Lanthanum permeability of tight junctions along the collecting duct of the rat

Lanthanum permeability of tight junctions along the collecting duct of the rat. The permeability of the tight junctions (zonulae occludentes) was evaluated along the entire length of the collecting duct of the rat using a lanthanum tracer technique. Nine rats with hereditary hypothalamic diabetes insipidus were studied using standard micropuncture and clearance techniques. Glomerular filtration rate (GFR) estimated from inulin clearance, urine and plasma osmolality (U/Posm) and urine flow rate (V) were determined in eight of nine animals. During either sustained diuresis (five animals) or vasopressin-induced antidiuresis (four animals), individual surface convolutions of distal convoluted tubules or early cortical collecting ducts were preserved for ultrastructural examination by intraluminal microperfusion with a glutaraldehyde-formaldehyde fixative followed by a second microperfusion with a lanthanum tracer. Mean GFR during diuresis was 6.31 ± SE 0.63 ml/min/kg of body wt and V = 797 ± SE 108 µl/min/kg or 13.6 ± SE 2.2% of the filtered load of water. After administration of exogenous vasopressin, V fell to 311 ± 157 µl/min/kg or 5.2 ± SE 3.8% of the filtered load of water and U/Posm rose from 0.658 ± SE 0.043 to 2.124 ± 0.454. Tight junctions of cortical and outer medullary segments of the collecting duct resisted lanthanum penetration. Tight junctions of the inner medullary and papillary segments of the collecting duct were freely permeable to lanthanum suggesting the presence of a paracellular shunt pathway for solute and water movement. The results were independent of the presence or absence of vasopressin. Physiological studies have previously demonstrated that cortical and outer medullary segments of the collecting duct have a low urea permeability while inner medullary and papillary segments of the collecting duct have a relatively high urea permeability. The possibility is suggested that urea movement across the inner medullary and papillary segments of the collecting duct may occur, at least in part, via a paracellular pathway formed by the nonoccluding tight junction and the lateral intercellular space.Perméabilité au lanthane des jonctions serrées le long du canal collecteur du rat. La perméabilité des jonctions serrées (zonulae occludentes) a été evaluée tout au long du canal collecteur du rat, au moyen d'une technique utilisant le lanthane comme traceur. Neuf rats atteints de diabète insipide hypothalamique héréditaire ont été étudiés au moyen des techniques habituelles de microponction et de clearance. La filtration glomérulaire (GFR) mesurée par la clearance de l'inuline, le rapport des osmolarités urine/plasma (U/Posm) et le débit urinaire (V) ont été obtenus chez huit des neuf animaux. Au cours de la diurèse entretenue (cinq animaux) ou de l'antidiurèse induite par la vasopressine (quatre animaux) des convolutions superficielles de tubes contournés distaux ou des canaux collecteurs précoces ont été préservés aux fins d'étude ultrastructurale par la microperfusion intraluminale d'un fixateur glutaraldéhyde-formaldéhyde suivie d'une deuxième microperfusion de lanthane. Le GFR moyen au cours de la diurèse était de 6,31 ± SE 0,63 ml/min/kg poids corporel et V = 797 ± SE 108 µl/min/kg ou 13,6 ± SE 2,2% de la charge d'eau filtrée. Après l'administration de vasopressine, V a diminué à 311 ± 157 µl/min/kg ou 5,2 ± SE 3,8 % de la charge d'eau filtrée et U/Posm est passé de 0,658 ± SE 0,043 à 2,124 ± 0,454. Les jonctions serrées des segments corticaux et médullaire externe des canaux collecteurs ont résisté à la pénétration de lanthane. Les jonctions serrées des segments médullaire interne et papillaire des canaux collecteurs ont été librement perméables au lanthane ce qui suggère une voie de shunt paracellulaire pour les mouvements d'ezu et de substances dissoutes. Les résultats sont indépendants de la présence ou de l'absence de vasopressine. Les études physiologiques antérieures ont montré que les segments corticaux et médullaires externes des canaux collecteurs ont une perméabilité faible pour l'urée, cependant que les segments médullaires internes et papillaires ont une perméabilité élevée. Il est suggéré que des mouvements d'urée à travers les segments médullaires internes et papillaires des canaux collecteurs puissent avoir lieu, au moins en partie, par une voie paracellulaire formée par les jonctions serrées non occlusives et l'espace intercellulaire latéral

Regulation of manganese superoxide dismutase in glomerular epithelial cells: Mechanisms for interleukin 1 induction

Regulation of manganese superoxide dismutase in glomerular epithelial cells: Mechanisms for interleukin 1 induction. Reactive oxygen species have been implicated as mediators of tissue injury in glomerular inflammation. The expression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD), was examined in primary cultures of rat glomerularepithelial cells (GEC) in response to inflammatory mediators. The results demonstrate that GEC respond to interleukin-1 (IL-1) and bacterial lipopolysaccharride (LPS) with an increase in MnSOD steady-state mRNA levels. The IL-1α-mediated induction of MnSOD mRNA levels was both time- and dose-dependent. Maximal levels, approximately 40-fold above controls, were observed at 12 hours with 2 ng/ml of IL-1α. MnSOD protein levels were also markedly elevated by IL-1α. The induction of MnSOD mRNA by IL-1α required de novo transcription as well as some degree of protein synthesis. To elucidate the potential intracellular signal that mediates IL-1α-dependent MnSOD expression, three classical signaling pathways were examined. We found no evidence that MnSOD induction by IL-lα is mediated by either the cyclooxygenase or lipoxygenase pathway or via activation of protein kinase C. Based on the presence of IL-lα in several forms of glomerular inflammation, the observed increase in MnSOD expression by this immunoregulatory cytokine must have an important role in the antioxidant defense of glomerular epithelial cells

Structural and functional response of toad urinary bladder to LiCl

AbstractStructural and functional response of toad urinary bladder to LiCl. The physiological and morphological response of toad urinary bladder was examined during mucosal exposure of LiCl both with and without vasopressin (VP). With 20 or 100 mU/ml of VP in the serosal bath there was a decrease in Jv between the first and second VP stimulation in LiCl-treated bladders (VP20, -14 ± 6%; VP100, -16 ± 5%) that was not different from that observed without LiCl (VP20, -8 ± 3%, P = NS). However, with 1 mU/ml of VP, a significant decrease in Jv was evident in LiCl-treated (-30 ± 10%) versus control sacs (+6 ± 8%; P < 0.02). At all VP concentrations tested, a significant decrease in SCC and PD was observed between the first stimulation without LiCl and the second stimulation with LiCl. Both osmotic (Pf) and diffusional water permeability (Pd) were increased significantly with 11 mM LiCl only, while neither basal nor VP-stimulated urea permeability (Pu) was affected. Morphological changes paralleled the physiological alterations induced by LiCl. These data demonstrate that LiCl interferes with the osmotic response of the toad bladder to low concentrations of VP, and increases both Pf and Pd while leaving Pu unaffected. These findings coupled with the cell swelling and intracellular vacuolization suggest the presence of a defect in transepithelial water movement somewhere beyond the apical membrane of the granular cell exposed to LiCl.Réponse structurelle et fonctionnelle de la vessie de crapaud au LiCl. La réponse physiologique et morphologique de la vessie de crapaud a été examinée pendant exposition de la muqueuse à du LiCl en présence ou en l'absence de vasopressine (VP). Pour 20 ou 100 mU/ml de VP dans le bain séreux, il y avait une diminution de Jv entre la première et la seconde stimulation par VP dans les vessies traitées par le LiCl (VP20, -14 ± 6%; VP100, -16 ± 5%), qui n'étaient pas différentes de celles observées sans LiCl (VP20, -8 ± 3%; P = NS). Cependant, avec 1 mU/ml de VP, une diminution significative de Jv était évidente dans les sacs traités au LiCl (-30 ± 10%) par rapport aux sacs contrôles (+6 ± 8%; P < 0,02). Pour toutes les concentrations de VP testées, une diminution significative du SCC et de PD a été observée entre la première stimulation sans LiCl, et la seconde stimulation avec LiCl. Les perméabilités osmotiques (Pf) et diffusionnelles (Pd) à l'eau étaient augmentées significativement avec 11 mM de LiCl seulement tandis que la perméabilité à l'urée basale ou stimulée par la VP (Pu) n'était pas affectée. Des modifications morphologiques allaient de pair avec les altérations physiologiques induites par le LiCl. Ces données démontrent que LiCl interfère avec la réponse osmotique de la vessie de crapaud pour de faibles concentrations de VP, augmente Pf et Pd, mais laisse Pu inchangé. Ces résultats, couplés avec le gonflement cellulaire et la vacuolisation intracellulaire suggèrent la présence d'un défaut du mouvement transépithélial d'eau quelque part au delà de la membrane apicale de la cellule granulaire exposée au LiCl

Effect of low molecular weight proteins and dextran on renal cathepsin B and L activity

Effect of low molecular weight proteins and dextran on renal cathepsin B and L activity. Renal extraction of low molecular weight proteins (LMWP) accounts for 30% to 80% of their total metabolic clearance. Extraction includes glomerular filtration, proximal tubular uptake, and intralysosomal proteolysis. To characterize the anatomic sites and enzymes involved in digestion of reabsorbed LMWP, the lysosomal proteases, cathepsin B and L, were measured by ultramicroassay in isolated S1, S2 and S3 segments of the proximal tubule of proteinuric rats. Increased glomerular filtration and tubular uptake of LMWP were induced by i.v. and i.p. injections of myoglobin and cationic and anionic lysozyme. Both cationic lysozyme and myoglobin increased cathepsin B and L activities in the proximal tubule, while anionic lysozyme had no effect. Morphologic examination of kidney tissue suggested that proximal tubular uptake of anionic lysozyme was negligible in comparison with the cationic form. Hence, only LMWP absorbed by the proximal tubule cells stimulated cathepsin B and L activities. Proximal tubular uptake of cationic lysozyme was determined by measurement of lysozyme activities in S1, S2, and S3. S1 segments contained the highest lysozyme activity, while S2 and S3 had much lower activities, and cathepsin B and L activity following cationic lysozyme injection was stimulated only in S1 segments. These results suggest that cathepsin B and L participate in lysosomal digestion of certain LMWP. Furthermore, the activities of cathepsin B and L adapt to increased uptake of LMWP. To gain additional insight into the mechanism of cathepsin adaptation, the cathepsin B and L activities were measured following injection of dextran with a similar low molecular weight. Dextran uptake in proximal tubules was confirmed by morphologic examination of kidney tissue. Dextran increased cathepsin B and L activities in the proximal tubule. Hence, increased endocytic activity of proximal tubule cells or increased lysosomal load of macromolecules or both rather than direct protein-enzyme interaction seem to be involved in cathepsin stimulation

Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies