ABSTRACT
The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/ non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl-/HCO3- exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.
Subject(s)
Animals , Male , Anion Exchange Protein 1, Erythrocyte/metabolism , Callithrix/metabolism , Gene Expression Profiling/veterinary , Gene Expression Regulation , Immunohistochemistry/veterinary , Kidney Tubules/cytology , Kidney Tubules, Collecting/cytology , Microscopy, Electron, Transmission/veterinaryABSTRACT
Phospholipase D (PLD) is an enzyme involved in signal transduction and widely distributed in mammalian cells. The signal transduction pathways and role for phospholipid metabolism during hormonal response in cortical collecting duct remain partly undefined. It has been reported that dexamethasone increases transepithelial transport in M-1 cells that are derived from the mouse cortical collecting duct. We investigated the expression and activity of PLD in M-1 cells. Basal PLD activity of M-1 cells cultured in the presence of dexamethasone (5 microM) was higher than in the absence of dexamethasone. Dexamethasone and ATP activated PLD in M-1 cells but phorbol ester did not stimulate PLD activity. Vasopressin, bradykinin, dibutyryl cyclic AMP, and ionomycin were ineffective in activating PLD of the cells. The PLD2 isotype was detected by immunoprecipitation but PLD1 was not detected in M-1 cells. Addition of GTPgammaS and ADP-ribosylation factor or phosphatidylinositiol 4,5-bisphosphate to digitonin-permeabilized cells did not augment PLD activity. In intact cells PLD activity was increased by sodium oleate but there was no significant change between dexamethasone treated- and untreated cells by oleate. These results suggest that at least two types of PLD are present in M-1 cells and PLD plays a role in the corticosteroid-mediated response of cortical collecting duct cells.
Subject(s)
Mice , Animals , Biological Transport/drug effects , Dexamethasone/pharmacology , Dose-Response Relationship, Drug , Drug Interactions , Glycerophospholipids/analysis , Isoenzymes/drug effects , Kidney Cortex/cytology , Kidney Tubules, Collecting/drug effects , Kidney Tubules, Collecting/cytology , Mice, Transgenic , Oleic Acid/pharmacology , Phospholipase D/drug effectsABSTRACT
The luminal membrane of collecting duct cells, specially the intercalated cells, is normally exposed to active kallikrein. This is due to the specific localization of renal kallikrein in the connecting tubule cells. We have previously reported inhibition of distal bicarbonate secretion by renal kallikrein. The present study was performed to evaluate the participation of basolateral Cl-/HCO3- exchanger and luminal H(+)-ATPase activity of cortical collecting duct segments (CCD) in the mechanism involved in the inhibition of bicarbonate secretion induced by the enzyme. The effect of orthograde injections of 1 microgram/ml (250 U/6.3 mg) pig pancreatic kallikrein, in the absence and presence of 1 mM DIDS (stilbene-disulfonic acid) in the renal tubule system, was evaluated. Urine fractions were collected after two-minutes stop-flow. Changes in the urine fraction (Fr) related to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/Fr:Ff polyfructosan) increased significantly in the first 120 microliters urine fraction collected after glandular 1 microgram/ml kallikrein, P < 0.05, (first stop-flow) and after glandular 1 microgram/ml kallikrein plus 1 mM. DIDS P < 0.05 (second stop flow). Bicarbonate secretion rate (Fr:Ff HCO3-/Fr:Ff polyfructosan) of collecting ducts was significantly reduced in the first 120 microliters urine fraction collected, related to control, during the first and second stop-flow periods. No difference was shown in bicarbonate excretion between the first 120 microliters urine fractions collected after administration of glandular kallikrein and glandular kallikrein plus DIDS. To measure H(+)-ATPase activity, rat microdissected cortical collector tubules (CCD) were incubated in the presence of increasing glandular kallikrein doses (A: 93, B: 187 and C: 375 mU/200 microL) in the presence of ouabain (4 microM) and omeprazole (100 microM) to inhibit Na(+)-K(+)-ATPase and H(+)-K(+)-ATPase, respectively. In CCD, bafilomycin-sensitive H(+)-ATPase activity (pmol/mm/min) after increasing kallikrein doses did not differ significantly from control...
Subject(s)
Animals , Female , Rats , Antiporters , Proton-Translocating ATPases/metabolism , Bicarbonates , Biological Transport , Kallikreins/pharmacology , Chloride-Bicarbonate Antiporters , Coagulants , Rats, Inbred WKY , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/enzymologyABSTRACT
El riñon juega un papel central en el mantenimiento del volumen y la composición del líquido corporal. En condiciones fisiológicas, la ingesta de sal y agua, día con día, es más o menos constante, por lo que la excreción urinaria de sodio es determinante en el mantenimiento del líquido corporal. esta función la lleva a cabo el riñon gracias a la integración de la ultrafiltración, en el glomérulo, y de la reabsorción tubular a lo largo de la nefrona. El control fino de la excreción urinaria de sodio se lleva a cabo gracias a la reabsorción tubular en la nefrona distal. En este sitio, con base en el requerimiento de iones (CI- y K+) y la sensibilidad a diuréticos, tres diferentes mecanismos de transporte de sodio se han identificado en la membrana apical: 1) los cotransportadores de Na+:K+:2CI- y Na+:CI- sensibles a derivados del ácido sulfamoílbenzoico (furosemide o bumetanida) en el asa ascendente de Henle; 2) el contrasportador de Na+:CI- sensible a la benzotiadiazina (o tiazidas) en el tíbulo distal; y 3) los canales de Na+ sensibles a amilorida en el túbulo colector. La inhibición de esta proteínas con diuréticos aumenta la excreción urinaria de Na+. Gracias a técnicas avanzadas en biología molecular, como la clonación por expresión funcional en ovocitos de Xenopus laevis, se ha identificado actualmente DNAc que codifica para miembros de las familias de estos receptores. En este trabajo se presenta una revisión sobre los avances en el conocimiento de la estructura primaria y de la relación entre la estructura y función de los receptores para diuréticos. Se discuten también las posibles consecuencias que estos hallazgos tendrán para el entendimiento en la fisiología y la fisiopatología del manejo renal del sodio