND: Netrin-1 was recently identified as an early diagnostic biomarker of acute kidney injury. However, its usefulness for early diagnosis of chronic kidney disease (CKD) is unknown. The current study evaluated whether these proteins are increased in urine from experimental animals with diabetes. METHODS: The current study evaluated whether netrin-1 is increased in urine from diabetic rats and mice, and whether netrin-1 correlated with development of nephropathy. RESULTS: In rats, urinary netrin-1 excretion was significantly (p<0.001) higher in the diabetic group at 4 and 10 weeks after induction of diabetes as compared with the control group. Similarly, netrin-1 was increased significantly (p<0.001) in urine from hypertensive rats at 4 weeks as compared with controls. Likewise, urinary albumin excretion rates were increased in diabetic rats at 4 and 10 weeks as compared with controls and were increased in hypertensive rats at 4 weeks. Consistent with the diabetic model in rats, netrin-1 excretion was also increased early in diabetic mice's urine, and peak levels correlated with disease severity. CONCLUSION: Netrin-1 can be detected in urine from diabetic and hypertensive rats and may serve as a useful early diagnostic biomarker for development of CKD.

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.