The RenTg Mice: A Powerful Tool to Study Renin-Dependent Chronic Kidney Disease

Background

Several studies have shown that activation of the renin-angiotensin system may lead to hypertension, a major risk factor for the development of chronic kidney disease (CKD). The existing hypertension-induced CDK mouse models are quite fast and consequently away from the human pathology. Thus, there is an urgent need for a mouse model that can be used to delineate the pathogenic process leading to progressive renal disease. The objective of this study was dual: to investigate whether mice overexpressing renin could mimic the kinetics and the physiopathological characteristics of hypertension-induced renal disease and to identify cellular and/or molecular events characterizing the different steps of the progression of CKD.

Methodology/Principal Findings

We used a novel transgenic strain, the RenTg mice harboring a genetically clamped renin transgene. At 3 months, heterozygous mice are hypertensive and slightly albuminuric. The expression of adhesion markers such as vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 are increased in the renal vasculature indicating initiation of endothelial dysfunction. At 5 months, perivascular and periglomerular infiltrations of macrophages are observed. These early renal vascular events are followed at 8 months by leukocyte invasion, decreased expression of nephrin, increased expression of KIM-1, a typical protein of tubular cell stress, and of several pro-fibrotic agents of the TGFβ family. At 12 months, mice display characteristic structural alterations of hypertensive renal disease such as glomerular ischemia, glomerulo- and nephroangio-sclerosis, mesangial expansion and tubular dilation.

Conclusions/Significance

The RenTg strain develops CKD progressively. In this model, endothelial dysfunction is an early event preceding the structural and fibrotic alterations which ultimately lead to the development of CKD. This model can provide new insights into the mechanisms of chronic renal failure and help to identify new targets for arresting and/or reversing the development of the disease.     

Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival

Brain-derived neurotrophic factor (BDNF), a major neuronal growth factor, is also known to exert an antiapoptotic effect in myeloma cells. Whereas BDNF secretion was described in B lymphocytes, the ability of B cells to produce sortilin, its transport protein, was not previously reported. We studied BDNF production and the expression of its receptors, tyrosine protein kinase receptor B and p75 neurotrophin receptor in the human pre-B, mature, and plasmacytic malignant B cell lines under normal and stress culture conditions (serum deprivation, Fas activation, or their combination). BDNF secretion was enhanced by serum deprivation and exerted an antiapoptotic effect, as demonstrated by neutralization experiments with antagonistic Ab. The precursor form, pro-BDNF, also secreted by B cells, decreases under stress conditions in contrast to BDNF production. Stress conditions induced the membranous expression of p75 neurotrophin receptor and tyrosine protein kinase receptor B, maximal in mature B cells, contrasting with the sequestration of both receptors in normal culture. By blocking Ab and small interfering RNA, we evidenced that BDNF production and its survival function are depending on sortilin, a protein regulating neurotrophin transport in neurons, which was not previously described in B cells. Therefore, in mature B cell lines, an autocrine BDNF production is up-regulated by stress culture conditions and exerts a modulation of apoptosis through the sortilin pathway. This could be of importance to elucidate certain drug resistances of malignant B cells. In addition, primary B lymphocytes contained sortilin and produced BDNF after mitogenic activation, which suggests that sortilin and BDNF might be implicated in the survival and activation of normal B cells also.     

A new epithelial-like cell line from eye muscle of catla Catla catla (Hamilton): development and characterization

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major carp ( Catla catla ), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz's L-15 supplemented with 15% foetal bovine serum (FBS). These cells have been subcultured >80 times over a period of 1·5 years. This cell line has been designated SICE. The SICE cell line consists predominantly of epithelial-like cells. These cells are strongly positive for epithelial markers such as pancytokeratin and cytokeratin 19. The cells were able to grow at temperatures between 25 and 32° C with optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or 20% FBS. Six marine fish viruses (fish nervous necrosis virus, marine birnavirus-NC1, chum salmon virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus-Sp and hirame rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial-like morphology and reached confluency on the fourth day after subculture. Polymerase chain reaction amplification of mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence-activated cell sorter revealed that most of the cells on the second day of culture were in S-phase, indicating a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the SICE cell line can be a useful tool for transgenic and genetic manipulation studies.     

Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production

Helminth exposure appears to protect hosts from inappropriate inflammatory responses, such as those causing inflammatory bowel disease. A recently identified, strongly proinflammatory limb of the immune response is characterized by T cell IL-17 production. Many autoimmune type inflammatory diseases are associated with IL-17 release. Because helminths protect from these diseases, we examined IL-17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL-17 production by lamina propria mononuclear cells (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL-17A mRNA by MLN cells and inhibits IL-17 production by cultured LPMC and MLN cells. Helminth exposure augments IL-4 and IL-10 production. Blocking both IL-4 and IL-10, but not IL-10 alone, restores IL-17 production in vitro. Colonization of colitic IL-10-deficient mice with H. polygyrus suppresses LPMC IL-17 production and improves colitis. Ab-mediated blockade of IL-17 improves colitis in IL-10-deficient mice. Thus, helminth-associated inhibition of IL-17 production is most likely an important mechanism mediating protection from inappropriate intestinal inflammation.     

Causal inference in biomolecular pathways using a Bayesian network approach and an Implicit method

We introduce here the concept of Implicit networks which provide, like Bayesian networks, a graphical modelling framework that encodes the joint probability distribution for a set of random variables within a directed acyclic graph. We show that Implicit networks, when used in conjunction with appropriate statistical techniques, are very attractive for their ability to understand and analyze biological data. Particularly, we consider here the use of Implicit networks for causal inference in biomolecular pathways. In such pathways, an Implicit network encodes dependencies among variables (proteins, genes), can be trained to learn causal relationships (regulation, interaction) between them and then used to predict the biological response given the status of some key proteins or genes in the network. We show that Implicit networks offer efficient methodologies for learning from observations without prior knowledge and thus provide a good alternative to classical inference in Bayesian networks when priors are missing. We illustrate our approach by an application to simulated data for a simplified signal transduction pathway of the epidermal growth factor receptor (EGFR) protein.     

The potential role of carbon dioxide in the neuroimmunoendocrine changes following cerebral ischemia

Carbon dioxide (CO(2)) interacts in complex ways with the brain and the endocrine and immune systems. Arterial CO(2) may be elevated or decreased following cerebral ischemia-reperfusion injury or stroke. The aim of the present review is to delineate potential changes in the neuroimmunoendocrine system following cerebral ischemia-reperfusion injury and to provide evidence for the modulatory role of carbon dioxide in this setting. It appears that lesions of the right and left cerebral hemispheres are associated with different patterns of immune activation and cytokine release. Changes in arterial CO(2) can profoundly alter the neuroimmunoendocrine system, especially the hypothalamic-pituitary-adrenal (HPA) axis and the production of pro-inflammatory cytokines. Hypercapnia activates the HPA axis, exerts antiinflammatory and antioxidant effects, and can alter the secretion and function of various brain neurotransmitters. There is conflicting evidence surrounding arterial CO(2): its effects on the ischemic brain may be either beneficial or deleterious. Mild hypercapnia may exert some neuroprotection following cerebral ischemia, but severe hypercapnia may aggravate neuronal injury by extra- and intra-cellular acidification and/or impairment of cellular calcium hemostasis. Future studies are required to delineate the potential relationship between arterial CO(2) and prognosis and long-term survival following cerebral ischemia-reperfusion injury. "Therapeutic hypercapnia" seems to be a promising approach to the treatment of stroke patients, and its use should be justified by further experimental and clinical studies.     

Thymoquinone protects renal tubular cells against tubular injury

In this work the effect of angiotensin II (AT II) on proximal tubular epithelial cells (pTECs) in vitro was studied. AT II was found to activate the nuclear factor kappaB (NF-kappaB) and its controlled genes, for example, interleukin 6 (IL-6) of pTECs in a time-dependent manner. Two points with maximum NF-kappaB activation were found, the first after 12 h and the second after 3.5 days. The first point may be due to activation of NF-kappaB in pTECs in response to AT II while the second may be due to activation of the advanced glycation end product (AGE)/receptor of the AGE (RAGE) system. Thymoquinone (TQ) was found to decrease NF-kappaB activation in a dose-dependant manner with maximum inhibitory effect at a concentration of 500 nM. Also, pre-incubation of pTECs with TQ leads to disappearance of the second peak of NF-kappaB. These data are consistent with results obtained from IL-6 enzyme-linked immunosorbent assay (ELISA) and transient transfection experiments. The results explain the therapeutic value of TQ which can be used to delay end stage renal diseases in diabetics.     

Identification of Residues Surrounding the Active Site of Type A Botulinum Neurotoxin Important for Substrate Recognition and Catalytic Activity

Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain (LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar residues resulted in drastic lowering of the catalytic rate constant (k _cat), but had less effect on substrate affinity (K _m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k _cat/K _m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation to Asn resulted in 140-fold decrease in k _cat/K _m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163, Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165 with Leu resulted in fourfold increase in k _cat/K _m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe, respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC competent for catalysis or provide conformational selection of the substrate.     

New bioactive clerodane diterpenoids from the roots of Casearia membranacea

Bioassay-guided fractionation of the acetone extract of the roots of Casearia membranacea furnished three new clerodane diterpenes, caseamembrins S-U (1-3) and the known caseamembrin Q (4). Their structures were established by extensive spectroscopic analyses, especially 2D-NMR. Compounds 1-4 were tested against human tumor cells, including HeLa (cervical epitheloid carcinoma), DLD-1 (colon carcinoma), Daoy (medulloblastoma), and KB (oral epidermoid carcinoma) cell lines. Caseamembrin T (2) exhibited the most potent activity against Daoy cells (ED(50)=10 ng/ml), superior to that of the standard drug mitomycin.     

Bioactive marine prostanoids from octocoral Clavularia viridis

Chemical investigation of the nonpolar extract of soft coral Clavularia viridis resulted in isolation of five new prostanoids, designated as claviridic acids A-E (1-5, resp.), in addition to the known clavulones I-III. Their structures were determined on the basis of spectroscopic techniques, especially HR-ESI-MS, CD, and 2D-NMR experiments. The isolated marine prostanoids exhibited potent inhibitory effect on PHA-induced proliferation of peripheral blood mononuclear cells (PBMC), as well as significant cytotoxic activity against human gastric cancer cells (AGS).