Abstract-Chronic renal failure (CRF) is associated with oxidative stress which promotes production of reactive carbonyl compounds and lipoperoxides leading to the accumulation of advanced glycation and lipoxidation end products. Reactive oxygen species (ROS) avidly reacts with nitric oxide (NO) producing cytotoxic reactive nitrogen species capable of nitrating proteins and damaging other molecules. This study tested the hypothesis that CRF results in enhanced ROS-mediated NO inactivation and protein nitration which can be ameliorated with antioxidant therapy. Male Sprague Dawley rats were randomized to CRF (5/6 nephrectomy) and sham-operated controls and fed either a regular diet (vitamin E, 40 U/Kg food) or an antioxidant-fortified diet (vitamin E, 5000 U/Kg food) for 6 weeks. Blood pressure, plasma malondialdehyde (MDA), tissue NO synthase (NOS) isoforms, tissue nitrotyrosine (the footprint of NO interaction with ROS), and vascular tissue NO production were determined. CRF resulted in marked elevations of blood pressure, plasma MDA, and tissue nitrotyrosine abundance, but did not change plasma L-arginine level. This was coupled with depressed vascular tissue NO production and reduced immunodetectable NOS proteins in the vascular, renal, and cardiac tissues. Antioxidant therapy ameliorated the CRF-induced hypertension, improved vascular tissue NO production, lowered tissue nitrotyrosine burden, and reversed downregulations of NOS isoforms. In contrast, antioxidant therapy had no effects in the controls. CRF is associated with oxidative stress which promotes NO inactivation by ROS leading to functional NO deficiency, hypertension, and widespread accumulation of protein nitration products. Amelioration of oxidative stress by high-dose vitamin E enhances NO availability, improves hypertension, lowers protein nitration products, and increases NOS expression and vascular NO production in CRF animals. Key Words: uremia Ⅲ nitric oxide synthase Ⅲ hypertension, renal Ⅲ oxidative stress Ⅲ oxygen O xidative stress occurs when generation of the reactive oxygen species (ROS) exceeds the natural antioxidant capacity of the organism. In the presence of oxidative stress, uncontained ROS attack, denature, or modify lipids, proteins, carbohydrates, DNA, and other molecules. Oxidative stress is a known feature of chronic renal failure (CRF), and its presence is evidenced by the reported elevation of lipid peroxidation product, malondialdehyde, depressed antioxidant capacity, and impaired antioxidant enzyme activities in the plasma and erythrocytes of CRF patients and animals. [1][2][3][4][5][6][7] Increased ROS activity results in the nonenzymatic production of reactive carbonyl compounds and lipoperoxides which, in turn, react with and modify structural and functional proteins leading to formation of advanced glycation end products (AGE) and advanced lipoxidation end products (ALE). 8,9 Accumulation of these products is thought to contribute to the cardiovascular and other complications of CRF. The present study extends the prev...

Significant reduction of renal mass triggers a chain of events that result in glomerular hypertension/hyperfiltration, proteinuria, glomerulosclerosis, tubulointerstitial injury, and end-stage renal disease. These events are mediated by a constellation of hemodynamic, oxidative, and inflammatory reactions that are, in part, driven by local AT 1 receptor (AT 1 r) activation by angiotensin II (Ang II). Here we explored the effects of 5/6 nephrectomy with and without AT 1 r blockade (losartan for 8 weeks) on AT 1 r and AT 2 r and Ang II-positive cell count, pathways involved in oxidative stress and inflammation [NAD(P)H oxidase, nuclear factor B (NFB), 12-lipooxygenase, cyclooxygenase (COX)-1, COX-2, monocyte chemoattractant protein (MCP)-1, plasminogen activator inhibitor (PAI)-1, renal T cell, and macrophage infiltration] as well as renal function and structure. The untreated group exhibited hypertension, deterioration of renal function and structure, reduced or unchanged plasma renin activity, aldosterone concentration, marked up-regulations of AT 1 r (250%), Ang II-expressing cell count (Ͼ20-fold), NAD(P)H oxidase subunits (gp91 phox, p22 phox , and P47 phox ; 20 -40%), COX-2 (250%), 12-lipooxygenase (100%), MCP-1 (400%), and PAI-1 (Ͼ20-fold), activation of NFB, and interstitial infiltrations of T cells and macrophages in the remnant kidneys. AT 1 r blockade attenuated the biochemical and histological abnormalities, prevented hypertension, and decelerated deterioration of renal function and structure. Thus, the study demonstrated a link between up-regulation of Ang II/AT 1 r system and oxidative stress, inflammation, hypertension, and progression of renal disease in rats with renal mass reduction.Significant reduction in renal mass by subtotal nephrectomy or by various disease processes triggers a chain of events that culminates in progressive glomerulosclerosis, tubulointerstitial injury, proteinuria, and end-stage renal disease (Remuzzi et al., 2006). Progressive deterioration of the remnant/diseased kidney function and structure is associated with and largely mediated by profound alteration of renal hemodynamics, inflammation, and oxidative stress (Mackenzie et al., 2000). In this context, renal mass reduction results in glomerular capillary hypertension and glomerular hyperfiltration, which play a major role in the pathogenesis of proteinuria and glomerulosclerosis (Hostetter et al., 2001). This assertion is supported by the observations that prevention/alleviation of the maladaptive hemodynamic alterations by lowering dietary protein or blocking renin-angiotensin system (RAS) decelerates progression of renal disease (Nickenig and Harrison, 1994;Mackenzie et al., 2000). In addition to the hemodynamic factors, accumulation of the inflammatory cells plays a major part in progression of renal disease (Chow et al., 2004;Rodríguez-Iturbe et al., 2004b). This supposition is consistent with the demonstration that inhibition of leukocyte recruitment by chemokine receptor antagonists and treatment with immun...

