The Ngal reporter mouse detects the response of the kidney to injury in real time

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.     

A comparison of statistical methods for the detection of hepatocellular carcinoma based on serum biomarkers and clinical variables

Background

Currently, a surgical approach is the best curative treatment for those with hepatocellular carcinoma (HCC). However, this requires HCC detection and removal of the lesion at an early stage. Unfortunately, most cases of HCC are detected at an advanced stage because of the lack of accurate biomarkers that can be used in the surveillance of those at risk. It is believed that biomarkers that could detect HCC early will play an important role in the successful treatment of HCC.

Methods

In this study, we analyzed serum levels of alpha fetoprotein, Golgi protein, fucosylated alpha-1-anti-trypsin, and fucosylated kininogen from 113 patients with cirrhosis and 164 serum samples from patients with cirrhosis plus HCC. We utilized two different methods, namely, stepwise penalized logistic regression (stepPLR) and model-based classification and regression trees (mob), along with the inclusion of clinical and demographic factors such as age and gender, to determine if these improved algorithms could be used to increase the detection of cancer.

Results and discussion

The performance of multiple biomarkers was found to be better than that of individual biomarkers. Using several statistical methods, we were able to detect HCC in the background of cirrhosis with an area under the receiver operating characteristic curve of at least 0.95. stepPLR and mob demonstrated better predictive performance relative to logistic regression (LR), penalized LR and classification and regression trees (CART) used in our prior study based on three-fold cross-validation and leave one out cross-validation. In addition, mob provided unparalleled intuitive interpretation of results and potential cut-points for biomarker levels. The inclusion of age and gender improved the overall performance of both methods among all models considered, while the stratified male-only subset provided the best overall performance among all methods and models considered.

Conclusions

In addition to multiple biomarkers, the incorporation of age and gender into statistical models significantly improved their predictive performance in the detection of HCC.

     

Association of Perioperative Plasma Neutrophil Gelatinase-Associated Lipocalin Levels with 3-Year Mortality after Cardiac Surgery: A Prospective Observational Cohort Study

Background

Higher levels of plasma neutrophil gelatinase-associated lipocalin (pNGAL) are an early marker of acute kidney injury and are associated with increased risk of short-term adverse outcomes. The independent association between pNGAL and long-term mortality is unknown.

Methods

In this prospective observational cohort study, we studied 1191 adults who underwent cardiac surgery between 2007 and 2009 at 6 centers in the TRIBE-AKI cohort. We measured the pNGAL on the pre-operative and first 3 post-operative days and assessed the relationship of peri-operative pNGAL concentrations with all-cause mortality.

Results

During a median follow-up of 3.0 years, 139 participants died (50/1000 person-years). Pre-operative levels of pNGAL were associated with 3-year mortality (unadjusted HR 1.96, 95% CI 1.34,2.85) and the association persisted after adjustment for pre-operative variables including estimated glomerular filtration rate (adjusted HR 1.48, 95% CI 1.04–2.12). After adjustment for pre- and intra-operative variables, including pre-operative NGAL levels, the highest tertiles of first post-operative and peak post-operative pNGAL were also independently associated with 3-year mortality risk (adjusted HR 1.31, 95% CI 1.0–1.7 and adjusted HR 1.78, 95% CI 1.2–2.7, respectively). However, after adjustment for peri-operative changes in serum creatinine, there was no longer an independent association between the first post-operative and peak post-operative pNGAL and long-term mortality (adjusted HR 0.98,95% CI 0.79–1.2 for first pNGAL and adjusted HR 1.19, 95% CI 0.87–1.61 for peak pNGAL).

Conclusions

Pre-operative pNGAL levels were independently associated with 3-year mortality after cardiac surgery. While post-operative pNGAL levels were also associated with 3-year mortality, this relationship was not independent of changes in serum creatinine. These findings suggest that while pre-operative pNGAL adds prognostic value for mortality beyond routinely available serum creatinine, post-operative pNGAL measurements may not be as useful for this purpose.     

NGAL-Siderocalin in kidney disease

Kidney damage induces the expression of a myriad of proteins in the serum and in the urine. The function of these proteins in the sequence of damage and repair is now being studied in genetic models and by novel imaging techniques. One of the most intensely expressed proteins is lipocalin2, also called NGAL or Siderocalin. While this protein has been best studied by clinical scientists, only a few labs study its underlying metabolism and function in tissue damage. Structure-function studies, imaging studies and clinical studies have revealed that NGAL-Siderocalin is an endogenous antimicrobial with iron scavenging activity. This review discusses the "iron problem" of kidney damage, the tight linkage between kidney damage and NGAL-Siderocalin expression and the potential roles that NGAL-Siderocalin may serve in the defense of the urogenital system. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis?

