RESUMEN

INTRODUCTION: The aim of this study was to determine whether mobile phones and mobile phone locating devices are associated with improved ambulance response times in central London. PATIENTS AND METHODS: All calls from the London Ambulance Service database since 1999 were analysed. In addition, 100 consecutive patients completed a questionnaire on mobile phone use whilst attending the St Thomas's Hospital Emergency Department in central London. RESULTS: Mobile phone use for emergencies in central London has increased from 4007 (5% of total) calls in January 1999 to 21,585 (29%) in August 2004. Ambulance response times for mobile phone calls were reduced after the introduction of the mobile phone locating system (mean 469 s versus 444 s; P = 0.0195). The proportion of mobile phone calls made from mobile phones for life-threatening emergencies was higher after injury than for medical emergencies (41% versus 16%, P = 0.0063). Of patients transported to the accident and emergency department by ambulance, 44% contacted the ambulance service by mobile phone. Three-quarters of calls made from outside the home or work-place were by mobile phone and 72% of patients indicated that it would have taken longer to contact the emergency services if they had not used a mobile. CONCLUSIONS: Since the introduction of the mobile phone locating system, there has been an improvement in ambulance response times. Mobile locating systems in urban areas across the UK may lead to faster response times and, potentially, improved patient outcomes.

Asunto(s)

Teléfono Celular/estadística & datos numéricos , Sistemas de Comunicación entre Servicios de Urgencia/estadística & datos numéricos , Ambulancias , Servicios Médicos de Urgencia , Humanos , Londres , Factores de Tiempo , Estudios de Tiempo y Movimiento

RESUMEN

OBJECTIVES: To review the presentation, treatment and response of those runners from the London Marathon who presented to St Thomas' Hospital with exercise induced hyponatraemia. DESIGN: Observational case series. SETTING: St Thomas' Hospital, a tertiary hospital situated near the finish line of the 2003 London Marathon. PARTICIPANTS: All runners who presented to St Thomas' Hospital on the day of the 2003 London Marathon with altered mental state whose serum sodium concentration was less than 135 mmol/L. MAIN OUTCOME MEASURES: Presenting symptoms, volume and type of fluids administered and response to treatment (biochemical and clinical). RESULTS: Fourteen patients were diagnosed with exercise associated hyponatraemia with serum sodium concentrations ranging from 116 to 133 mmol/L. Eleven presented with confusion. There were long delays between the finish time and presentation time for some runners. Anecdotal descriptions suggested some runners finished the race with normal mental state then became confused. There was no correlation between running time and serum sodium level. All patients received 0.9% saline and six received 1.8% saline. Despite this, some patients demonstrated falls in serum sodium concentrations. Thirteen to fourteen patients were symptomatically well the following morning, with the remaining patient significantly improved. CONCLUSION: Presentation of exercise associated hyponatraemia may be delayed. Optimal treatment is controversial, but the use of isotonic saline may not result in rises of serum sodium and we would suggest the early use of hypertonic fluids in symptomatic patients.

Asunto(s)

Hiponatremia/etiología , Carrera , Adulto , Confusión/etiología , Ingestión de Líquidos , Femenino , Humanos , Hiponatremia/diagnóstico , Hiponatremia/terapia , Masculino , Persona de Mediana Edad , Solución Salina Hipertónica/uso terapéutico

