RESUMEN
The water channel aquaporin-1 (AQP1) is the principal water pathway for isotonic water reabsorption in the kidney proximal tubule (PT). We investigated flow-mediated fluid (Jv) and [Formula: see text] ([Formula: see text]) reabsorption in PTs of the mouse kidney by microperfusion in wild-type (WT) and AQP1 knockout (KO) mice. Experiments were simulated in an adaptation of a mathematical model of the rat PT. An increase in perfusion rate from 5 to 20 nL/min increased Jv and [Formula: see text] in PTs of WT mice. AQP1 KO mice significantly decreased Jv at low and high flow rates compared with control. In contrast, [Formula: see text] was not reduced at either low or high flow rates. Cell volume showed no significant difference between WT and AQP1 KO mice. Renal clearance experiments showed significantly higher urine flow in AQP1 KO mice, but there was no significant difference in either Na+ and K+ or [Formula: see text] excretion. Acid-base parameters of blood pH, Pco2, [Formula: see text], and urine pH were the same in both WT and KO mice. In model calculations, tubules whose tight junction (TJ) water permeability (Pf) was that assigned to the rat TJ, showed no difference in Jv between WT and KO, whereas TJ Pf set to 25% of the rat predicted Jv concordant with our observations from AQP1 KO. These results affirm the dominance of AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrate that flow-stimulated [Formula: see text] reabsorption is intact and independent of AQP1. With reference to the model, the findings also suggest that TJ water flux in the PT is less prominent in the mouse than in the rat kidney.NEW & NOTEWORTHY We found an absence of flow-dependent modulation of fluid absorption but no effect on either proximal tubule (PT) [Formula: see text] absorption or acid-base parameters in the aquaporin 1 (AQP1) knockout mouse. We affirmed the dominance of the water channel AQP1 in mediating isotonic water reabsorption by the mouse PT and demonstrated that flow-stimulated [Formula: see text] reabsorption is independent of AQP1. With reference to the model, the findings also suggest that tight junctional water flux in the PT is less prominent in the mouse than rat kidney.
Asunto(s)
Acuaporina 1 , Túbulos Renales Proximales , Ratones , Ratas , Animales , Acuaporina 1/genética , Acuaporina 1/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones Noqueados , Tamaño de la Célula , Agua/metabolismoRESUMEN
Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.
Asunto(s)
Síndrome de Bartter/genética , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Animales , Células Cultivadas , Eliminación de Gen , Activación del Canal Iónico , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Ratones , Fenotipo , Canales de Potasio de Rectificación Interna/genética , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Flow-modulated salt and water transport in proximal tubules has been recognized for more than four decades. Recent work has made major progress in defining the underlying cellular mechanisms. First, we demonstrated that perfusion-absorption balance is present in the isolated perfused proximal tubule of the mouse kidney, and thus is independent of neuronal control and systemic hormonal regulation. In proximal tubule, higher axial flow rates stimulate sodium and bicarbonate absorption by increased apical membrane Na+/H+-transporter and H-ATPase activity. It is also evident that fluid shear stress stimulates Na+/H+ exchanger isoform 3 (NHE3) exocytosis and trafficking to the apical membrane of the proximal tubule cells. Second, experimental data and modeling calculations provide strong evidence that brush border microvilli function as flow sensors in the proximal tubule. Flow-induced changes of proximal tubule absorption depend on the changes of torque (bending moment) on the microvilli, and that an intact actin cytoskeleton is required to transduce signals from the brush border to cell and alter transport activity, NHE3 expression and trafficking. Third, the increased NHE3 exocytosis by dopamine blockers enhanced tubule sensitivity to torque, and the IP3 receptor-mediated intracellular Ca2+ signaling is a critical step in transduction of fluid drag on microvillus drag tips in modulating Na+ and HCO3 - transport. Finally, in all of our experimental studies, flow-dependent transport in mouse tubules was achieved with virtually no change in tubule cell volume. Our model calculations suggest that this observation is strong evidence for proportional luminal and peritubular effects of flow on transporter density.
RESUMEN
BACKGROUND: Clomiphene is the current first-line infertility treatment in women with the polycystic ovary syndrome, but aromatase inhibitors, including letrozole, might result in better pregnancy outcomes. METHODS: In this double-blind, multicenter trial, we randomly assigned 750 women, in a 1:1 ratio, to receive letrozole or clomiphene for up to five treatment cycles, with visits to determine ovulation and pregnancy, followed by tracking of pregnancies. The polycystic ovary syndrome was defined according to modified Rotterdam criteria (anovulation with either hyperandrogenism or polycystic ovaries). Participants were 18 to 40 years of age, had at least one patent fallopian tube and a normal uterine cavity, and had a male partner with a sperm concentration of at least 14 million per milliliter; the women and their partners agreed to have regular intercourse with the intent of conception during the study. The primary outcome was live birth during the treatment period. RESULTS: Women who received letrozole had more cumulative live births than those who received clomiphene (103 of 374 [27.5%] vs. 72 of 376 [19.1%], P=0.007; rate ratio for live birth, 1.44; 95% confidence interval, 1.10 to 1.87) without significant differences in overall congenital anomalies, though there were four major congenital anomalies in the letrozole group versus one in the clomiphene group (P=0.65). The cumulative ovulation rate was higher with letrozole than with clomiphene (834 of 1352 treatment cycles [61.7%] vs. 688 of 1425 treatment cycles [48.3%], P<0.001). There were no significant between-group differences in pregnancy loss (49 of 154 pregnancies in the letrozole group [31.8%] and 30 of 103 pregnancies in the clomiphene group [29.1%]) or twin pregnancy (3.4% and 7.4%, respectively). Clomiphene was associated with a higher incidence of hot flushes, and letrozole was associated with higher incidences of fatigue and dizziness. Rates of other adverse events were similar in the two treatment groups. CONCLUSIONS: As compared with clomiphene, letrozole was associated with higher live-birth and ovulation rates among infertile women with the polycystic ovary syndrome. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00719186.).
Asunto(s)
Clomifeno/uso terapéutico , Fármacos para la Fertilidad Femenina/uso terapéutico , Infertilidad Femenina/tratamiento farmacológico , Nitrilos/uso terapéutico , Síndrome del Ovario Poliquístico/complicaciones , Triazoles/uso terapéutico , Adulto , Clomifeno/efectos adversos , Clomifeno/farmacología , Método Doble Ciego , Femenino , Fármacos para la Fertilidad Femenina/efectos adversos , Fármacos para la Fertilidad Femenina/farmacología , Humanos , Infertilidad Femenina/etiología , Estimación de Kaplan-Meier , Letrozol , Nacimiento Vivo , Fase Luteínica , Masculino , Nitrilos/efectos adversos , Nitrilos/farmacología , Ovulación/efectos de los fármacos , Embarazo , Calidad de Vida , Triazoles/efectos adversos , Triazoles/farmacologíaRESUMEN
OBJECTIVE: To summarize baseline characteristics from a large multicenter infertility clinical trial. DESIGN: Cross-sectional baseline data from a double-blind randomized trial of two treatment regimens (letrozole vs. clomiphene). SETTING: Academic Health Centers throughout the United States. PATIENT(S): Seven hundred fifty women with polycystic ovary syndrome (PCOS) and their male partners took part in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Historic, biometric, biochemical, and questionnaire parameters. RESULT(S): Females averaged 30 years and were obese (body mass index [BMI] 35) with â¼20% from a racial/ethnic minority. Most (87%) were hirsute and nulligravid (63%). Most of the women had an elevated antral follicle count and enlarged ovarian volume on ultrasound. Women had elevated mean circulating androgens, LH-to-FSH ratio (â¼2), and antimüllerian hormone levels (8.0 ng/mL). In addition, women had evidence for metabolic dysfunction with elevated mean fasting insulin and dyslipidemia. Increasing obesity was associated with decreased LH-to-FSH levels, antimüllerian hormone levels, and antral follicle counts but increasing cardiovascular risk factors, including prevalence of the metabolic syndrome. Men were obese (BMI 30) and had normal mean semen parameters. CONCLUSION(S): The treatment groups were well matched at baseline. Obesity exacerbates select female reproductive and most metabolic parameters. We have also established a database and sample repository that will eventually be accessible to investigators. CLINICAL TRIAL REGISTRATION NUMBER: NCT00719186.
Asunto(s)
Obesidad/diagnóstico , Obesidad/epidemiología , Síndrome del Ovario Poliquístico/diagnóstico , Síndrome del Ovario Poliquístico/epidemiología , Embarazo , Adulto , Método Doble Ciego , Femenino , Fármacos para la Fertilidad Femenina/farmacología , Fármacos para la Fertilidad Femenina/uso terapéutico , Humanos , Masculino , Obesidad/tratamiento farmacológico , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Embarazo/efectos de los fármacos , Adulto JovenRESUMEN
Underlying glomerulotubular balance (GTB) is the impact of axial flow to regulate Na(+) and HCO(3)(-) transport by modulating Na(+)-H(+) exchanger 3 (NHE3) and H-ATPase activity. It is not known whether the cascade of events following a change in flow relies on local angiotensin (ANG II) generation or receptor availability. Mouse tubules were microperfused in vitro at flows of 5 and 20 nl/min, and net fluid (J(v)) and HCO(3)(-) (J(HCO3)) absorption and cell height were measured. Na(+) (J(Na)) and Cl(-) (J(Cl)) absorption and changes in microvillous torque were estimated. Raising flow increased Na(+) and HCO(3)(-) reabsorption but did not change either Cl(-) transport or cell volume. Losartan reduced absolute Na(+) and HCO(3)(-) absorption at both low and high flows but did not affect fractional flow-stimulated transport. Compared with controls, in AT(1a) knockout (KO) mouse tubules, 53% of flow-stimulated Na(+) absorption was abolished, but flow-stimulated HCO(3)(-) absorption was retained at similar levels. The remaining flow-stimulated J(HCO3) was eliminated by the H-ATPase inhibitor bafilomycin. Inhibition of the AT(2) receptor by PD123319 increased both J(Na) and J(HCO3) but did not affect flow-mediated fractional changes. NHE3 expression at the protein level was reduced in AT(1a) KO mice kidneys. We conclude that 1) although the AT(1a) receptor is necessary for flow to impact NHE3, the effect on H(+)-ATPase is independent of AT(1a); 2) the small flow-mediated changes in cell volume suggest a coordinate flow effect on both luminal and basolateral transporters; and 3) there is no evidence of flow-dependent Cl(-) transport, and thus no evidence for convective paracellular Cl(-) transport in mouse tubules.
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Angiotensina II/fisiología , Bicarbonatos/metabolismo , Hemostasis/fisiología , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , Sodio/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Transporte Biológico/fisiología , Inhibidores Enzimáticos/farmacología , Femenino , Hemostasis/efectos de los fármacos , Técnicas In Vitro , Glomérulos Renales/efectos de los fármacos , Túbulos Renales/efectos de los fármacos , Losartán/farmacología , Macrólidos/farmacología , Ratones , Ratones Noqueados , Modelos Animales , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , Receptor de Angiotensina Tipo 1/deficiencia , Receptor de Angiotensina Tipo 1/genética , Receptor de Angiotensina Tipo 1/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure-related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure-modifying mechanism in a setting of impaired NO/cGMP signaling.
Asunto(s)
Genoma Humano , Guanilato Ciclasa/genética , Hipertensión/genética , Sitios de Carácter Cuantitativo , Receptores Citoplasmáticos y Nucleares/genética , Sistema Renina-Angiotensina/genética , Sistemas de Mensajero Secundario/genética , Vasodilatación/genética , Animales , GMP Cíclico/genética , GMP Cíclico/metabolismo , Endotelio Vascular/enzimología , Femenino , Ligamiento Genético , Guanilato Ciclasa/metabolismo , Humanos , Hipertensión/enzimología , Masculino , Ratones , Ratones Noqueados , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Renina/genética , Renina/metabolismo , Guanilil Ciclasa Soluble , Especificidad de la EspecieRESUMEN
In response to volume expansion, locally generated dopamine decreases proximal tubule reabsorption by reducing both Na/H-exchanger 3 (NHE3) and Na-K-ATPase activity. We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na(+) and HCO(3)(-) reabsorption and have suggested that this observation underlies glomerulotubular balance. In the present work, we investigate the impact of dopamine on the sensitivity of reabsorptive fluxes to changes in luminal flow. Mouse proximal tubules were microperfused in vitro at low and high flow rates, and volume and HCO(3)(-) reabsorption (J(v) and J(HCO3)) were measured, while Na(+) and Cl(-) reabsorption (J(Na) and J(Cl)) were estimated. Raising luminal flow increased J(v), J(Na), and J(HCO3) but did not change J(Cl). Luminal dopamine did not change J(v), J(Na), and J(HCO3) at low flow rates but completely abolished the increments of Na(+) absorption by flow and partially inhibited the flow-stimulated HCO(3)(-) absorption. The remaining flow-stimulated HCO(3)(-) absorption was completely abolished by bafilomycin. The DA1 receptor blocker SCH23390 and the PKA inhibitor H89 blocked the effect of exogenous dopamine and produced a two to threefold increase in the sensitivity of proximal Na(+) reabsorption to luminal flow rate. Under the variety of perfusion conditions, changes in cell volume were small and did not always parallel changes in Na(+) transport. We conclude that 1) dopamine inhibits flow-stimulated NHE3 activity by activation of the DA1 receptor via a PKA-mediated mechanism; 2) dopamine has no effect on flow-stimulated H-ATPase activity; 3) there is no evidence of flow stimulation of Cl(-) reabsorption; and 4) the impact of dopamine is a coordinated modulation of both luminal and peritubular Na(+) transporters.
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Dopamina/farmacología , Glomérulos Renales/metabolismo , Túbulos Renales/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Algoritmos , Animales , Benzazepinas/farmacología , Bicarbonatos/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Cloruros/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Femenino , Isoquinolinas/farmacología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Macrólidos/farmacología , Ratones , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , ATPasas de Translocación de Protón/antagonistas & inhibidores , Receptores de Dopamina D1/antagonistas & inhibidores , Sodio/metabolismo , Sulfonamidas/farmacología , Sulpirida/farmacologíaRESUMEN
Nogo-B is a member of the reticulon family of proteins that has been implicated in diverse forms of vascular injury. Although Nogo-B is expressed in renal tissues, its localization and function in the kidney have not been examined. Here, we report that Nogo-B is expressed specifically in the epithelial cells of the distal nephron segments in the murine kidney. After unilateral ureteral obstruction (UUO) and ischemia/reperfusion, Nogo-B gene and protein levels increased dramatically in the kidney. This increase was driven in part by injury-induced de novo expression in proximal tubules. Examination of Nogo-B immunostaining in human biopsy specimens from patients with acute tubular necrosis showed similar increases in Nogo-B in cortical tubules. Mice genetically deficient in Nogo-A/B were indistinguishable from wild-type (WT) mice based on histological appearance and serum analyses. After UUO, there was a significant delay in recruitment of macrophages to the kidney in the Nogo-A/B-deficient mice. However, measurements of fibrosis, inflammatory gene expression, and histological damage were not significantly different from WT mice. Thus, Nogo-B is highly expressed in murine kidneys in response to experimental injuries and may serve as a marker of diverse forms of renal injury in tissues from mice and humans. Furthermore, Nogo-B may regulate macrophage recruitment after UUO, although it does not greatly affect the degree of tissue injury or fibrosis in this model.
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Células Epiteliales/metabolismo , Túbulos Renales/metabolismo , Proteínas de la Mielina/genética , Animales , Movimiento Celular/genética , Células Epiteliales/patología , Células Epiteliales/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Necrosis de la Corteza Renal/genética , Necrosis de la Corteza Renal/metabolismo , Necrosis de la Corteza Renal/patología , Médula Renal/metabolismo , Médula Renal/patología , Necrosis Papilar Renal/genética , Necrosis Papilar Renal/metabolismo , Necrosis Papilar Renal/patología , Túbulos Renales/patología , Túbulos Renales/fisiología , Macrófagos/metabolismo , Macrófagos/patología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Mielina/metabolismo , Proteínas Nogo , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patologíaRESUMEN
Myosin VI (Myo6) is an actin-based molecular motor involved in clathrin-mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snell's waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid-base parameters, and plasma concentrations and urinary excretions of Na(+) and K(+). To assess the integrity of endocytosis-mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4-fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 min after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled-2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis-mediated protein absorption in the mouse kidney proximal tubule.
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Endocitosis/fisiología , Túbulos Renales Proximales/fisiología , Cadenas Pesadas de Miosina/deficiencia , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Transdiferenciación Celular , Peroxidasa de Rábano Silvestre/metabolismo , Túbulos Renales Proximales/patología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Mutantes Neurológicos , Proteínas de Microfilamentos/metabolismo , Microvellosidades/genética , Microvellosidades/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Olfactory-like chemosensory signaling occurs outside of the olfactory epithelium. We find that major components of olfaction, including olfactory receptors (ORs), olfactory-related adenylate cyclase (AC3) and the olfactory G protein (G(olf)), are expressed in the kidney. AC3 and G(olf) colocalize in renal tubules and in macula densa (MD) cells which modulate glomerular filtration rate (GFR). GFR is significantly reduced in AC3(-/-) mice, suggesting that AC3 participates in GFR regulation. Although tubuloglomerular feedback is normal in these animals, they exhibit significantly reduced plasma renin levels despite up-regulation of COX-2 expression and nNOS activity in the MD. Furthermore, at least one member of the renal repertoire of ORs is expressed in a MD cell line. Thus, key components of olfaction are expressed in the renal distal nephron and may play a sensory role in the MD to modulate both renin secretion and GFR.
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Túbulos Renales Distales/química , Riñón/química , Mucosa Olfatoria/química , Transducción de Señal , Adenilil Ciclasas/análisis , Animales , Subunidades alfa de la Proteína de Unión al GTP/análisis , Tasa de Filtración Glomerular , Riñón/fisiología , Ratones , Ratones Noqueados , Neuronas Receptoras Olfatorias , Renina/sangre , Renina/metabolismoRESUMEN
In this study, we demonstrate that fluid shear stress (FSS)-induced actin cytoskeletal reorganization and junctional formation in renal epithelial cells are nearly completely opposite the corresponding changes in vascular endothelial cells (ECs) [Thi MM et al. (2004) Proc Natl Acad Sci USA 101:16483-16488]. Mouse proximal tubule cells (PTCs) were subjected to 5 h of FSS (1 dyn/cm(2)) to investigate the dynamic responses of the cytoskeletal distribution of filamentous actin (F-actin), ZO-1, E-cadherin, vinculin, and paxillin to FSS. Immunofluorescence analysis revealed that FSS caused basal stress fiber disruption, more densely distributed peripheral actin bands (DPABs), and the formation of both tight junctions (TJs) and adherens junctions (AJs). A dramatic reinforcement of vinculin staining was found at the cell borders as well as the cell interior. These responses were abrogated by the actin-disrupting drug, cytochalasin D. To interpret these results, we propose a "junctional buttressing" model for PTCs in which FSS enables the DPABs, TJs, and AJs to become more tightly connected. In contrast, in the "bumper-car" model for ECs, all junctional connections were severely disrupted by FSS. This "junctional buttressing" model explains why a FSS of only 1/10 of that used in the EC study can cause a similarly dramatic, cytoskeletal response in these tall, cuboidal epithelial cells; and why junctional buttressing between adjacent cells may benefit renal epithelium in maximizing flow-activated, brush border-dependent, transcellular salt and water reabsorption.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Túbulos Renales Proximales/metabolismo , Uniones Estrechas/metabolismo , Absorción/efectos de los fármacos , Animales , Cadherinas/metabolismo , Células Cultivadas , Citocalasina D/farmacología , Citoesqueleto/patología , Células Epiteliales/patología , Túbulos Renales Proximales/patología , Proteínas de la Membrana/metabolismo , Ratones , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Paxillin/metabolismo , Fosfoproteínas/metabolismo , Resistencia al Corte , Estrés Mecánico , Uniones Estrechas/patología , Vinculina/metabolismo , Proteína de la Zonula Occludens-1RESUMEN
ROMK null mice with a high survival rate and varying severity of hydronephrosis provide a good model to study type II Bartter syndrome pathophysiology (26). During the development of such a colony, we found that more male than female null mice survived, 58.7% vs. 33.3%. To investigate the possible mechanism of this difference, we compared the survival rates, renal functions, degree of hydronephrosis, as well as PGE(2) and TXB(2) production between male and female ROMK wild-type and null mice. We observed that female ROMK Bartter's mice exhibited lower GFR (0.37 vs. 0.54 ml.min(-1).100 g BW(-1), P < 0.05) and higher fractional Na(+) excretion (0.66% vs. 0.48%, P < 0.05) than male Bartter's. No significant differences in acid-base parameters, urinary K(+) excretion, and plasma electrolyte concentrations were observed between sexes. In addition, we assessed the liquid retention rate in the kidney to evaluate the extent of hydronephrosis and observed that 67% of male and 90% of female ROMK null mice were hydronephrotic mice. Urinary PGE(2) excretion was higher in both sexes of ROMK null mice: 1.35 vs. 1.10 ng/24 h in males and 2.90 vs. 0.87 ng/24 h in females. TXB(2) excretion was higher in female mice in both wild-type and ROMK null mice. The increments of urinary PGE(2) and TXB(2) were significantly higher in female null mice than males, 233.33% vs. 22.74% of PGE(2) and 85.67% vs. 20.36% of TXB(2). These data demonstrate a more severe Bartter phenotype in female ROMK null mice, and higher PGE(2) and TXB(2) production may be one of the mechanisms of this manifestation.
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Síndrome de Bartter/metabolismo , Síndrome de Bartter/fisiopatología , Dinoprostona/metabolismo , Canales de Potasio de Rectificación Interna/genética , Caracteres Sexuales , Factores de Edad , Animales , Síndrome de Bartter/mortalidad , Peso Corporal , Femenino , Tasa de Filtración Glomerular , Hidronefrosis/metabolismo , Hidronefrosis/mortalidad , Hidronefrosis/fisiopatología , Masculino , Ratones , Ratones Noqueados , Ratones Mutantes , Fenotipo , Potasio/orina , Canales de Potasio de Rectificación Interna/metabolismo , Índice de Severidad de la Enfermedad , Sodio/orina , Tasa de Supervivencia , Tromboxano B2/metabolismo , OrinaRESUMEN
ROMK-deficient (Romk(-/-)) mice exhibit polyuria, natriuresis, and kaliuresis similar to individuals with type II Bartter's form of hyperprostaglandin E syndrome (HPS; antenatal Bartter's syndrome). In the present study, we utilized both metabolic and clearance studies to define the contributions of specific distal nephron segments to the renal salt wasting in these mice. The effects of furosemide, hydrochlorothiazide, and benzamil on urinary Na(+) and K(+) excretion in both wild-type (Romk(+/+)) and Romk(-/-) mice were used to assess and compare salt transport by the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-expressing thick ascending limb (TAL), the Na(+)-Cl(-) cotransporter (NCC)-expressing distal convoluted tubule (DCT1/DCT2), and the epithelial Na(+) channel (ENaC)-expressing connecting segment (CNT) and collecting duct (CD), respectively. Whole kidney glomerular filtration rate was reduced by 47% in Romk(-/-) mice. Furosemide-induced increments in the fractional excretion rate of Na(+) and K(+) and absolute excretion of Na(+) and K(+) were significantly blunted in Romk(-/-) mice, consistent with a major salt transport defect in the TAL. In contrast, hydrochlorothiazide produced an exaggerated natriuresis in Romk(-/-) mice, indicating upregulation of salt absorption by the DCT. Benzamil resulted in a similar increment in absolute Na excretion in both Romk(-/-) and Romk(+/+), indicating no significant upregulation of Na(+) transport by ENaC in ROMK null mice. Moreover, hydrochlorothiazide increased the fractional K(+) excretion rate in Romk(-/-) mice, confirming our recent observation that maxi-K channels contribute to distal K(+) secretion in the absence of ROMK.
Asunto(s)
Síndrome de Bartter/metabolismo , Cloruros/metabolismo , Diuréticos/farmacología , Hidroclorotiazida/farmacología , Canales de Potasio de Rectificación Interna/genética , Sodio/metabolismo , Adaptación Fisiológica/fisiología , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Síndrome de Bartter/genética , Síndrome de Bartter/fisiopatología , Proteínas de Transporte de Catión/metabolismo , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/metabolismo , Femenino , Furosemida/farmacología , Tasa de Filtración Glomerular/efectos de los fármacos , Tasa de Filtración Glomerular/fisiología , Asa de la Nefrona/metabolismo , Masculino , Ratones , Ratones Mutantes , Canales de Potasio de Rectificación Interna/metabolismo , Embarazo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of Na(+)-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient (Romk(-/-)) mice. Plasma aldosterone was 40% higher and urinary PGE(2) excretion was 1.5-fold higher in Romk(-/-) compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule Na(+)/H(+) exchanger (NHE3) and Na(+)-P(i) cotransporter (NaPi-IIa) and TAL-specific Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2/BSC1) in Romk(-/-) mice, while the distal convoluted tubule (DCT)-specific Na(+)-Cl(-) cotransporter (NCC/TSC) was markedly increased. The abundance of the alpha-,beta-, and gamma-subunits of the epithelial Na(+) channel (ENaC) was slightly increased, although only differences for gamma-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of Romk(-/-) mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent Na(+) absorption in the TAL is compensated predominately by upregulation of Na(+) transport in downstream DCT cells. These adaptive changes in Romk(-/-) mice may help to limit renal Na(+) loss, and thereby, contribute to survival of these mice.
Asunto(s)
Síndrome de Bartter/metabolismo , Síndrome de Bartter/fisiopatología , Proteínas Portadoras/metabolismo , Asa de la Nefrona/metabolismo , Canales de Potasio de Rectificación Interna/genética , Sodio/metabolismo , Adaptación Fisiológica/fisiología , Animales , Síndrome de Bartter/genética , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Dinoprostona/sangre , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Mutantes , Canales de Potasio de Rectificación Interna/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIa/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Regulación hacia Arriba/fisiología , Agua/metabolismoRESUMEN
Tight junctions play a key role in mediating paracellular ion reabsorption in the kidney. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a human disorder caused by mutations in the tight junction protein claudin-16. However, the molecular mechanisms underlining the renal handling of magnesium and its dysfunction causing FHHNC are unknown. Here we show that claudin-16 plays a key role in maintaining the paracellular cation selectivity of the thick ascending limbs of the nephron. Using RNA interference, we have generated claudin-16-deficient mouse models. Claudin-16 knock-down (KD) mice exhibit chronic renal wasting of magnesium and calcium and develop renal nephrocalcinosis. Our data suggest that claudin-16 forms a non-selective paracellular cation channel, rather than a selective Mg(2+)/Ca(2+) channel as previously proposed. Our study highlights the pivotal importance of the tight junction in renal control of ion homeostasis and provides answer to the pathogenesis of FHHNC. We anticipate our study to be a starting point for more sophisticated in vivo analysis of tight junction proteins in renal functions. Furthermore, tight junction proteins could be major targets of drug development for electrolyte disorders.
Asunto(s)
Riñón/metabolismo , Magnesio/metabolismo , Proteínas de la Membrana/fisiología , Interferencia de ARN , Animales , Secuencia de Bases , Claudinas , Cartilla de ADN , Lentivirus/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microscopía ConfocalRESUMEN
The mathematical model of rat proximal tubule has been extended to include calculation of microvillous torque and to incorporate torque-dependent solute transport in a compliant tubule. The torque calculation follows that of Du Z, Yan Q, Duan Y, Weinbaum S, Weinstein AM, and Wang T (Am J Physiol 290: F289-F296, 2006). In the model calculations, torque-dependent scaling of luminal membrane transporter density [either as an ensemble or just type 3 Na(+)/H(+) exchanger (NHE3) alone] had a relatively small impact on overall Na(+) reabsorption and could produce a lethal derangement of cell volume; coordinated regulation of luminal and peritubular transporters was required to represent the overall impact of luminal flow on Na(+) reabsorption. When the magnitude of torque-dependent Na(+) reabsorption in the model agrees with that observed in mouse proximal tubules, the model tubule shows nearly perfect perfusion-absorption balance at high luminal perfusion rates, but enhanced sensitivity of reabsorption at low flow. With a slightly lower coefficient for torque-sensitive transporter insertion, perfusion-absorption balance in the model tubule is closer to observations in the rat over a broader range of inlet flows. In simulation of hyperglycemia, torque-dependent transport attenuated the diuretic effect and brought the model tubule into closer agreement with experimental observation in the rat. The model was also extended to represent finite rates of hydration and dehydration of CO(2) and H(2)CO(3). With carbonic anhydrase inhibition, torque-dependent transport blunted the diuretic effect and enhanced the shift from paracellular to transcellular NaCl reabsorption. The new features of this model tubule are an important step toward simulation of glomerulotubular balance.
