RESUMEN

The expression patterns of plasma membrane transporters that specify the epithelial cell type are acquired with ontogeny. To study this process during metanephrogenic mesenchyme-to-epithelium transition, branching ureteric buds with their adjacent mesenchymal blastema (mouse embryonic day E14) were dissected and explanted on a collagen matrix. In culture, induced mesenchymal cells condensed, aggregated, and converted to the comma- and S-shaped body. During in vitro condensation and aggregation, transcription factor Pax-2 protein was downregulated while the epithelial markers E-cadherin and beta-catenin proteins were upregulated. In addition, Wilms' tumor suppressor protein WT-1 was detectable upon condensation and downregulated in the S stage, where expression persisted in the long arm of the S. Patch-clamp, whole cell conductance (G, in nS/10 pF) of pre-epithelial condensed mesenchymal cells (n = 7) was compared with that of tubular proximal S-shaped-body epithelium (n = 6). Both stages expressed E-cadherin and WT-1 mRNA, as demonstrated by single-cell RT-PCR, testifying further to the epithelial as well as the nephrogenic commitment of the recorded cells. Mesenchymal cells exhibited whole cell currents (G = 6.7 +/- 1.3) with reversal potentials (V(rev), in mV) near equilibrium potential for Cl(-) (E(Cl)) (V(rev) = -40 +/- 7) suggestive of a high fractional Cl(-) conductance. Currents of the S-shaped-body cells (G = 4.0 +/- 1.1), in sharp contrast, had a V(rev) at E(K) (V(rev) = -82 +/- 6) indicating a high fractional K(+) conductance. Further, analysis of K(+)-selective whole cell tail currents and single-channel recording revealed a change in K(+) channel expression. Also, Kir6.1 K(+) channel mRNA and protein were downregulated between both stages, whereas K(v)LQT K(+) channel mRNA was abundant throughout. In conclusion, metanephrogenic mesenchyme-to-epithelium transition is accompanied by a profound reorganization of plasma membrane ion channel conductance.

Asunto(s)

Células Epiteliales/citología , Regulación del Desarrollo de la Expresión Génica , Canales Iónicos/metabolismo , Riñón/embriología , Riñón/metabolismo , Mesodermo/citología , Canales de Potasio de Rectificación Interna , Canales de Potasio con Entrada de Voltaje , Transactivadores , Animales , Cadherinas/genética , Cadherinas/metabolismo , Agregación Celular , Diferenciación Celular , Membrana Celular/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Conductividad Eléctrica , Células Epiteliales/metabolismo , Inmunohistoquímica , Canales Iónicos/genética , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Riñón/citología , Mesodermo/metabolismo , Ratones , Factor de Transcripción PAX2 , Potasio/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Cloruro de Sodio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas WT1 , beta Catenina

RESUMEN

Embryonic metanephroi, differentiating into the adult kidney, have come to be a generally accepted model system for organogenesis. Nephrogenesis implies a highly controlled series of morphogenetic and differentiation events that starts with reciprocal inductive interactions between two different primordial tissues and leads, in one of two mainstream processes, to the formation of mesenchymal condensations and aggregates. These go through the intricate process of mesenchyme-to-epithelium transition by which epithelial cell polarization is initiated, and they continue to differentiate into the highly specialized epithelial cell populations of the nephron. Each step along the developmental metanephrogenic pathway is initiated and organized by signaling molecules that are locally secreted polypeptides encoded by different gene families and regulated by transcription factors. Nephrogenesis proceeds from the deep to the outer cortex, and it is directed by a second, entirely different developmental process, the ductal branching of the ureteric bud-derived collecting tubule. Both systems, the nephrogenic (mesenchymal) and the ductogenic (ureteric), undergo a repeat series of inductive signaling that serves to organize the architecture and differentiated cell functions in a cascade of developmental gene programs. The aim of this review is to present a coherent picture of principles and mechanisms in embryonic renal epithelia.

Asunto(s)

Riñón/embriología , Urotelio/embriología , Animales , Diferenciación Celular , Polaridad Celular , Embrión de Mamíferos , Humanos , Riñón/citología , Mesodermo/citología , Mesodermo/fisiología , Morfogénesis , Urotelio/citología

RESUMEN

Bradykinin (BK)-stimulated colonic Cl- secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic free [Ca2+] ([Ca2+]i), membrane conductance and transepithelial ion transport. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+]i as assessed by fura-2 ratio imaging. In cell-attached, patch-clamp recordings, BK transiently activated low-conductance K channels. These channels were activated/inactivated reversibly in inside-out patches by switching [Ca2+]i in the bath between 30 nM and 100 nM. Excised channels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10+/-1 pS and 34+/-4 pS (n=10) at positive and negative voltages, respectively, and a 15-fold lower permeability for Na+ than for K+. The mean open probability of these channels did not depend on voltage but increased with increasing [Ca2+]i with an apparent concentration for a half-maximal response (EC50) of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transcriptase-polymerase chain reaction technique showed hSK4 messenger ribonucleic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 cells showed a similar dependence on [Ca2+]i. Whole cell conductance (in nS/10pF) increased from 0.5+/-0. 1 (n=6) at 10 nM [Ca2+]i in the pipette solution to 1.5+/-0.2 (n=7) at 100 nM, and to 2.0+/-0.5 (n=7) at 1 microM due to activation of a K+ conductance. In Ussing-chambered T84 monolayers grown on filters, BK did not evoke a short-circuit current (Isc). When, however, the monolayers were pre-stimulated by forskolin (1 microM), BK further enhanced Cl-secretion (DeltaIsc=21+/-5 microA/cm2, n=10) transiently and biphasically. In conclusion, BK enhances cyclic adenosine monophosphate-stimulated Cl- secretion in T84 cells, probably via basolateral, Ca2+-liganded activation of low-conductance hSK4-type K+ channels.

Asunto(s)

Adenocarcinoma/metabolismo , Bradiquinina/farmacología , Cloruros/metabolismo , Neoplasias del Colon/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Señalización del Calcio/fisiología , Fura-2 , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

The NaCl-reabsorbing collecting duct epithelium develops by budding and branching of the embryonic ureter. The expression of Na+ channels during this branching morphogenesis was studied in the outermost branches of rat ureteric buds (UB; embryonic day E15 to postnatal day P6) and in cortical collecting ducts (CCD; days P7-P28) in primary monolayer culture. Expression of both Na+ channel mRNA and of Na+-selective membrane conductance were estimated by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and by patch-clamp recording, respectively. UB and CCD uniformly represented a principal-like cell type in culture. Messenger RNA encoding the alpha-ENaC subunit was detected in oligo-dT primed cDNA (5 ng) of embryonic UB cells (E15-17) after 30 PCR cycles. The abundance of alpha-ENaC mRNA, when normalized by reference to beta-actin, was higher by a factor of 2 in postnatal (P1-6) UB and by a factor of 5 in CCD cells (P7-14) compared with the embryonic stage. Highly Na+-selective, low-conductance channels were identified in apical patches from both UB and CCD monolayers, but only CCD cells exhibited macroscopic, amiloride-sensitive Na+ currents in whole-cell patch-clamp recordings. We conclude that alpha-ENaC mRNA and functional Na+ channel protein are expressed already before morphogenesis of the CCD is completed and prior to the onset of epithelial NaCl reabsorption.

Asunto(s)

Expresión Génica , Corteza Renal/embriología , Túbulos Renales Colectores/embriología , Canales de Sodio/genética , Amilorida/farmacología , Animales , Diferenciación Celular , Conductividad Eléctrica , Epitelio/embriología , Epitelio/metabolismo , Edad Gestacional , Corteza Renal/metabolismo , Túbulos Renales Colectores/metabolismo , Microscopía Electrónica de Rastreo , Morfogénesis , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Uréter/embriología , Uréter/metabolismo

RESUMEN

Developmental expression of ion channels possibly participating in regulatory volume decrease was studied in rat embryonic (day E17) and perinatal (days P1-6) ureteric bud and in postnatal (P9-14) cortical collecting duct cells in primary monolayer culture. In isotonic bath solution, whole cell conductance (in nS/10 pF) was highest in E17 (4.0 +/- 0.5, n = 31) compared with P1-6 (2.0 +/- 0.1, n = 16) and P9-14 (1.3 +/- 0.2, n = 12) due to a decreasing contribution of a DIDS-sensitive Cl conductance, from E17 (2.8 +/- 0. 7, n = 12) to P1-6 (0.53 +/- 0.07, n = 9) and P9-14 (0.05 +/- 0.1, n = 7). Cl conductance in E17 exhibited a permselectivity of I approximately Cl approximately Br >> gluconate, and it activated time dependently. Hypotonic bath solution induced a large increase of whole cell conductance in P1-6 and in P9-14 but not in E17 (by 20. 0 +/- 3.7, 21.5 +/- 5.5, and 4.9 +/- 1.7; n = 11, 12, and 25, respectively) due to the activation of a time-dependently inactivating Cl conductance with a permselectivity of I >/= Br > Cl >> gluconate. In conclusion, the expression of Cl channels, as studied in vitro, appears to shift from an apparently constitutively active embryonic to a hypotonic swelling-activated type during late embryonic development of the collecting duct.

Asunto(s)

Envejecimiento/fisiología , Canales de Cloruro/fisiología , Desarrollo Embrionario y Fetal/fisiología , Células Epiteliales/fisiología , Túbulos Renales Colectores/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Animales Recién Nacidos , Aniones/metabolismo , Canales de Cloruro/efectos de los fármacos , Cloruros/farmacología , Ácido Egtácico/farmacología , Conductividad Eléctrica , Células Epiteliales/ultraestructura , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Soluciones Hipotónicas , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/crecimiento & desarrollo , Potenciales de la Membrana , Microscopía Electrónica de Rastreo , Concentración Osmolar , Ratas , Uréter/embriología , Uréter/crecimiento & desarrollo , Uréter/fisiología

RESUMEN

Ontogeny of apical-basolateral epithelial cell polarity was studied by a comparison of embryonic ureteric bud (UB) and mature cortical collecting duct (CCD) primary cultures. Patch-clamp techniques were applied to identify apical ion channels and to measure specific conductance (in nS/10 pF) of the apical membrane (ga) and of the whole cell (Gw). In UB, Gw (1.3 +/- 0.17; n = 32) and ga (1.4 +/- 0.15; n = 9) were not different, whereas they differed greatly in CCD (Gw: 0.79 +/- 0.07, n = 35; ga: 2.3 +/- 0.38, n = 9). In UB, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (100 microM) similarly reduced ga and Gw by 0.66 +/- 0.21 (n = 9) and 0.48 +/- 0.07 (n = 7), respectively, whereas DIDS had no effect in CCD. Amiloride (1 mM) did not alter UB conductance, whereas, in CCD, ga was more reduced than Gw (0.75 +/- 0.18, n = 8, vs. 0.22 +/- 0.10, n = 7). In addition to this Na(+)-selective conductance, a nonselective cation (NSC) conductance was enriched sixfold in the apical membrane of CCD, possibly due to apical 9 +/- 1-pS (n = 7) NSC channels, whereas UB apical NSC conductance was low. Apical 16 +/- 2-pS (n = 6) K+ channels were identified in CCD but not in UB. The comparison of both developmental states suggests a differentiation of the apical membrane during UB to CCD ontogeny.

Asunto(s)

Animales Recién Nacidos/crecimiento & desarrollo , Desarrollo Embrionario y Fetal , Canales Iónicos/metabolismo , Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Aldosterona/farmacología , Amilorida/farmacología , Animales , Calcio/metabolismo , Membrana Celular/fisiología , Células Cultivadas , Colforsina/farmacología , Citosol/metabolismo , Conductividad Eléctrica , Embrión de Mamíferos/fisiología , Embrión de Mamíferos/ultraestructura , Canales Iónicos/fisiología , Túbulos Renales Colectores/crecimiento & desarrollo , Microscopía Electrónica , Técnicas de Placa-Clamp , Ratas/embriología

RESUMEN

Urinary osmotic concentration capacity during renal ontogeny is subject to changes of medullary cytoarchitecture and of segmental epithelial transport characteristics. Osmotic equilibrium between interstitial and tubular fluid of the terminal nephron segment in response to vasopressin is an absolute essential of maximal urinary osmotic concentration. The regulation of osmotic water permeability (Pf) in this terminal epithelial segment during ontogenetic differentiation has not been documented. The inner medullary collecting duct (IMCD), the terminal 40% of total segmental length, was dissected at two stages of postnatal ontogenetic differentiation from immature (days 7-15) and from mature (days 33-37) rat kidneys and perfused in vitro. Pf (micron/s) was measured (bath hyperosmotic) in the absence and presence of arginine vasopressin (AVP, 230 pM). Basal Pf was 32.3 +/- 4.03 (n = 26) in the immature IMCD (IMCDi) and 111.5 +/- 20.6 (n = 15) in the mature segment (IMCDm). AVP increased Pf in IMCDi from 46.4 +/- 10.5 to 102 +/- 25.7 micron/s, whereas in IMCDm the AVP-dependent change of Pf was from 104.2 +/- 41.2 to 693 +/- 176 micron/s. AVP (2,300 pM) did not further increase Pf in IMCDi. Forskolin (50 microM) changed Pf in IMCDi from 34.9 +/- 6.3 to 104.1 +/- 16 micron/s; the corresponding change in IMCDm was from 150 +/- 32 to 985.8 +/- 133 micron/s. An analogue of adenosine 3',5'-cyclic monophosphate (cAMP; 10(-3) M) increased Pf in IMCDi from 35.5 +/- 11.4 to 138.5 +/- 32.6 and in IMCDm from 79.6 +/- 32.3 to 702.2 +/- 283 micron/s.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Túbulos Renales Colectores/metabolismo , Agua/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Arginina Vasopresina/farmacología , Femenino , Médula Renal , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/crecimiento & desarrollo , Masculino , Ósmosis , Permeabilidad , Ratas , Ratas Endogámicas

RESUMEN

Specific binding of corticosteroids in cultured CCT cells was measured as a function of time, of temperature, of pH, and of concentration. Scatchard analysis revealed the existence of two species of binding sites for both, 3H-aldosterone type I A: KD = 2.3.10(-9) M, N = 33.10(-17) mol/10(4) cells; type I B: KD = 51.10(-9) M, N = 55.10(-17) mol/10(4) cells) and dexamethasone (type II A: KD = 4.7.10(-9) M, N = 2.3.10(17) mol/10(4) cells; type II B: KD = 22.10(-9) M, N = 6.5.10(-17) mol/10(4) cells). The data demonstrate that CCT cells in primary monolayer culture express corticosteroid binding sites similar to cells of the CCT in vivo.

Asunto(s)

Aldosterona/metabolismo , Dexametasona/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Concentración de Iones de Hidrógeno , Cinética , Conejos , Temperatura

Asunto(s)

Túbulos Renales/citología , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Diferenciación Celular , División Celular , Células Cultivadas , Técnicas de Cultivo/métodos , Disección/métodos , Células Epiteliales , Epitelio/metabolismo , Túbulos Renales/anatomía & histología , Túbulos Renales/metabolismo , Túbulos Renales Proximales/citología , Asa de la Nefrona/citología , Técnica de Dilución de Radioisótopos , Sodio/metabolismo , Radioisótopos de Sodio , Intercambiadores de Sodio-Hidrógeno

RESUMEN

Primary monolayer cultures of polarized epithelia were derived from single cortical collecting tubules (CCT) of the mammalian kidney (rabbit). The expression of Na-K-ATPase activity (mol 10(-15)/min.cell) and of the transepithelial voltage (VT) in monolayers on membranes was measured in response to defined hormonal supplements of the nephron culture medium (NCM). Hormones were: triiodothyronine (T3; 10(-11) M), dexamethasone (10(-8) M), and aldosterone (10(-9) M). Control was NCM plus fetal calf serum (FCS; 3% v/v). Data are mean (SEM). Na-K-ATPase activity was 10.4 (6.2) in control (n = 27); 18.8 (5.2) in T3 (n = 35); 21.2 (5.5) in dexamethasone (n = 26), and 30.9 (6.5) in aldosterone (n = 24). VT (mV) was 4.2 (3.2) in control (n = 12); 12.7 (3.7) in T3 (n = 12); 16.8 (2.5) in dexamethasone (n = 12), and 28.4 (3.6) in aldosterone (n = 14). Data are evidence that thyroid and corticosteroid hormones selectively induce the postmitotic expression of the sodium carrier activity and other functions related to vectorial solute transport in this polarized epithelium.

Asunto(s)

Hormonas/fisiología , Corteza Renal/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , Sodio/metabolismo , Aldosterona/fisiología , Animales , Transporte Biológico Activo , Células Cultivadas , Dexametasona/fisiología , Epitelio/metabolismo , Potenciales de la Membrana , Conejos , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Triyodotironina/fisiología

RESUMEN

The ontogenetic differentiation of transepithelial chloride transport was evaluated in the cortical collecting tubule of the rabbit kidney. Tubules from four control groups (I-IV) were studied during in vitro perfusion. I: body weight 150-280 g; II: 330-480 g; III: 530-880 g; IV: 980-1610 g. In each group, aldosterone (100 micrograms/100 g body weight/day) was given subcutaneously in three doses daily, for 6 days (IA-IVA). Transepithelial net chloride flux (pmol cm2 s1) increased by a factor of almost 3 from group I to group IV (p less than 0.01). Aldosterone induces net chloride flux by 103% (P = 0.03) in IA and by 78% (P = 0.01) in IIA; changes in groups III (21%) and IV (27%) were small. Therefore, the mineralocorticoid induces transepithelial chloride transport in cortical collecting tubule during early transport differentiation. The inducing action decreases with natural differentiation. Moreover, aldosterone alone suffices to induce the complete expression of transepithelial chloride transport in the cortical collecting tubule.

Asunto(s)

Aldosterona/farmacología , Cloruros/metabolismo , Corteza Renal/crecimiento & desarrollo , Túbulos Renales/crecimiento & desarrollo , Envejecimiento , Animales , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Cinética , Ouabaína/farmacología , Perfusión , Conejos , Valores de Referencia

RESUMEN

The effect of storage media, which are routinely used in replantation, upon the proliferative capacity of periodontal ligament fibroblasts, was compared with the effect of a tissue culture medium. The periodontal tissue was obtained from mandibular central incisors of White New Zealand rabbits. The experiments were performed in fibroblasts derived during second subculture. The storage media were physiologic salt solution, Ringer's solution and Rivanol; the tissue culture medium was alpha-minimum essential medium without nucleosides. The incubation period was 1 hour. [3H]-thymidine incorporation and cell counts were taken to indicate changes in the proliferative capacity of the fibroblasts. The tissue culture experiments showed that the proliferative ability of the periodontal ligament fibroblasts was dependent upon the composition of the storage medium. Physiologic salt solution, Ringer's solution and Rivanol were unable to maintain the metabolism of the fibroblasts. alpha-MEM medium, however, was capable of stimulating proliferation of the periodontal ligament fibroblasts.

Asunto(s)

Medios de Cultivo/farmacología , Ligamento Periodontal/citología , Animales , Recuento de Células , División Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ligamento Periodontal/metabolismo , Conejos , Timidina/metabolismo , Tritio

