Characterization of an in vitro system of human renal papillary collecting duct cells

Summary We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10⁴ live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOsm for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and γ-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins. Part of this work was presented at a Symposium of the Center for Alternatives to Animal Testing, April 4–5, 1989, Johns Hopkins Medical Institutions, Baltimore, MD 21205. This work was supported in part by grants R01-AI24179, PO1-A804393 for the Public Health Service, U.S. Department of Health and Human Services, and by a grant from the National Kidney Foundation, Baltimore, MD affiliate.     

Biological diversity and cultural heritage

In this paper some examples of the development of communities of microorganisms and plants on historic buildings and montiments are shown. When the building stones differ from the surrounding natural substrata, an increase in the biological diversity of the area is produced. In some cases, monuments can come to constitute a true refuge for a few species when the natural habitat is threatened. It is suggested that biological diversity, when it does not represent a threat for the cultural heritage, should be considered worthy of preservation.     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Rapid regulation of electrolyte absorption in sweat duct

Even though the same Cl channel (CFTR) is common to certain fluid transport functions that are oppositely directed, i.e., secretion and absorption, only fluid secretion has clearly been shown to be acutely regulated. It is now clear that fluid secretion activated by -adrenergic stimulation is controlled by cAMP-mediated opening and closing of CFTR-Cl channels. Since the conductance of the human sweat duct is almost wholly due to CFTR-Cl conductance (CFTR-G_Cl), we sought to determine whether salt absorption via CFTR-Cl channels could also be subject to acute regulation in this purely absorptive epithelium. After -toxin permeabilization, we found that addition of cAMP resulted in a large increase in Cl diffusion potentials across the apical membrane and a more than twofold increase in the average membrane conductance. Since the cAMP effects were dependent on Cl alone, not on Na, and since apical Cl conductance appears to be almost exclusively comprised of CFTR-G_Cl, we surmise that this form of electrolyte absorption like secretion is also subject to acute control through CFTR-G_Cl. Acute regulation of absorption involves both activation by phosphorylation (PKA) and inactivation by dephosphorylation (unknown endogenous phosphatase) of CFTR. Phosphorylation of CFTR was shown by the facts that CFTR-G_Cl could be activated by cAMP and inhibited by the kinase antagonist staurosporine, or by removal of either substrate ATP or Mg²⁺ cofactor. Inactivation of CFTR-G_Cl by endogenous phosphatase(s) was indicated by a spontaneous but reversible loss of CFTR-G_Cl upon removal of cAMP. Such loss of CFTR-G_Cl activity could be prevented either by application of phosphatase inhibitors or by using phosphatase-resistant ATP--S as substrate to phosphorylate CFTR. We surmise that absorptive function is subject to rapid regulation which can be switched on and off acutely by a control system that is common to both absorptive and secretory processes and that this control is crucial to switching between conductive and nonconductive transport mechanisms during salt absorption.The authors are grateful to Mr. Kirk Taylor for expert technical assistance, and to numerous volunteer human subjects for participating in the experiments with informed consent. Supported by grants from the National Institutes of Health, DK-41329-04 and the National Cystic Fibrosis Foundation, Z 439.     

Testicular main ducts and spermatic ducts in some cyprinid fishes I. Morphology, fine structure and histochemistry

Alburnus alburnus, Leuciscus cephalus and Vimba vimba efferent duct systems of the male gonads consist of testicular main ducts and spermatic ducts. These have similar histological, fine structural and (enzyme–) histochemical characteristics and function in (1) storage and (2) nutrition of spermatozoa, (3) synthesis of steroid glucuronides, (4) secretion of proteins and enzymes (5) formation of an ionic gradient in the seminal fluid and (6) they have auto– and heterophagocytotic activities. Therefore testicular main ducts and spermatic ducts are important in the formation of the seminal fluid.     

Gene disruption in Lactobacillus plantarum strain 80 by site-specific recombination: isolation of a mutant strain deficient in conjugated bile salt hydrolase activity

A chloramphenicol-resistance gene (cml) was introduced into the Lactobacillus plantarum gene encoding conjugated bile acid hydrolasc (cbh) on a ColEl replicon. This plasmid which is nonreplicative in Lactobacillus was used to transform L. plantarum strain 80. A homologous double cross-over recombination event resulted in replacement of the chromosomal cbh gene by the cml-containing cbh gene. The transformants obtained were unable to synthesize active conjugated bile acid hydrolase (Cbh). The Cbh-Cml^R phenotype was stably maintained for more than 100 generations under nonselective conditions.This paper is dedicated with great appreciation to Dr. Frits Berends on the occasion of his retirement as Head of the Biochemistry Department of the TNO Medical Biological Laboratory     

Rare earth cerium oxide nanoparticles attenuated liver fibrosis in bile duct ligation mice model

Liver fibrosis is one of the major liver complications which eventually progresses to liver cirrhosis and liver failure. Cerium oxide nanoparticles, also known as nanoceria (NC) are nanoparticles with potential antioxidant and anti-inflammatory activities. Herein, we evaluated the hepatoprotective and anti-fibrotic effects of nanoceria (NC) against bile duct ligation (BDL) induced liver injury. NC were administered i.p. for 12 days (0.5 and 2 mg/kg) to C57BL/6J mice. The biochemical markers of liver injury, oxidative and nitrosative stress markers, inflammatory cytokines were evaluated. Fibrosis assessment and mechanistic studies were conducted to assess the hepatoprotective effects of NC. Administration of NC proved to significantly ameliorate liver injury as evident by reduction in SGOT, SGPT, ALP and bilirubin levels in the treated animals. NC treatment significantly reduced the hydroxyproline levels and expression of fibrotic markers. In summary, our findings establish the hepatoprotective and anti-fibrotic effects of NC against BDL induced liver injury and liver fibrosis. These protective effects were majorly ascribed to their potential ROS inhibition and antioxidant activities through catalase, superoxide dismutase (SOD)-mimetic properties and auto-regenerating capabilities.     

Quantitative Detection of 15 Serum Bile Acid Metabolic Products by LC/MS/MS in the Diagnosis of Primary Biliary Cholangitis

To determine 15 bile acid metabolic products in human serum by liquid chromatography-tandem mass spectrometry (LC/MS/MS) and value their diagnostic outcome in primary biliary cholangitis (PBC). Serum from 20 healthy controls and 26 patients with PBC were collected and went LC/MS/MS analysis of 15 bile acid metabolic products. The test results were analyzed by bile acid metabolomics, and the potential biomarkers were screened and their diagnostic performance was judged by statistical methods such as principal component and partial least squares discriminant analysis and area under curve (AUC). 8 differential metabolites can be screened out: Deoxycholic acid (DCA), Glycine deoxycholic acid (GDCA), Lithocholic acid (LCA), Glycine ursodeoxycholic acid (GUDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurodeoxycholic acid (TDCA), Glycine chenodeoxycholic acid (GCDCA). The performance of the biomarkers was evaluated by the AUC, specificity and sensitivity. In conclusion, DCA, GDCA, LCA, GUDCA, TLCA, TUDCA, TDCA and GCDCA were identified as eight potential biomarkers to distinguish between healthy people and PBC patients by multivariate statistical analysis, which provided reliable experimental basis for clinical practice.     

人体胆汁液晶光学性质的研究

在单偏光和正交偏光显微镜下对人体胆汁液晶的光学性质进行研究。结果表明：人体胆汁中的液晶以球形的微粒分散于胆汁中,其多色性、吸收性不明显,但边缘和贝克线很清楚,折射率大于胆汁折射率;在正交偏光镜下,呈现出特殊的干涉图样,载物台在３６０度转动过程中呈现出四次消光现象,光性符号是单轴正性的;干涉色级是一级灰白,双折射率△ｎ＝０．０１１－０．０１５。     

Structure of the female reproductive tract of the apple snail. II. Scanning electron microscopy

The albumen gland of Pomacea paludosa, a prosobranch gastropod, contains two main ducts. The albumen gland duct consists of a single layer of secretory and non-secretory cells. The surface of the non-secretory cells is covered with cilia. Microvilli are associated with the luminal edges of the secretory cells. Globules of secretory products appear at the cell surfaces. The capsule gland duct coils through the albumen gland and is composed of two opposing faces each of two layers of cells. The upper layer consists of ciliated non-secretory cells and the microvilli covered necks of the goblet-shaped secretory cells. The bases of the secretory cells comprises the lower layer of cells. Differences in the arrangement of cellular processes and number exists between the duct epithelia.