Ganesan Ramesh

Netrin-1 regulates the inflammatory response of neutrophils and macrophages, and suppresses ischemic acute kidney injury by inhibiting COX-2-mediated PGE2 production

Netrin-1 regulates inflammation but the mechanism by which this occurs is unknown. Here we explore the role of netrin-1 in regulating the production of the prostanoid metabolite PGE2 from neutrophils in in vitro and in vivo disease models. Ischemia reperfusion in wild-type and RAG-1 knockout mice induced severe kidney injury that was associated with a large increase in neutrophil infiltration and COX-2 expression in the infiltrating leukocytes. Administration of netrin-1 suppressed COX-2 expression, PGE2 and thromboxane production, and neutrophil infiltration into the kidney. This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function. Treatment with the PGE2 receptor EP4 agonist enhanced neutrophil infiltration and renal injury which was not inhibited by netrin-1. Consistent with in vivo data, both LPS and IFNγ-induced inflammatory cytokine production in macrophages and IL-17-induced IFNγ production in neutrophils were suppressed by netrin-1 in vitro by suppression of COX-2 expression. Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation. Thus, netrin-1 regulates the inflammatory response of neutrophils and macrophages through suppression of COX-2 mediated PGE2 production. This could be a potential drug for treating many inflammatory immune disorders.

NADPH Oxidase 4 Is Expressed in Pulmonary Artery Adventitia and Contributes to Hypertensive Vascular Remodeling

Objective— Pulmonary hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PAs) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PAs and determine the functional relevance of Nox4 in PH. Approach and Results— Elevated expression of Nox4 was detected in hypertensive PAs from 3 rat PH models and human PH using qualititative real-time reverse transcription polymerase chain reaction, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia, and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of reactive oxygen species production. Small-molecule inhibitors of Nox4 reduced adventitial reactive oxygen species generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and noninvasive indices of PA stiffness in monocrotaline-treated rats as determined by morphometric analysis and high-resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PAs. In fibroblasts, Nox4 overexpression stimulated migration and proliferation and was necessary for matrix gene expression. Conclusion— These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling, and development of PH.

Netrin-1-treated macrophages protect the kidney against ischemia-reperfusion injury and suppress inflammation by inducing M2 polarization

Improper macrophage activation is pathogenically linked to various metabolic, inflammatory, and immune disorders. Therefore, regulatory proteins controlling macrophage activation have emerged as important new therapeutic targets. We recently demonstrated that netrin-1 regulates inflammation and infiltration of monocytes and ameliorates ischemia-reperfusion-induced kidney injury. However, it was not known whether netrin-1 regulates the phenotype of macrophages and the signaling mechanism through which it might do this. In this study, we report novel mechanisms underlying netrin-1s effects on macrophages using in vivo and in vitro studies. Overexpression of netrin-1 in spleen and kidney of transgenic mice increased expression of arginase-1, IL-4, and IL-13 and decreased expression of COX-2, indicating a phenotypic switch in macrophage polarization toward an M2-like phenotype. Moreover, flow cytometry analysis showed a significant increase in mannose receptor-positive macrophages in spleen compared with wild type. In vitro, netrin-1 induced the expression of M2 marker expression in bone marrow-derived macrophages, peritoneal macrophages, and RAW264.7 cells, and suppressed IFNγ-induced M1 polarization and production of inflammatory mediators. Adoptive transfer of netrin-1-treated macrophages suppressed inflammation and kidney injury against ischemia-reperfusion. Netrin-1 activated PPAR pathways and inhibition of PPAR activation abolished netrin-1-induced M2 polarization and suppression of cytokine production. Consistent with in vitro studies, administration of PPAR antagonist to mice abolished the netrin-1 protective effects against ischemia-reperfusion injury of the kidney. These findings illustrate that netrin-1 regulates macrophage polarization through PPAR pathways and confers anti-inflammatory actions in inflammed kidney tissue.

MicroRNA-150 protects the mouse heart from ischaemic injury by regulating cell death

AIMSnCardiac injury is accompanied by dynamic changes in the expression of microRNAs (miRs). For example, miR-150 is down-regulated in patients with acute myocardial infarction, atrial fibrillation, dilated and ischaemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than traditional clinical markers such as brain natriuretic peptide. We recently showed using the β-arrestin-biased β-blocker, carvedilol that β-arrestin1-biased β1-adrenergic receptor cardioprotective signalling stimulates the processing of miR-150 in the heart. However, the potential role of miR-150 in ischaemic injury and HF is unknown.nnnMETHODS AND RESULTSnHere, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodelling after MI. The cardioprotective roles of miR-150 during ischaemic injury were in part attributed to direct repression of the pro-apoptotic genes egr2 (zinc-binding transcription factor induced by ischaemia) and p2x7r (pro-inflammatory ATP receptor) in cardiomyocytes.nnnCONCLUSIONnThese findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury.