RESUMEN

The study tool of cultured tubule epithelia has been applied to new areas in nephron cell biology, such as the evolution of epithelial membrane asymmetry. Studies utilizing monoclonal antibodies against plasma membrane glycoproteins in MDCK revealed that the development of surface cell polarity is a continuous process requiring intact tight junctions and their electrical resistor function [101]. The role of the junctional complex to establish and maintain distinct membrane protein domains had been suggested earlier from work utilizing the apical aminopeptidase [102] and fluorescent membrane probes [103]. Cultured tubule epithelia lend themselves for the evaluation of cell-specific membrane protein synthesis [104] and antigenic determinants [105]. Human renal epithelia, from normal [106, 107] and defined abnormal kidney [108], have been maintained functional in primary and passage culture [106]. Pathophysiological mechanisms may be examined in cultured tubule epithelia, as shown first [109] by studies on the recovery from ischemic failure, where anoxia and substrate deprivation resulted in cell swelling which was prevented in culture by an oncotic agent. This article has not attempted to give an exhaustive account of the studies in which cultured tubule cells have served as a tool. Instead, the investigations quoted herein represent some principal lines of study, as seen from renal physiology, which may disclose details in culture of complex in vivo phenomena. It was Bernard [110] who, in 1865, suggested that "physiological events must be isolated outside the organism . . . to better understand the deepest associations of the phenomena."

Asunto(s)

Túbulos Renales/metabolismo , Aldosterona/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Epinefrina/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Glucosa/metabolismo , Hormonas/farmacología , Insulina/farmacología , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Fosfatos/metabolismo , Sodio/metabolismo , Vasopresinas/farmacología

RESUMEN

Protein content per cell in epithelial monolayers derived from cortical collecting tubules (CCT) of the rabbit kidney decreases with increasing cell density during growth in culture. When expressed per 500 cells in culture (this number of cells is present per mm of segmental length in vivo, a total protein concentration of 149 +/- 18 ng was measured. In the in vivo CCT nephron segment, total protein concentration was 172 +/- 16 ng per mm of segmental length.

Asunto(s)

Corteza Renal/citología , Túbulos Renales/citología , Proteínas/análisis , Animales , División Celular , Células Cultivadas , Células Epiteliales , Femenino , Cinética , Masculino , Conejos

RESUMEN

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.

Asunto(s)

Adenilil Ciclasas/metabolismo , Hormonas/farmacología , Nefronas/enzimología , Animales , Arginina Vasopresina/farmacología , Calcitonina/farmacología , Técnicas de Cultivo , Epitelio/enzimología , Histocitoquímica , Isoproterenol/farmacología , Hormona Paratiroidea/farmacología , Conejos

RESUMEN

Cultured nephron epithelia offer an advantage as they are available in a completely controlled monolayer environment. Some of the culture systems retain differentiated properties for a time span sufficient to study long term chemical and physical events in epithelia of defined nephronal origin. The characteristics expressed in culture include transepithelial voltage and resistance in cells derived from distal nephron segments, and the hormonal dependence of these parameters. Na-K-ATPase activity and its selective regulation by steroid and thyroid hormones has been evaluated in defined epithelial monolayers. Further, the acute stimulation of adenylate cyclase activity has been demonstrated in human nephron culture, and the Na-coupled glucose transport is present in cortical cell cultures. Transepithelial voltage and Na-K-ATPase activity may be considered complex and highly differentiated expressions of function as they are indicative of several apical and basal transporter systems in cultured polarized nephron cells. The intention of current work is to establish functional lines from defined segments of the nephron, using strategies of passage, cloning, and transfection.

Asunto(s)

Epitelio/fisiología , Nefronas/fisiología , Animales , Adhesión Celular , Ciclo Celular , Diferenciación Celular , División Celular , Células Cultivadas , Medios de Cultivo , Técnicas de Cultivo/instrumentación , Técnicas de Cultivo/métodos , Células Epiteliales , Cinética , Nefronas/citología , Fenotipo

RESUMEN

The renal medullary countercurrent system differentiates into its final segmental nephron function and geometry during perinatal development. The influence of these changes on the medullary longitudinal osmotic gradient cannot be evaluated by experimental studies. Therefore, a computation analysis using a differential equation model of the renal countercurrent system was applied to quantitate the effect of medullary architecture and solute transport on the concentration profiles for salt and urea in tubules (loop of Henle and collecting duct) and in the central core along the entire medulla during ontogeny. The results indicate that both the changing distribution of loop segments within the medulla and the increase in active salt transport of the individual thick ascending loop determine the magnitude and slope of the axial medullary solute gradients.

Asunto(s)

Médula Renal/crecimiento & desarrollo , Ósmosis , Animales , Animales Recién Nacidos/fisiología , Transporte Biológico , Médula Renal/anatomía & histología , Médula Renal/metabolismo , Modelos Biológicos , Ratas , Cloruro de Sodio/metabolismo

RESUMEN

Forskolin is a unique diterpene that may directly activate the catalytic subunit of adenylate cyclase. We therefore examined the effect of 50 microM forskolin on osmotic water permeability in rabbit cortical collecting tubules perfused in vitro. Forskolin increased net volume flux (Jv, from 0.30 to 1.22 nl/mm/min, P less than 0.02) in all tubules. The hydro-osmotic effect of forskolin was similar with respect to magnitude and time course to that produced by a maximal dose (250 microU/ml) of arginine vasopressin. An additive effect on Jv and Lp was not observed when maximal concentrations of forskolin and arginine vasopressin were given simultaneously. The compound d(CH2)5Tyr(Et) VAVP, which noncompetitively inhibits the vasopressin receptor, significantly reduced collecting tubular hydro-osmotic response to arginine vasopressin. In contrast, the hydro-osmotic response to forskolin was maintained in the presence of d(CH2)5 Tyr(Et)VAVP. However, the hydro-osmotic response to forskolin could be inhibited by 1.0 microM guanine 5'-(beta,gamma-imido) triphosphate (GppNHp) and by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). These results demonstrate that forskolin exerts an hydro-osmotic effect in the mammalian nephron which occurs independent of the vasopressin receptor. Guanine nucleotide regulatory proteins may modulate the osmotic water permeability effect of forskolin. Finally, calmodulin is required for full expression of the effect of forskolin to increase osmotic water flux.

Asunto(s)

Diterpenos/farmacología , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales/efectos de los fármacos , Agua/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Colforsina , Diterpenos/antagonistas & inhibidores , Guanilil Imidodifosfato/farmacología , Túbulos Renales Colectores/enzimología , Túbulos Renales Colectores/metabolismo , Ósmosis , Perfusión , Permeabilidad , Conejos , Sulfonamidas/farmacología

RESUMEN

Microenzymatic methods have been utilized in the past to quantify the activity of Na-K-ATPase in tubular segments of the mammalian nephron. The assay reported here measures the precipitated inorganic phosphate liberated by the hydrolysis of gamma-32P-ATP. Activity data in single nephron segments of the cortical collecting tubule (CCT) confirm previous work; specifically, first data on enzyme activity in small cultured cell populations derived from CCT demonstrate that the Na-carrier enzyme can be quantified in nephron cell cultures.

Asunto(s)

Túbulos Renales Colectores/enzimología , Túbulos Renales/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Amoníaco/farmacología , Animales , Células Cultivadas , Nefronas/enzimología , Conejos

RESUMEN

The study of distribution and quantitation of a fluorescent probe in living epithelia with the aid of an inverted microscope requires that individual cells can be analysed without optical interference from adjacent cells. This report describes the application of fluorescence microscopy and fluorometry to a recently developed in vitro culture system of renal epithelial cells. Epithelial cells derived from the mammalian renal cortical collecting tubule (CT) and the thick ascending loop of Henle (TAL) are cultivated as continuous monolayers in serum-free, hormone-supplemented media. A specific mitochondrial marker (DASPMI) is added to the medium and incorporated into the cytoplasm. The microscopic image reveals that the mitochondrial fluorescence distribution differs between CT and TAL cultures. The fluorometric quantitation shows a normally distributed histogram of medium-range intensity in TAL cell cultures while CT cultures exhibit a two-peak pattern of mitochondrial fluorescence distribution among epithelial cells.

Asunto(s)

Técnicas Histológicas , Riñón/citología , Microscopía Fluorescente/métodos , Compuestos de Piridinio , Animales , Células Cultivadas , Células Epiteliales , Mitocondrias/ultraestructura , Espectrometría de Fluorescencia