SUMMARYThe placenta is a transient organ that is necessary for proper fetal development. Its main functional component is the trophoblast, which is derived from extra-embryonic ectoderm. Little is known about early trophoblast differentiation in the human embryo, owing to lack of a proper in vitro model system. Human embryonic stem cells (hESCs) differentiate into functional trophoblast following BMP4 treatment in the presence of feeder-conditioned media; however, this model has not been widely accepted, in part owing to a lack of proof for a trophoblast progenitor population. We have previously shown that p63, a member of the p53 family of nuclear proteins, is expressed in proliferative cytotrophoblast (CTB), precursors to terminally differentiated syncytiotrophoblast (STB) in chorionic villi and extravillous trophoblast (EVT) at the implantation site. Here, we show that BMP4-treated hESCs differentiate into bona fide CTB by direct comparison with primary human placental tissues and isolated CTB through gene expression profiling. We show that, in primary CTB, p63 levels are reduced as cells differentiate into STB, and that forced expression of p63 maintains cyclin B1 and inhibits STB differentiation. We also establish that, similar to in vivo events, hESC differentiation into trophoblast is characterized by a p63 + /KRT7 + CTB stem cell state, followed by formation of functional KLF4 + STB and HLA-G + EVT. Finally, we illustrate that downregulation of p63 by shRNA inhibits differentiation of hESCs into functional trophoblast. Taken together, our results establish that BMP4-treated hESCs are an excellent model of human trophoblast differentiation, closely mimicking the in vivo progression from p63 + CTB stem cells to terminally differentiated trophoblast subtypes.

Hyperglycosylated human chorionic gonadotropin (HhCG) is a glycoprotein hormone secreted during embryonic implantation and trophoblast invasion of the uterine wall and is an early marker of pregnancy (1 ). Relative to hCG, HhCG has a higher molecular mass (38.5-40 kDa, depending on the amount of carbohydrate) and a higher number of asparagine (N)-linked triantennary carbohydrates and serine (O)-linked tetrasaccharide core structures in the ␤-subunit (2 ). Although both are secreted from the placenta and choriocarcinoma, HhCG is produced by mononucleated cytotrophoblasts, and hCG is produced by syncytiotrophoblast cells (3)(4)(5)(6). Because the cytotrophoblasts are primitive and invasive in nature, HhCG is also called invasive trophoblast antigen (ITA) (5 ).Birken et al. (7 ) described a monoclonal antibody (B152) specific for the ␤-subunit C-terminal peptide and the O-linked oligosaccharide of HhCG. Although the epitope for this antibody does not require sialic acid, the presence of the O-linked tetrasaccharide core structure is essential (1 ).Using IRMAs and ELISAs, investigators showed that (a) HhCG rapidly increases in early pregnancy, attaining substantially higher concentrations and decreasing earlier than hCG (1,8 ); (b) HhCG is increased in Down syndrome-affected pregnancies in both the first and second trimesters (9 -11 ); and (c) the HhCG:hCG ratio appears to be higher in those with invasive vs noninvasive trophoblastic disease (12 ).The above HhCG assays were performed manually using large sample volumes (200 L) and long incubation times (turnaround time, 1-2 days). We therefore developed an automated immunochemiluminometric assay (ICMA) that uses two monoclonal antibodies: the HhCGspecific B152 antibody described above and a hCG ␤-subunit-specific antibody (B207). Both antibodies were purified from cell lines provided by Dr. O'Connor (Columbia University, New York, NY). B152 was biotinylated with long-chain NHS-biotin (13 ), and B207 was conjugated with acridinium ester (14 ).The Nichols Institute Diagnostics Advantage ® instrument automatically pipetted 15 L of sample into a cuvette, followed by 25 L of streptavidin-coated magnetic particles (4 g/L Dynal M-270), 70 L of capture antibody (6 mg/L B152), and 260 L of buffer [0.1 mol/L phosphate-buffered saline (PBS), pH 8.2, containing 50 g/L bovine serum albumin (BSA)]. During a 30-min incubation at 37°C, HhCG in the sample bound to the B152 capture antibody, which in turn bound to the magnetic particles. The magnetic particles were automatically washed three times to remove unbound materials. Detection antibody [300 L of 1 mg/L B207 in 0.5 mol/L PBS (pH 7.4) with 5 g/L protease-free BSA, 60 mL/L normal mouse serum, and 1 g/L mouse ␥-globulin] was then added to the washed magnetic particles. During this 10-min incubation at 37°C, the B207 antibody bound to a hCG-shared epitope on the HhCG molecule, forming a sandwich complex. After another three washes, the magnetic particle-containing wells were transferred to the on-board luminometer. Hydrogen perox...

Objective To determine if hyperglycosylated hCG (hhCG), produced by invasive trophoblasts, measured as early as 9 days after egg retrieval can predict ongoing pregnancies (OP) after in vitro fertilization and fresh embryo transfer (IVF-ET). Design Cohort Setting Academic ART center Patients Consecutive patients undergoing IVF-ET Interventions Serum hhCG and hCG levels measured 9 (D9) and 16 (D16) days after egg retrieval Outcome Ongoing pregnancy (OP) beyond 9 weeks of gestation Results OP (62 of 112 participants) was associated with higher D9 levels of hhCG and hCG However, hhCG was detectable in all D9 OP samples, while hCG was detectable in only 22%. D9 hhCG levels >110 pg/mL was 96% specific for OP, yielding a positive predictive value of 95%. Compared to D9 hCG levels, hhCG was more sensitive and had a larger area under the curve (0.87 vs. 0.67). Diagnostic test characteristics were similar between D16 hhCG and hCG levels. Conclusions In patients undergoing assisted reproduction, a test to detect pregnancy early and predict outcomes is highly desirable. HhCG is detectable in serum 9 days after egg retrieval IVF-ET cycles. At this early assessment, hhCG is superior to traditional hCG and highly predictive of ongoing pregnancies.

ObjectiveTo determine the sensitivity and specificity of hyperglycosylated hCG (hhCG) measurements for the diagnosis of clinical pregnancies in the IVF setting and how soon post embryo transfer (ET) a pregnancy can be detected using an ultrasensitive (hhCG) assay. To determine if a single, early hhCG measurement can discriminate between biochemical and clinical pregnancies.DesignA 4 center prospective blinded clinical trial was performed with patients undergoing IVF-ET. Patients had blood drawn and submitted for hhCG analysis on the day of ET and at days 4, 6, 8, and 12 thereafter. First morning urines were collected and submitted for hhCG analysis on days 0, 4, 6, 8, 10 and 12.SettingFertility CentersOutcome MeasuresClinical pregnancies were defined as an ultrasound study demonstrating a gestational sac and/or heart beat at appropriate gestational ages.ResultsFifty-six of 58 enrolled patients completed the study. There were 25 clinical and 6 biochemical pregnancies. For blastocyst transfers, a single serum or urine hhCG measurement identified pregnancies (both biochemical and clinical) at 6 days post ET with 100% sensitivity and specificity. There were 6 biochemical pregnancies, all following blastocyst transfers. All of these pregnancies were identified by lower values.

Background: Tumor necrosis factor-α (TNF-α) and its receptors play important roles in the development and persistence of psoriatic plaques. The antithyroid thioureylenes, propylthiouracil and methimazole, are effective in the treatment of patients with psoriasis with a significant number of patients showing clearing or near clearing of their lesions after a several weeks of treatment.

Objective To examine the association of vitamin D insufficiency and risk of pregnancy-induced hypertension (PIH) among human immunodeficiency virus (HIV)-infected pregnant women. Study Design This is a retrospective cohort study evaluating the impact of low maternal vitamin D levels on PIH and perinatal outcomes among HIV-infected pregnant women receiving care at an urban HIV center from 1991 to 2014. Results A total of 366 pregnant women were included, of which 11% developed PIH. Lower levels of 25-hydroxyvitamin D (25(OH)D) and bioactive 1,25-dihydroxyvitamin D (1,25(OH)2D) were associated with increased HIV disease activity. 25(OH)D levels were not significantly associated with the incidence of PIH. Higher 1,25(OH)2D levels were associated with reduced incidence of PIH in univariate (odds ratio, OR: 0.87 [95% confidence interval, CI: 0.79–0.95], p = 0.004) and multivariate (OR: 0.88 [95% CI: 0.80–0.97], p = 0.010) analyses. No association was found between 25(OH)D levels and other obstetric outcomes. Lower 1,25(OH)2D levels were associated with group B Streptococcus colonization (OR: 0.92 [95% CI: 0.86–0.99]) and low birth weight (LBW) (OR: 0.90 [95% CI: 0.83–0.98]) on multivariate analysis. Mean 1,25(OH)2D levels were significantly lower in women with preterm delivery and LBW infants. Conclusion Lower bioactive vitamin D levels are related to PIH in HIV-infected women. This association may be related to the coexistence of abnormal placental vitamin D metabolism and abnormal placental implantation.