PURPOSE OF REVIEW: During the past decade, paraoxonase 1, a HDL-associated protein, has been demonstrated to be an important contributor to the antioxidant capacity of HDL. Studies using paraoxonase 1 null mice by gene targeting and transgenic mice corroborated the hypothesis that paraoxonase 1 protects against atherosclerosis. In contrast to paraoxonase 1, the other two members of the paraoxonase gene family, namely paraoxonase 2 and paraoxonase 3, are either undetectable (paraoxonase 2) or detected at very low levels (paraoxonase 3) on HDL, and are considered to participate in intracellular antioxidant mechanisms. In this review, we summarize studies reported in the past 2 years suggesting a protective role for paraoxonase 2 and paraoxonase 3 in the development of atherosclerosis in mice. RECENT FINDINGS: Adenovirus-mediated expression of human paraoxonase 2 or paraoxonase 3 proteins protects against the development of atherosclerosis in apolipoprotein E-deficient mice. Paraoxonase 2-deficient mice develop significantly larger atherosclerotic lesions than their wild-type and heterozygous counterparts on an atherogenic diet despite having lower levels of apolipoprotein B-containing lipoproteins. Atherosclerotic lesions were significantly lower in male hPON3Tg/LDLR null mice than in LDLR null mice on a western diet. SUMMARY: We conclude that, in addition to paraoxonase 1, both paraoxonase 2 and paraoxonase 3 proteins are protective against the development of atherosclerosis in mice. These findings underscore the utility of all members of the paraoxonase gene family as therapeutic targets for the treatment of atherosclerosis.     

Nonnegative matrix factorization: an analytical and interpretive tool in computational biology

In the last decade, advances in high-throughput technologies such as DNA microarrays have made it possible to simultaneously measure the expression levels of tens of thousands of genes and proteins. This has resulted in large amounts of biological data requiring analysis and interpretation. Nonnegative matrix factorization (NMF) was introduced as an unsupervised, parts-based learning paradigm involving the decomposition of a nonnegative matrix V into two nonnegative matrices, W and H, via a multiplicative updates algorithm. In the context of a pxn gene expression matrix V consisting of observations on p genes from n samples, each column of W defines a metagene, and each column of H represents the metagene expression pattern of the corresponding sample. NMF has been primarily applied in an unsupervised setting in image and natural language processing. More recently, it has been successfully utilized in a variety of applications in computational biology. Examples include molecular pattern discovery, class comparison and prediction, cross-platform and cross-species analysis, functional characterization of genes and biomedical informatics. In this paper, we review this method as a data analytical and interpretive tool in computational biology with an emphasis on these applications.     

Design and Characterization of Metformin-Loaded Solid Lipid Nanoparticles for Colon Cancer

Colorectal cancer is a global concern, and its treatment is fraught with non-selective effects including adverse side effects requiring hospital visits and palliative care. A relatively safe drug formulated in a bioavailability enhancing and targeting delivery platform will be of significance. Metformin-loaded solid lipid nanoparticles (SLN) were designed, optimized, and characterized for particle size, zeta potential, drug entrapment, structure, crystallinity, thermal behavior, morphology, and drug release. Optimized SLN were 195.01?±?6.03 nm in size, ?17.08?±?0.95 mV with regard to surface charge, fibrous in shape, largely amorphous, and release of metformin was controlled. The optimized size, charge, and shape suggest the solid lipid nanoparticles will migrate and accumulate in the colon tumor preventing its proliferation and subsequently leading to tumor shrinkage and cell death.     

The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways

Previous studies have reported a protective role for the von Hippel-Lindau (VHL) gene products against pro-apoptotic cellular stresses, but the mechanisms remain unclear. In this study, we examined the role of VHL in renal cells subjected to chemical hypoxia, using four VHL-negative and two VHL-positive cell lines. VHL-negative renal carcinoma cells underwent apoptosis following chemical hypoxia (short-term glucose deprivation and antimycin treatment), as evidenced by morphologic changes and internucleosomal DNA cleavage. Reintroduction of VHL expression prevented this apoptosis. VHL-negative cells displayed a significant (greater than 5-fold) activation of caspase 9 and release of cytochrome c into the cytosol following chemical hypoxia. In contrast, VHL-positive cells showed minimal caspase 9 activation, and absence of cytochrome c release under the same conditions. Caspase 8 was only minimally activated in both VHL-negative and -positive cells. In addition, VHL-positive cells displayed a striking up-regulation of Bcl-2 expression (5-fold) following chemical hypoxia. Antisense oligonucleotides to Bcl-2 significantly down-regulated Bcl-2 protein expression in VHL-positive cells and rendered them sensitive to apoptosis. Overexpression of Bcl-2 in VHL-negative cells conferred resistance to apoptosis. Our results suggest that VHL protects renal cells from apoptosis via Bcl-2-dependent pathways.     

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.