Direct nucleation of calcium oxalate dihydrate crystals onto the surface of living renal epithelial cells in culture

Direct nucleation of calcium oxalate dihydrate crystals onto the surface of living renal epithelial cells in culture.BackgroundThe interaction of the most common crystal in human urine, calcium oxalate dihydrate (COD), with the surface of monkey renal epithelial cells (BSC-1 line) was studied to identify initiating events in kidney stone formation.MethodsTo determine if COD crystals could nucleate directly onto the apical cell surface, a novel technique utilizing vapor diffusion of oxalic acid was employed. Cells were grown to confluence in the inner four wells of 24-well plates. At the start of each experiment, diethyloxalate in water was placed into eight adjacent wells, and the plates were sealed tightly with tape so that oxalic acid vapor diffused into a calcium-containing buffer overlying the cells.ResultsSmall crystals were visualized on the cell surface after two hours, and by six hours the unambiguous habitus of COD was confirmed. Nucleation onto cells occurred almost exclusively via the (001) face, one that is only rarely observed when COD crystals nucleate onto inanimate surfaces. Similar results were obtained when canine renal epithelial cells (MDCK line) were used as a substrate for nucleation. Initially, COD crystals were internalized almost as quickly as they formed on the apical cell surface.ConclusionsFace-specific COD crystal nucleation onto the apical surface of living renal epithelial cells followed by internalization is a heretofore unrecognized physiological event, suggesting a new mechanism to explain crystal retention within the nephron, and perhaps kidney stone formation when this process is dysregulated or overwhelmed

Role of Calcium Oxalate Monohydrate Crystal Interactions with Renal Epithelial Cells in the Pathogenesis of Nephrolithiasis: A Review

Renal tubular fluid in the distal nephron is supersaturated with calcium and oxalate ions that nucleate to form crystals of calcium oxalate monohydrate (COM), the most common crystal in renal stones. How these nascent crystals are retained in the nephron to form calculi in certain individuals is not known. Recent studies from this laboratory have demonstrated that COM crystals can bind within seconds to the apical surface of renal epithelial cells, suggesting one mechanism whereby crystals could be retained in the tubule. Adherence of crystals to cells along the nephron may be opposed by specific urinary anions such as glycosaminoglycans, uropontin, nephrocalcin, and citrate. In culture, adherent crystals are quickly internalized by renal cells, and reorganization of the cytoskeleton, alterations in gene expression, and initiation of proliferation can ensue. Each of these cellular events appears to be regulated by extra-cellular factors. Identification of molecules in tubular fluid and on the cell surface that determine whether a crystal-cell interaction results in retention of the crystal or its passage out of the nephron appears critical for understanding the pathogenesis of nephrolithiasis

Amino acid-mediated stimulation of renal phospholipid biosynthesis after acute tubular necrosis

Amino acid-mediated stimulation of renal phospholipid biosynthesis after acute tubular necrosis. The mechanism by which amino acid infusion stimulates membrane phospholipid biosynthesis during renal regeneration after mercuric-chloride-induced acute tubular necrosis was studied in the rat. Amino acids can act directly on regenerating renal tissue to enhance net phospholipid synthesis because preincubation of cortical slices with amino acids induced an increase in [14C]-choline incorporation into phospholipid without altering the rate of breakdown. This amino acid stimulation of phospholipid biosynthesis was studied further by measuring [14C]-choline accumulation and its sequential conversion to phosphorylcholine, cytidine diphosphocholine (CDP-choline), and phosphatidylcholine via the Kennedy pathway in regenerating renal tissue. [14C]-Choline accumulation was increased after amino acid infusion, compared to glucose infusion. There were also increments in the Vmax of the choline kinase reaction, which converts entering [14C]-choline into [14C]-phosphorylcholine, and of the cholinephosphotransferase reaction in which [14C]-CDP-choline is incorporated into [14C]-phosphatidylcholine, whereas the apparent Km of each reaction was unchanged. Thus, amino acids infused after tubular necrosis can act directly on regenerating renal cells to increase precursor availability and augment two reactions of the phospholipid bio-synthetic pathway.Stimulation par les acides aminés de la biosynthèse rénale de phospholipides après nécrose tubulaire aiguë. Le mécanisme par lequel la perfusion d'acides aminés stimule la biosynthèse de phospholipides membranaires au cours de la régénération rénale consécutive à la nécrose tubulaire aiguë déterminée par le chlorure mercurique a été étudié chez le rat. Les acides aminés peuvent agir directement sur le tissu rénal en régénération pour accroître la synthèse nette de phospholipides puisque la pré-incubation de tranches de cortex avec des acides aminés détermine une augmentation de l'incorporation de [14C]-choline dans les phospholipides sans modification du catabolisme. Cette stimulation par les acides aminés de la synthèse des phospholipides a été étudiée par la mesure de l'accumulation de [14C]-choline et de sa conversion séquentielle en phosphorylcholine, cytidine diphosphocholine (CDP-choline) et phosphatidylcholine, selon la voie de Kennedy, dans le tissu rénal en cours de régénération. L'accumulation de [14C]-choline est augmentée après perfusion d'acides aminés, par comparaison avec celle de glucose. On observe aussi des augmentations des Vmax de la réaction de la choline kinase, qui transforme la [14C]-choline entrante en [14C]-phosphorylcholine, et de la réaction de la cholinephosphotransferase, dans laquelle la [14C]-CDP-choline est incorporée dans la [14C]-phosphatidylcholine, alors que le Km de ces réactions est inchangé. Ainsi les acides aminés perfusés après une nécrose tubulaire peuvent agir directement sur les cellules rénales en régénération pour augmenter la disponibilité en précurseur et accélérer deux réactions de la voie de biosynthèse des phospholipides

AMP peptide targets tight junctions to protect and heal barrier structure and function in models of IBD.

Background: A peptide derived from Antrum Mucosal Protein (AMP)-18 (gastrokine-1) reduces the extent of mucosal erosions and clinical severity in mice with dextran sulfate sodium (DSS)-induced colonic injury. The present study set out to determine if AMP peptide was also therapeutic for immune- and cytokine-mediated mouse models of intestinal injury and inflammatory bowel diseases (IBD) by enhancing and stabilizing tight junctions (TJs). Methods: Therapeutic effects of AMP peptide were examined in interleukin-10 deficient and a T cell adoptive transfer models of colitis in immunodeficient recombinase activating gene-1 knock-out (RAG-1−/−) mice. Mechanisms by which AMP peptide enhances barrier function and structure were studied ex vivo using intestine and colon from mice given lipopolysaccharide (LPS), and in AMP-18 deficient mice given DSS. Results: In interleukin-10 deficient mice given piroxicam, AMP peptide enhanced recovery after weight loss, protected against colon shortening and segmental dilation, and reduced the colitis activity score. In the T cell transfer model, treatment with the peptide protected against colon shortening. In mice given LPS in vivo to induce gut injury, AMP peptide prevented the onset of, and reversed established intestinal hyperpermeability by targeting TJ proteins and perijunctional actin

A Novel Peptide for Simultaneously Enhanced Treatment of Head and Neck Cancer and Mitigation of Oral Mucositis

We have characterized a novel 21 amino acid-peptide derived from Antrum Mucosal Protein (AMP)-18 that mediates growth promotion of cultured normal epithelial cells and mitigates radiation-induced oral mucositis in animal models, while suppressing in vitro function of cancer cells. The objective of this study was to evaluate these dual potential therapeutic effects of AMP peptide in a clinically relevant animal model of head and neck cancer (HNC) by simultaneously assessing its effect on tumor growth and radiation-induced oral mucositis in an orthotopic model of HNC. Bioluminescent SCC-25 HNC cells were injected into the anterior tongue and tumors that formed were then subjected to focal radiation treatment. Tumor size was assessed using an in vivo imaging system, and the extent of oral mucositis was compared between animals treated with AMP peptide or vehicle (controls). Synergism between AMP peptide and radiation therapy was suggested by the finding that tumors in the AMP peptide/radiation therapy cohort demonstrated inhibited growth vs. radiation therapy-only treated tumors, while AMP peptide-treatment delayed the onset and reduced the severity of radiation therapy-induced oral mucositis. A differential effect on apoptosis appears to be one mechanism by which AMP-18 can stimulate growth and repair of injured mucosal epithelial cells while inhibiting proliferation of HNC cells. RNA microarray analysis identified pathways that are differentially targeted by AMP-18 in HNC vs. nontransformed cells. These observations confirm the notion that normal cells and tumor cells may respond differently to common biological stimuli, and that leveraging this finding in the case of AMP-18 may provide a clinically relevant opportunity

Study of CP violation in Dalitz-plot analyses of B0 --> K+K-KS, B+ --> K+K-K+, and B+ --> KSKSK+

We perform amplitude analyses of the decays $B^0 \to K^+K^-K^0_S$, $B^+ \rightarrow K^+K^-K^+$, and $B^+ \to K^0_S K^0_S K^+$, and measure CP-violating parameters and partial branching fractions. The results are based on a data sample of approximately $470\times 10^6$ $B\bar{B}$ decays, collected with the BABAR detector at the PEP-II asymmetric-energy $B$ factory at the SLAC National Accelerator Laboratory. For $B^+ \to K^+K^-K^+$, we find a direct CP asymmetry in $B^+ \to \phi(1020)K^+$ of $A_{CP}= (12.8\pm 4.4 \pm 1.3)$, which differs from zero by $2.8 \sigma$. For $B^0 \to K^+K^-K^0_S$, we measure the CP-violating phase $\beta_{\rm eff} (\phi(1020)K^0_S) = (21\pm 6 \pm 2)^\circ$. For $B^+ \to K^0_S K^0_S K^+$, we measure an overall direct CP asymmetry of $A_{CP} = (4 ^{+4}_{-5} \pm 2)$. We also perform an angular-moment analysis of the three channels, and determine that the $f_X(1500)$ state can be described well by the sum of the resonances $f_0(1500)$, $f_2^{\prime}(1525)$, and $f_0(1710)$.Comment: 35 pages, 68 postscript figures. v3 - minor modifications to agree with published versio

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE