Vascular Endothelial Growth Factor Augments Arginine Transport and Nitric Oxide Generation via a KDR Receptor Signaling Pathway.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is an endothelium-specific peptide that stimulates angiogenesis via two receptor tyrosine kinases, Flt-1 and KDR. Endothelial nitric oxide synthase (eNOS) plays a major role in VEGF signaling. Delivery of arginine to membrane bound eNOS by the cationic amino acid transporter-1 (CAT-1) has been shown to modulate eNOS activity. The current studies were designed to test the hypothesis that VEGF enhances eNOS activity via modulation of arginine transport by CAT-1. METHODS: Using radio-labeled arginine, {[3H] L-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with VEGF with and without silencing the VEGF receptors Flt-1 or KDR. Subsequently, western blotting for CAT-1, PKCα, ERK 1/2, JNK, and their phosphorylated forms were performed. NO generation was measured by the Griess reaction. RESULTS: VEGF (50 and 100 ng/ml) significantly augmented endothelial arginine transport in a time dependent manner, an effect which was prevented by Sunitinib (2 µM), a multi targeted receptor tyrosine kinase inhibitor. The increase in arginine transport velocities by VEGF was not affected by silencing Flt-1 while silencing KDR abrogated VEGF effect. Furthermore, incubating cells with 50 and 100 ng of VEGF for 30 minutes significantly augmented CAT-1 abundance. The expression of PKC-α, JNK, and ERK1/2 and their phosphorylated forms were unchanged following incubation of HUVEC with VEGF. The concentration of NO2/NO3 following incubation with VEGF was significantly higher than from untreated cells. This increase was significantly attenuated by silencing KDR. CONCLUSIONS: VEGF increases arginine transport via modulation of CAT-1 in endothelial cells. This effect is exclusively dependent on KDR rather than Flt-1.

"General deterioration": a diagnosis that is a marker for risk of mortality upon re-admission.

BACKGROUND: Predicting mortality is important in treatment planning and professional duty towards patients and their families. OBJECTIVES: To evaluate the predictive value regarding patients' survival once the diagnosis of "general deterioration" replaces an ICD-9 diagnosis upon re-admission. METHODS: In this retrospective cohort case-control study, we screened the records of patients re-admitted at least three times during the past 2 years. For each patient's death during the third hospitalization, we matched (for age and gender) a patient who survived the third hospitalization. We evaluated 14 parameters potentially accountable for increased risk of mortality, e.g., length of stay at each admission, interval to re-admission, etc. We applied a multifactorial analysis using logistic regression to predict the risk of mortality during the third hospitalization as potentially affected by the aforementioned parameters. RESULTS: The study included 81 study patients and 81 controls. Of the 14 parameters potentially explaining an increased risk of mortality during the third hospitalization, several were found to be statistically significant. The most significant was the diagnostic switch from a specific ICD-9 diagnosis on first admission to the non-specific diagnosis of "general deterioration" at the second hospitalization. In such cases, the risk of death during the third hospitalization was increased by 5300% (odds ratio = 54, P = 0.008). The increased risk of mortality was not restricted to patients with malignancy as their background diagnosis. CONCLUSIONS: At re-admission a switch from disease-specific diagnosis to the obscure diagnosis "general deterioration" increases the subsequent risk of mortality.

Renal Tubular CD24 Upregulation Aggravates Folic Acid Induced Acute Kidney Injury: A Possible Role for T Regulatory Cells Inhibition in Mice.

Acute kidney injury (AKI) is characterized by cell death and inflammation. CD24 is a protein induced during tissue damage and is not expressed in mature renal tissue. We explored the role of CD24 in the pathogenesis of folic acid-induced AKI (FA-AKI) in mice. A single Intraperitoneal (IP) injection of folic acid induced AKI in WT and CD24-/- mice. Renal function tests, histological analysis, immunohistochemistry, Western blot analysis, and ELISA were performed to assess the severity of renal damage and the intensity of the inflammatory response. FA-AKI induced CD24 in the distal tubular epithelial cells. Compared to WT mice, FA-AKI CD24-/- mice exhibited an attenuated reduction in renal function and histological injury, lower serum IL-10 and interferon γ, and decreased expression of renal TNFα. In contrast, renal and systemic IL-33 upregulation were augmented. CD24-/- FA-AKI animals exhibited increased splenic margination and renal infiltration of regulatory T cells (Tregs). At day 7, FA-AKI CD24-/- mice exhibited increased expression of tubular pro-apoptotic and decreased anti-apoptotic proteins compared to WT animals. Anti-CD24 antibody administration to FA-AKI mice attenuated the decrease in renal function as well as the histological injury. Renal biopsies from patients with ATN stained strongly for CD24 in the distal tubules. In conclusion, during AKI, upregulation of CD24 promotes renal inflammation through inhibition of Treg infiltration and diversion of cell death towards necrosis rather than apoptosis. Neutralization of CD24 may prove a target for future therapies in AKI.