Bax and Bak have critical roles in ischemic acute kidney injury in global and proximal tubule–specific knockout mouse models

Bax and Bak, two pro-apoptotic Bcl-2 family proteins, have been implicated in acute kidney injury following renal ischemia/reperfusion; however, definitive evidence for a role of these genes in the disease process is lacking. Here we first examined two Bax-deficient mouse models and found that only conditional Bax-deletion specifically from proximal tubules could ameliorate ischemic acute kidney injury. Global (whole mouse) knockout of Bax enhanced neutrophil infiltration without significant effect on kidney injury. In contrast, global knockout of Bak protected mice from ischemic acute kidney injury with improved renal function. Interestingly, in these models, Bax or Bak knockout attenuated renal tubular cell apoptosis without significantly affecting necrotic tubular damage. Cytochrome c release in ischemic acute kidney injury was also suppressed in conditional Bax or global Bak-knockout mice. In addition, Bak deficiency prevented mitochondrial fragmentation in ischemic acute kidney injury. Thus, our gene-knockout studies support a critical role of Bax and Bak in tubular cell apoptosis in ischemic acute kidney. Furthermore, necrosis and apoptosis have distinguishable regulatory functions.

Plasma netrin-1 is a diagnostic biomarker of human cancers

Context:u2002The axon guidance cues netrin-1 is a secreted protein overexpressed in many different cancer tissues. Objectives:u2002To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer. Materials and Methods:u2002A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry. Results:u2002Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples. Discussion and Conclusion:u2002Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.

Kidney proximal tubular epithelial-specific overexpression of netrin-1 suppresses inflammation and albuminuria through suppression of COX-2-mediated PGE2 production in streptozotocin-induced diabetic mice.

Inflammation plays a key role in the development and progression of diabetic kidney disease; however, the role of the anti-inflammatory molecule netrin-1 in diabetic kidney disease is unknown. We examined the role of netrin-1 in diabetes-induced kidney inflammation and injury using tubule-specific netrin-1 transgenic mice. Diabetes was induced using streptozotocin in wild-type and netrin-1 transgenic animals. Kidney function, fibrosis, glucose excretion, albuminuria, and inflammation were evaluated. The mechanism of netrin-1-induced suppression of inflammation was studied in vitro using a proximal tubular epithelial cell line. Diabetes was associated with increased infiltration of neutrophils and macrophages, chemokine expression, and tubular epithelial cell apoptosis in kidney. These changes were minimal in kidney of netrin-1 transgenic mice. In addition, diabetes induced a large increase in the excretion of prostaglandin E2 (PGE2) in urine, which was suppressed in netrin-1 transgenic mice. Netrin-1-induced suppression of PGE2 production was mediated through suppression of NFκB-mediated cyclooxygenase-2 (COX-2) in renal tubular epithelial cells. Furthermore, netrin-1 also increased albumin uptake by proximal tubular epithelial cells through the PI3K and ERK pathways without increasing glucose uptake. These findings suggest that netrin-1 is a major regulator of inflammation and apoptosis in diabetic nephropathy and may be a useful therapeutic molecule for treating chronic kidney diseases such as diabetic nephropathy.

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.

CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation

Organ cross talk exists in many diseases of the human and animal models of human diseases. A recent study demonstrated that inflammatory mediators can cause acute kidney injury and neutrophil infiltration in a mouse model of dextran sodium sulfate (DSS)-colitis. However, the chemokines and their receptors that may mediate distant organ effects in colitis are unknown. We hypothesized that keratinocyte chemoattractant (KC)/IL-8 receptor chemokine (C-X-C motif) ligand 2 (CXCL2) mediates DSS-colitis-induced acute kidney injury. Consistent with our hypothesis, wild-type (WT) mice developed severe colitis with DSS treatment, which was associated with inflammatory cytokine and chemokine expression and neutrophil infiltration in the colon. DSS-colitis in WT was accompanied by acute kidney injury and enhanced expression of inflammatory cytokines in the kidney. However, CXCR2 knockout mice were protected against DSS-colitis as well as acute kidney injury. Moreover, the expression of cytokines and chemokines and neutrophil infiltration was blunted in CXCR2 knockout mice in the colon and kidney. Administration of recombinant KC exacerbated DSS-colitis-induced acute kidney injury. Our results suggest that KC/IL-8 and its receptor CXCR2 are critical and major mediators of organ cross talk in DSS colitis and neutralization of CXCR2 will help to reduce the incidence of acute kidney injury due to ulcerative colitis and Crohns disease in humans.

Semaphorin 3A inactivation suppresses ischemia-reperfusion-induced inflammation and acute kidney injury.

Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia-reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia-reperfusion-induced AKI. In vitro, sema3A enhanced toll-like receptor 4-mediated inflammation in epithelial cells, macrophages, and dendritic cells. Moreover, administration of sema3A-treated, bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates AKI via promoting inflammation and epithelial cell apoptosis.