RESUMEN

OBJECTIVES: To assess whether initial patient consult by senior clinicians reduces numbers of patients waiting to be seen as an indirect measure of waiting time throughout the emergency department (ED). METHODS: An emergency medicine consultant and a senior ED nurse (G or F grade), known as the IMPACT team, staffed the triage area for four periods of four hours per week, Monday to Friday between 9 am to 5 pm for three months between December 2001 and February 2002 when staffing levels permitted. Patients normally triaged by a nurse in this area instead had an early consultation with the IMPACT team. Data were collected prospectively on all patients seen by the IMPACT team. The number of patients waiting to be seen (for triage, in majors and in minors) was assessed every two hours during the IMPACT sessions and at corresponding times when no IMPACT team was operational. RESULTS: There was an overall reduction in the number of patients waiting to be seen in the department from 18.3 to 5.5 (p<0.0001) at formal two hourly assessments. The largest difference was seen in minors. Of the patients seen at triage by the IMPACT team, 48.9% were discharged home immediately after assessment and treatment. With the IMPACT team present, no patient waited more than four hours for initial clinical consult. CONCLUSIONS: By using a senior clinical team for initial patient consultation, the numbers of patients waiting fell dramatically throughout the ED. Many patients can be effectively treated and discharged after initial consult by the IMPACT team.

Asunto(s)

Medicina de Emergencia , Enfermería de Urgencia , Servicio de Urgencia en Hospital/estadística & datos numéricos , Cuerpo Médico de Hospitales/organización & administración , Grupo de Atención al Paciente/organización & administración , Triaje/organización & administración , Citas y Horarios , Medicina de Emergencia/organización & administración , Enfermería de Urgencia/organización & administración , Servicio de Urgencia en Hospital/organización & administración , Investigación sobre Servicios de Salud , Hospitales de Enseñanza/organización & administración , Humanos , Londres , Evaluación de Procesos y Resultados en Atención de Salud , Satisfacción del Paciente , Admisión y Programación de Personal , Estudios Prospectivos , Derivación y Consulta , Estudios de Tiempo y Movimiento

Asunto(s)

Nitrito de Amila/envenenamiento , Inhibidores Enzimáticos/uso terapéutico , Metahemoglobinemia/inducido químicamente , Metahemoglobinemia/tratamiento farmacológico , Azul de Metileno/uso terapéutico , Trastornos Relacionados con Sustancias/complicaciones , Adulto , Femenino , Humanos , Masculino , Trastornos Relacionados con Sustancias/diagnóstico

RESUMEN

BACKGROUND: Over the last decade there has been a rapid increase in the number of visitors landing at wildlife sites on the Antarctic continent, and concern has been raised that tourists may transmit important pathogens to or between wildlife colonies. The aim of this study was to determine if tourist activities pose a potential threat to Antarctic wildlife, or possibly to human populations through carriage of pathogens on boots. METHODS: In two trips conducted to Antarctica in the summer season of 2000/2001, swabs were collected from tourist boots: prior to landing, to determine baseline level of bacterial flora on the boots (A isolates); immediately on return to the ship, to quantify the level of contamination (B isolates); and after the boots were washed in seawater to determine the recovery of the organisms after cleaning (C isolates). Swabs were cultured for coliforms, and isolates identified using the API system. RESULTS: Twenty organisms resembling coliforms were isolated from 15 of 72 pairs of boots. Two isolates were recovered from group A, 4 from group B, and 14 from group C. Of these 20 isolates, 11 could be identified using the API identification method. The remaining 9 isolates all produced an unknown but identical profile number. CONCLUSION: These results indicate that current practices for cleaning the boots of tourists visiting Antarctic wildlife colonies may not be sufficient to prevent the transmission of pathogens, and indicate that further studies are needed to define the best method of disinfection.

Asunto(s)

Bacterias/aislamiento & purificación , Control de Enfermedades Transmisibles/métodos , Zapatos , Viaje , Regiones Antárticas , Desinfección , Humanos , Pruebas de Sensibilidad Microbiana

RESUMEN

UT-A1 is an extremely hydrophobic 929-amino acid integral membrane protein, expressed in the renal inner medullary collecting duct, with a central role in the urinary concentrating mechanism. Previous immunoblotting studies in rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa monomeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medullary urea concentration. The present studies were carried out using protein chemistry techniques to determine the origin of the two forms. Peptide-directed polyclonal antibodies targeted to five sites along the polypeptide sequence from the NH2 to the COOH terminus labeled both forms, thus failing to demonstrate a significant deletion in the primary amino acid chain. The 97- and 117-kDa monomeric forms were both reduced to 88 kDa by deglycosylation with N-glycosidase F, indicating that a single polypeptide chain is glycosylated to two different extents. Studies using nonionic detergents for membrane solubilization or using homobifunctional cross-linkers demonstrated that UT-A1 exists as a 206-kDa protein complex in native kidney membranes. The mobility of this complex was also increased by deglycosylation. Both the 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunoprecipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein and that the two monomeric forms (97 and 117 kDa) in inner medullary collecting duct are the consequence of different states of glycosylation.

Asunto(s)

Proteínas Portadoras/análisis , Túbulos Renales Colectores/metabolismo , Glicoproteínas de Membrana/análisis , Proteínas de Transporte de Membrana , Animales , Anticuerpos/inmunología , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Membrana Celular/metabolismo , Reactivos de Enlaces Cruzados , Electroforesis , Epítopos/inmunología , Glicosilación , Hexosaminidasas/farmacología , Immunoblotting , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Pruebas de Precipitina , Isoformas de Proteínas/química , Ratas , Ratas Sprague-Dawley , Vasopresinas/farmacología , Transportadores de Urea

RESUMEN

OBJECTIVES: To determine if there were differences in practice or intubation mishap rate between anaesthetists and accident and emergency physicians performing rapid sequence induction of anaesthesia (RSI) in the prehospital setting. METHODS: All patients who underwent RSI by a Helicopter Emergency Medical Service (HEMS) doctor from 1 May 1997 to 30 April 1999 were studied by retrospective analysis of in-flight run sheets. Intubation mishaps were classified as repeat attempts at intubation, repeat drug administration and failed intubation. RESULTS: RSI was performed on 359 patients by 10 anaesthetists (202 patients) and nine emergency physicians (157 patients). Emergency physicians recorded a larger number of patients as having Cormack and Lehane grade 3 or 4 laryngoscopy than anaesthetists (p<0.0001) but were less likely to use a gum elastic bougie to assist intubation (p=0.024). Patients treated by emergency physicians did not have a significantly different pulse, blood pressure, oxygen saturation or end tidal CO2 to patients treated by anaesthetists at any time after intubation. Emergency physicians were more likely to anaesthetise patients with a Glasgow Coma Score >12 than anaesthetists (p=0.003). There were two failed intubations (1%) in the anaesthetist group and four (2.5%) in the emergency physician group. Repeat attempts at intubation and repeat drug administration occurred in <2% of each group. CONCLUSIONS: RSI performed by emergency physicians was not associated with a significantly higher failure rate or an increased number of intubation mishaps than RSI performed by anaesthetists. Emergency physicians were able to safely administer sedative and neuromuscular blocking drugs in the prehospital situation. It is suggested that emergency physicians can safely perform rapid sequence induction of anaesthesia and intubation.

Asunto(s)

Ambulancias Aéreas , Anestesia General , Servicios Médicos de Urgencia , Intubación Intratraqueal , Traumatismo Múltiple/terapia , Adulto , Anestesiología , Competencia Clínica , Medicina de Emergencia , Femenino , Humanos , Masculino , Grupo de Atención al Paciente , Estudios Retrospectivos , Seguridad , Insuficiencia del Tratamiento

RESUMEN

UT-A3 has recently been identified as a splicing variant transcript of the UT-A gene present in the kidney. To study the cellular and subcellular localization of UT-A3, we raised a new polyclonal antibody to its COOH-terminal end. Immunoblots identified bands at 44 and 67 kDa predominately in the inner medulla and showed that the antibody does not recognize UT-A1. Deglycosylation with PNGase decreased the molecular mass of both forms to 40 kDa. UT-A3 is most abundant in the inner third of the inner medulla and is present in membrane fractions. Cell fractionation studies showed that UT-A3 is only detectable in inner medullary collecting duct (IMCD) cells. These observations were confirmed with immunolocalization studies demonstrating an exclusive labeling of IMCD cells. Double-labeling studies with anti-Na-K-ATPase demonstrated UT-A3 in intracellular membranes and in the apical region but were incompatible with a basolateral site for UT-A3. Although the function of this isoform in the inner medulla is unknown, the large abundance suggests that it may be important in the renal handling of urea.

Asunto(s)

Proteínas Portadoras/metabolismo , Túbulos Renales Colectores/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Animales , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Transportadores de Urea

RESUMEN

The Na+-H+ exchanger NHE3 and the thiazide-sensitive Na+-Cl- cotransporter NCC are the major apical sodium transporters in the proximal convoluted tubule and the distal convoluted tubule of the kidney, respectively. We investigated the mechanism of compensation that allows maintenance of sodium balance in NHE3 knockout mice and in NCC knockout mice. We used a so-called 'targeted proteomics' approach, which profiles the entire renal tubule with regard to changes in Na+ transporter and aquaporin abundance in response to the gene deletions. Specific antibodies to the Na+ transporters and aquaporins expressed along the nephron were utilized to determine the relative abundance of each transporter. Semiquantitative immunoblotting was used which gives an estimate of the percentage change in abundance of each transporter in knockout compared with wild-type mice. In NHE3 knockout mice three changes were identified which could compensate for the loss of NHE3-mediated sodium absorption. (a) The proximal sodium-phosphate cotransporter NaPi-2 was markedly upregulated. (b) In the collecting duct, the 70 kDa form of the y-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. This is thought to be an aldosterone-stimulated form of y-ENaC. (c) Glomerular filtration was significantly reduced. In the NCC knockout mice, amongst all the sodium transporters expressed along the renal tubule, only the 70 kDa form of the y-subunit of the epithelial sodium channel, ENaC, exhibited an increase in abundance. In conclusion, both mouse knockout models demonstrated successful compensation for loss of the deleted transporter. More extensive adaptation occurred in the case of the NHE3 knockout, presumably because NHE3 is responsible for much more sodium absorption in normal mice than in NCC knockout mice.

Asunto(s)

Proteínas Portadoras/fisiología , Túbulos Renales/fisiología , Intercambiadores de Sodio-Hidrógeno/fisiología , Simportadores , Animales , Bicarbonatos/sangre , Presión Sanguínea , Proteínas Portadoras/genética , Cloruros/sangre , Canales Epiteliales de Sodio , Tasa de Filtración Glomerular , Frecuencia Cardíaca , Homocigoto , Ratones , Ratones Noqueados , Potasio/sangre , Proteoma , Sodio/sangre , Canales de Sodio/fisiología , Simportadores del Cloruro de Sodio , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética

Asunto(s)

Enfermeras Practicantes , Visita a Consultorio Médico , Satisfacción del Paciente , Humanos , Proyectos de Investigación , Administración del Tiempo

RESUMEN

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (alpha, beta, and gamma) in rat kidney. Seven-day infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of beta- and gamma-ENaC (to 238% and 288% of vehicle, respectively), whereas alpha-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of beta- and gamma- but no significant change in alpha-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

Asunto(s)

Desamino Arginina Vasopresina/farmacología , Riñón/efectos de los fármacos , Canales de Sodio/metabolismo , Simportadores , Animales , Acuaporina 2 , Acuaporina 6 , Acuaporinas/metabolismo , Benzotiadiazinas , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Desamino Arginina Vasopresina/administración & dosificación , Diuréticos , Canales Epiteliales de Sodio , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Immunoblotting , Transporte Iónico/efectos de los fármacos , Riñón/metabolismo , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Masculino , Conejos , Ratas , Ratas Endogámicas , Receptores de Droga/antagonistas & inhibidores , Receptores de Droga/metabolismo , Sodio/metabolismo , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Simportadores del Cloruro de Sodio , Miembro 3 de la Familia de Transportadores de Soluto 12 , Regulación hacia Arriba/efectos de los fármacos , Privación de Agua

RESUMEN

The renal urea transporter gene (UT-A) produces different transcripts in the inner medullary collecting ducts (UT-A1) and thin descending limbs of Henle's loop (UT-A2), coding for distinct proteins. Peptide-directed rabbit polyclonal antibodies were used to identify the UT-A2 protein in renal medulla of mouse and rat. In the inner stripe of outer medulla, an antibody directed to the COOH terminus of UT-A recognized a membrane protein of 55 kDa. The abundance of this 55-kDa protein was strongly increased in response to chronic infusion of the vasopressin analog 1-deamino-[8-D-arginine]vasopressin (DDAVP) in Brattleboro rats, consistent with previous evidence that UT-A2 mRNA abundance is markedly increased. Immunofluorescence labeling with the COOH-terminal antibody in Brattleboro rats revealed labeling in the lower portion of descending limbs from short-looped nephrons (in the aquaporin-1-negative portion of this segment). This UT-A labeling was increased in response to DDAVP. Increased labeling was also seen in descending limbs of long-looped nephrons in the base of the inner medulla. These results indicate that UT-A2 is expressed as a 55-kDa protein in portions of the thin descending limbs of Henle's loop and that the abundance of this protein is strongly upregulated by vasopressin.

Asunto(s)

Proteínas Portadoras/metabolismo , Túbulos Renales Proximales/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Vasopresinas/fisiología , Animales , Anticuerpos/inmunología , Acuaporina 1 , Acuaporinas/metabolismo , Proteínas Portadoras/inmunología , Desamino Arginina Vasopresina/farmacología , Técnica del Anticuerpo Fluorescente , Immunoblotting , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Isoformas de Proteínas/inmunología , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba , Transportadores de Urea

RESUMEN

In the renal inner medullary collecting duct (IMCD), vasopressin regulates two key transporters, namely aquaporin-2 (AQP2) and the vasopressin-regulated urea transporter (VRUT). Both are present in intracellular vesicles as well as the apical plasma membrane. Short-term regulation of AQP2 has been demonstrated to occur by vasopressin-induced trafficking of AQP2-containing vesicles to the apical plasma membrane. Here, we have carried out studies to determine whether short-term regulation of VRUT occurs by a similar process. Cell surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed that vasopressin causes a dose-dependent increase in the amount of AQP2 labeled at the cell surface, whereas VRUT labeled at the cell surface did not increase in response to vasopressin. Immunoperoxidase labeling of inner medullary thin sections from Brattleboro rats treated with 1-desamino-8-D-arginine vasopressin (DDAVP) for 20 min revealed dramatic translocation of AQP2 to the apical region of the cell, with no change in the cellular distribution of VRUT. In addition, differential centrifugation of inner medullary homogenates from Brattleboro rats treated with DDAVP for 60 min revealed a marked depletion of AQP2 from the low-density membrane fraction (enriched in intracellular vesicles) but did not alter the quantity of VRUT in this fraction. Finally, AQP2-containing vesicles immunoisolated from a low-density membrane fraction from renal inner medulla did not contain immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but not of VRUT, depends on regulated vesicular trafficking to the plasma membrane.

Asunto(s)

Acuaporinas/metabolismo , Proteínas Portadoras/metabolismo , Túbulos Renales Colectores/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Vasopresinas/fisiología , Animales , Acuaporina 2 , Acuaporina 6 , Biotina/metabolismo , Membrana Celular/metabolismo , Centrifugación , Desamino Arginina Vasopresina/farmacología , Inmunohistoquímica , Túbulos Renales Colectores/citología , Proteínas de la Membrana/metabolismo , Ratas , Ratas Brattleboro , Fármacos Renales/farmacología , Distribución Tisular/fisiología , Transportadores de Urea

RESUMEN

Vesicle targeting proteins ("SNAREs") have been proposed to direct vasopressin-induced trafficking of aquaporin-2 water channels in kidney collecting ducts. A newly identified SNARE protein, SNAP-23, is proposed to mediate vesicle targeting to the plasma membrane in diverse tissues. The current studies were done to determine whether SNAP-23 is expressed in collecting ducts with an intracellular distribution compatible with a role in aquaporin-2 trafficking. RT-PCR demonstrated SNAP-23 mRNA in microdissected collecting ducts and other tubular segments including the proximal tubule and thick ascending limb. Immunoblotting using a polyclonal antibody raised against a COOH-terminal peptide revealed a solitary band at an apparent molecular mass of 30 kDa in renal medullary membrane fractions and inner medullary collecting duct suspensions. Differential centrifugation revealed that SNAP-23 is present in membrane fractions including the low-density fraction enriched in intracellular vesicles. Immunocytochemistry revealed SNAP-23 labeling at both the apex and the cytoplasm of collecting duct principal cells. Immunoblotting of intracellular vesicles immunoisolated using an aquaporin-2 antibody revealed the presence of both SNAP-23 and synaptobrevin-2 (VAMP-2) in aquaporin-2-bearing vesicles. We conclude that SNAP-23 is strongly expressed in collecting duct principal cells, consistent with a role in vasopressin-regulated trafficking of aquaporin-2. However, localization of SNAP-23 in both intracytoplasmic vesicles and plasma membranes suggests a function different from that originally proposed for SNAP-25 in synaptic vesicle targeting.

Asunto(s)

Acuaporinas/metabolismo , Proteínas Portadoras/metabolismo , Túbulos Renales Colectores/metabolismo , Animales , Acuaporina 2 , Acuaporina 6 , Masculino , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley

RESUMEN

To test the hypothesis that the abundance of the apical urea transporter of the inner medullary collecting duct (IMCD) is regulated in vivo by factors associated with altered water balance, immunoblots of rat inner medullary membrane fractions were probed with rabbit polyclonal antibodies against the renal urea transporter (RUT) gene product. In inner medullas of Brattleboro rats, which manifest severe chronic water diuresis, a 117-kD band was seen, in addition to the previously described 97-kD band. These two bands were detectable by antibodies directed against two different regions of the RUT sequence. When Brattleboro rats were treated with a 5-d infusion of arginine vasopressin (AVP) by osmotic minipump, the 117-kD band was markedly diminished, whereas the 97-kD band was unchanged. Simultaneous infusion of the diuretic agent furosemide prevented the AVP-induced decrease in the 117-kD band. In AVP-infused Sprague Dawley rats, the 117-kD band was barely perceptible. However, when AVP-treated rats were infused with furosemide for 5 d, the 117-kD band was markedly accentuated, whereas the 97-kD band was unchanged. The abundance of the 117-kD RUT protein in the renal papilla was inversely correlated with dietary protein intake. Further immunoblotting studies revealed that the 117-kD protein is heavily expressed in IMCD cells and not in non-collecting duct components of the inner medulla, and is present in low-density microsome fractions from inner medulla. From this study, the following conclusions can be made: (1) The collecting duct urea transporter is present in at least two forms (97 and 117 kD) in the IMCD. (2) The expression level of the 117-kD urea transporter protein is regulated and is inversely correlated with medullary osmolality and urea concentration, but does not correlate with circulating AVP level. (3) Although AVP regulates RUT function on a short-term basis, long-term changes in AVP levels do not increase RUT abundance.

Asunto(s)

Proteínas Portadoras/metabolismo , Riñón/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Animales , Arginina Vasopresina/farmacología , Agua Corporal/metabolismo , Centrifugación , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/farmacología , Diuréticos/farmacología , Furosemida/farmacología , Riñón/efectos de los fármacos , Médula Renal/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Masculino , Conejos , Ratas , Ratas Brattleboro , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Transportadores de Urea

RESUMEN

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 +/- 3.4 vs. 4.1 +/- 0.3 mmHg, and reduced plasma sodium concentrations (142.2 +/- 1. 6 vs. 149.1 +/- 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 +/- 53%, n = 12) relative to sham-operated controls (100 +/- 13%, n = 14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective. Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 +/- 28% of sham levels, n = 6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF.

Asunto(s)

Acuaporinas , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Canales Iónicos/biosíntesis , Túbulos Renales Colectores/metabolismo , Animales , Acuaporina 2 , Acuaporina 6 , Presión Sanguínea , Expresión Génica , Corazón/fisiología , Corazón/fisiopatología , Insuficiencia Cardíaca/patología , Inmunohistoquímica , Riñón/fisiología , Riñón/fisiopatología , Túbulos Renales Colectores/patología , Masculino , Tamaño de los Órganos , Ratas , Ratas Wistar , Valores de Referencia , Sodio/sangre , Función Ventricular Izquierda

RESUMEN

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called "UT-1") responsible for facilitated urea transport across the apical plasma membrane of inner medullary collecting duct (IMCD) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.

Asunto(s)

Proteínas Portadoras/metabolismo , Túbulos Renales Colectores/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Urea/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos , Transporte Biológico , Proteínas Portadoras/efectos de los fármacos , Membrana Celular/metabolismo , Dieta con Restricción de Proteínas , Disección , Ensayo de Inmunoadsorción Enzimática/métodos , Médula Renal/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Concentración Osmolar , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Orina/química , Transportadores de Urea

RESUMEN

The aquaporins are molecular water channels that mediate transcellular water transport across water-permeable epithelia. To investigate the cause of the concentrating defect in the nephrotic syndrome, immunoblotting using membrane fractions from inner medulla was utilized to assess the level of expression of four aquaporin water channels in vehicle-treated versus puromycin aminonucleoside (PAN)-treated rats. Scanning electron microscopy demonstrating loss of glomerular foot processes and measurements of urinary protein excretion confirmed the efficacy of the PAN treatment. In rats receiving PAN, there was an increase in plasma vasopressin, without a change in plasma sodium concentration. Inner medullary tissue hypertonicity was sustained in PAN-treated rats while the urinary osmolality was low, pointing to defective osmotic equilibration across the collecting ducts in PAN-nephrosis. Among collecting duct aquaporins, there was an 87% decrease in aquaporin-2 expression and a 70% decrease in aquaporin-3 expression in the inner medulla, whereas aquaporin-4 expression was unaltered. Transmission electron microscopy of the inner medullary collecting ducts of PAN-treated rats showed normal-appearing cells. Thus, PAN-nephrosis is associated with an extensive downregulation of collecting duct water channel expression despite increased circulating vasopressin, providing an explanation for the concentrating defect associated with the nephrotic syndrome.

Asunto(s)

Antimetabolitos Antineoplásicos/toxicidad , Acuaporinas , Canales Iónicos/metabolismo , Médula Renal/metabolismo , Síndrome Nefrótico/metabolismo , Puromicina Aminonucleósido/toxicidad , Animales , Antimetabolitos Antineoplásicos/administración & dosificación , Acuaporina 2 , Acuaporina 6 , Densitometría , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Immunoblotting , Capacidad de Concentración Renal , Médula Renal/efectos de los fármacos , Médula Renal/ultraestructura , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/ultraestructura , Masculino , Microscopía Electrónica de Transmisión de Rastreo , Síndrome Nefrótico/inducido químicamente , Síndrome Nefrótico/patología , Puromicina Aminonucleósido/administración & dosificación , Ratas , Ratas Sprague-Dawley

RESUMEN

Two bumetanide-sensitive ion cotransporters that carry Na+, K+, and Cl- in a coupled fashion have been identified. One type, the "absorptive" isoform, carries these ions across the apical plasma membrane of the thick ascending limb of Henle's loop. Another isoform, the "secretory" cotransporter, has been identified in a number of epithelial tissues by physiological means, but its sites of expression in the kidney have not been fully characterized. Complementary DNA believed to code for the secretory isoform (called "BSC2" or "NKCC1") have recently been cloned. This study used a specific affinity-purified antipeptide antibody to this protein for immunolocalization in the rat kidney. Immunoblot studies using this antibody show abundant immunoreactivity against bands of 140-190 and 120 kd in the parotid gland, colon, and stomach, sites where the secretory form of the cotransporter has been identified by physiological techniques. This distribution supports the hypothesis that this isoform represents the secretory form of the cotransporter. Studies in the kidney revealed that the same bands are associated with membrane fractions chiefly in the outer medulla. Immunolocalizations show that immunoreactivity is selectively and intensely localized to the basolateral plasma membrane of a subfraction of outer medullary collecting duct cells. An independently produced monoclonal antibody (T4) specific for Na-K-Cl cotransporter displays the same localization. Dual localizations of cotransporter antibody with respect to antibody specific for principal cells (aquaporin-2) and intercalated cells (band 3 and H(+)-ATPase) show that cotransporter immunoreactivity is localized to alpha-intercalated cells of the outer medullary collecting duct in the rat. This distinctive localization suggests that the secretory form of the cotransporter may play a role in renal NH4+ and/or acid secretion by this cell type.

Asunto(s)

Proteínas Portadoras/metabolismo , Riñón/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Membrana Celular/metabolismo , Cloruros/metabolismo , Immunoblotting , Inmunohistoquímica , Transporte Iónico , Riñón/citología , Corteza Renal/citología , Corteza Renal/metabolismo , Médula Renal/citología , Médula Renal/metabolismo , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Datos de Secuencia Molecular , Potasio/metabolismo , Ratas , Sodio/metabolismo , Simportadores de Cloruro de Sodio-Potasio

RESUMEN

To investigate the role of the thick ascending limb (TAL) Na(+)-K(+)-2Cl- cotransporter in regulation of water excretion, we have prepared a peptide-derived polyclonal antibody based on the cloned cDNA sequence of the rat type 1 bumetanide-sensitive cotransporter, BSC-1 (also termed "NKCC-2"). Immunoblots revealed a single broad 161-kDa band in membrane fractions of rat renal outer medulla and cortex but not from rat colon or parotid gland. A similar protein was labeled in mouse kidney. Immunoperoxidase immunohistochemistry in rat kidney revealed labeling restricted to the medullary and cortical TAL segments. Because long-term regulation of urinary concentrating ability may depend on regulation of Na(+)-K(+)-2Cl- cotransporter abundance, we used immunoblotting to evaluate the effects of several in vivo factors on expression levels of BSC-1 protein in rat kidney outer medulla. Chronic oral saline loading with 0.16 M NaCl markedly increased BSC-1 abundance. However, long-term vasopressin infusion or thirsting of rats did not affect BSC-1 abundance. Chronic furosemide infusion caused a 9-kDa upward shift in apparent molecular mass and an apparent increase in expression level. These results support the previous identification of BSC-1 as the TAL Na(+)-K(+)-2Cl- transporter and demonstrate that the expression of this transporter is regulated.

Asunto(s)

Proteínas Portadoras/metabolismo , Riñón/metabolismo , Animales , Anticuerpos/inmunología , Arginina Vasopresina/farmacología , Proteínas Portadoras/inmunología , Furosemida/farmacología , Immunoblotting , Inmunohistoquímica , Riñón/citología , Ratones , Ratas , Ratas Brattleboro , Ratas Sprague-Dawley , Simportadores de Cloruro de Sodio-Potasio , Factores de Tiempo , Distribución Tisular