Profound Decrease in Glomerular Arginine Transport by CAT (Cationic Amino Acid Transporter)-1 Contributes to the FLT-1 (FMS-Like Tyrosine Kinase 1) Induced Preeclampsia in the Pregnant Mice.

Endothelial dysfunction because of nitric oxide inactivation has been suggested to play a role in the pathogenesis of preeclampsia. During pregnancy, L-arginine transport by CAT-1 (cationic amino acid transporter 1), the only transporter for eNOS (endothelial nitric oxide synthase) is inhibited. We hypothesize that maternal arginine deficiency contributes to the development of preeclampsia. Adenovirus-mediated overexpression of sFlt-1 (soluble fms-like tyrosine kinase 1) in virgin and pregnant mice resulted in glomerular endotheliosis, hypertension, and albuminuria. L-arginine prevented the increase in blood pressure and albuminuria in Flt-1 pregnant but not in Flt-1 virgin mice. Flt-1 augmented arginine transport in pregnant but not in virgin dames. Ex vivo inhibition of CAT-2 leaving exclusively CAT-1 activity, decreased arginine transport velocities in Flt-1 animals more prominently in pregnant dames. Phosphorylated CAT-1/CAT-1 increased in pregnant, sFlt-1-pregnant, and sFlt-1 virgin mice. CAT-2 increased in Flt-1-pregnant and Flt-1-virgin dames. L-arginine augmented arginine transport in pregnant and Flt-pregnant mice and prevented the increase in pCAT-1 and CAT-2 expression. Glomerular cGMP (cyclic guanosine monophosphate) generation as a measure of eNOS activity was decreased in all Flt-1 treated animals. L-arginine abolished the decrease in cGMP levels only in Flt-1-pregnant mice. In conclusion, glomerular endothelial NO generation is compromised in Flt-1-pregnant mice because of CAT-1 inhibition induced by a combined effect of pregnancy and preeclampsia which involves: phosphorylation of CAT-1 and induction of CAT-2. These processes contribute to the clinical syndrome of preeclampsia in mice and are prevented by L-arginine.

Garlic attenuates nitric oxide production in rat cardiac myocytes through inhibition of inducible nitric oxide synthase and the arginine transporter CAT-2 (cationic amino acid transporter-2).

It is now accepted that allicin, the main biologically active compound in garlic, exhibits antioxidant activity. The present study was designed to test the hypothesis that the antioxidant activity of garlic can be partially attributed to the inhibition of nitric oxide (NO) production by cytokine-induced NO synthase (iNOS). Cardiac myocytes cultured from neonatal Wistar rats were stimulated by lipopolysaccharide (LPS) and incubated for 24 h with various concentrations of allicin. This resulted in marked inhibition of nitrite production. Interestingly, a low concentration of allicin (10 microM) was significantly more potent in abrogating the effect of LPS on nitrite production than a higher concentration (40 microM). Allicin decreased steady-state iNOS mRNA levels, and this effect was maximal when a lower concentration was used (10 microM compared with 40 microM). In order to explore additional effects of allicin on NO generation that might counteract the effect on iNOS, we assessed the effects of higher allicin concentrations on arginine transport. Allicin inhibited the uptake of 1 mM extracellular arginine in a concentration-dependent manner. The expression of the two arginine transporters that are expressed in cardiac myocytes [CAT-1 (cationic amino acid transporter-1) and CAT-2] was studied using reverse transcription-PCR. A concentration of 200 microM allicin abolished the expression of CAT-2 mRNA, 100 microM significantly attenuated it, whereas 50 microM had no effect. Allicin had no effect on steady-state CAT-1 mRNA levels. Our results suggest that allicin inhibits iNOS activity through two different mechanisms: at lower concentrations it decreases iNOS mRNA levels, whereas at higher concentrations it inhibits arginine transport through down-regulation of CAT-2 mRNA.