Asunto(s)
Transporte Biológico/fisiología , Túbulos Renales Proximales/fisiología , Modelos Biológicos , Animales , Bicarbonatos/metabolismo , Anhidrasas Carbónicas/fisiología , Permeabilidad de la Membrana Celular , Simulación por Computador , Glucosa/metabolismo , Matemática , Ratas , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/fisiología , TorqueRESUMEN
We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ absorption (Du Z, Duan Y, Yan Q, Weinstein AM, Weinbaum S, and Wang T. Proc Natl Acad Sci USA 101: 13068-13073, 2004). It was hypothesized that brush-border microvilli function as a sensor to detect and amplify luminal hydrodynamic forces and transmit them to the actin cytoskeleton. In the present study we examine whether 1) flow-dependent HCO3- transport is proportional to flow-dependent variations in microvillous torque (bending moment); 2) both luminal membrane Na(+)/H+ exchange (NHE3) and H(+)-ATPase activity are modulated by axial flow; and 3) paracellular permeabilities contribute to the flux perturbations. HCO3- absorption is examined by microperfusion of mouse S2 proximal tubules in vitro, with varying perfusion rates, and in the presence of the Na/H-exchange inhibitor EIPA, the H(+)-ATPase inhibitor bafilomycin, and the actin cytoskeleton inhibitor cytochalasin D. Paracellular permeability changes are assessed with measurements of epithelial HCO3- permeability and transepithelial potential difference (PD). It is found that 1) an increase in perfusion rate enhances HCO3- absorption and microvillous torque, and the fractional changes of each are nearly identical; 2) inhibition of NHE3 by EIPA, or H(+)-ATPase by bafilomycin, produced only partial inhibition of flow-stimulated bicarbonate transport; 3) disruption of the actin cytoskeleton by cytochalasin D blocked the increment of HCO3- absorption by high flow; and 4) HCO3- permeability and transepithelial PD are not modulated by flow. We conclude that flow-dependent modulation of proximal tubule HCO3- reabsorption is due to changes in both NHE3 and H(+)-ATPase activity within the luminal cell membrane and this requires an intact actin cytoskeleton. Paracellular permeability changes do not contribute to this flow dependence. Perfusion-absorption balance in the proximal tubule is a direct effect of flow-induced torque on brush-border microvilli to regulate luminal cell membrane transporter activity.
Asunto(s)
Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , ATPasas de Translocación de Protón/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Bicarbonatos/metabolismo , Bicarbonatos/farmacocinética , Transporte Biológico/efectos de los fármacos , Citocalasina D/farmacología , Técnicas In Vitro , Túbulos Renales Proximales/fisiología , Macrólidos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades/fisiología , Perfusión , Sodio/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/genética , TorqueRESUMEN
Normal variations in glomerular filtration induce proportional changes in proximal tubule Na+ reabsorption. This "glomerulotubular balance" derives from flow dependence of Na+ uptake across luminal cell membranes; however, the underlying physical mechanism is unknown. Our hypothesis is that flow-dependent reabsorption is an autoregulatory mechanism that is independent of neural and hormonal systems. It is signaled by the hydrodynamic torque (bending moment) on epithelial microvilli. Such signals need to be transmitted to the terminal web to modulate Na+-H+-exchange activity. To investigate this hypothesis, we examined Na+ transport and tubular diameter in response to different flow rates during the microperfusion of isolated S2 proximal tubules from mouse kidneys. The data were analyzed by using a mathematical model to estimate the microvillous torque as function of flow. In this model, increases in luminal diameter have the effect of blunting the impact of flow velocity on microvillous shear stress and, thus, microvillous torque. We found that variations in microvillous torque produce nearly identical fractional changes in Na+ reabsorption. Furthermore, the flow-dependent Na+ transport is increased by increasing luminal fluid viscosity, diminished in Na+-H+ exchanger isoform 3 knockout mice, and abolished by nontoxic disruption of the actin cytoskeleton. These data support our hypothesis that the "brush-border" microvilli serve a mechanosensory function in which fluid dynamic torque is transmitted to the actin cytoskeleton and modulates Na+ absorption in kidney proximal tubules.
Asunto(s)
Túbulos Renales/fisiología , Mecanotransducción Celular/fisiología , Animales , Citoesqueleto/metabolismo , Interpretación Estadística de Datos , Túbulos Renales/ultraestructura , Ratones , Ratones Endogámicos C57BL , Microfluídica , Microvellosidades/fisiología , Modelos Biológicos , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Apical potassium recycling is crucial for salt transport by the thick ascending limb (TAL). Loss-of-function mutations in the K channel, ROMK (Kir1.1; KCNJ1), cause Bartter syndrome, a genetically heterogeneous disorder characterized by severe reduction in salt absorption by the TAL, Na wasting, polyuria, and hypokalemic alkalosis. ROMK(-/-) null mice exhibit a Bartter phenotype and lack the small-conductance (30-pS) apical K channel (SK) in the TAL. However, a distinct 70-pS K channel can also significantly contribute to the apical conductance of TAL. We now examine the effect of ROMK deletion on the functional expression of the 70-pS K channel in the TAL. Functional expression of the 70-pS K channel was low [average channel activity (NP(o)) = 0.02] in ROMK(+/+) mice on a control K diet but increased to 0.27 by high-K intake for 2 wk. In contrast, the high-K diet decreased NP(o) of SK by approximately 30%, from 2.04 to 1.44. In ROMK heterozygous (+/-) mice on a control K diet, SK activity was about one-half of that observed in ROMK(+/+) mice (0.95 vs. 2.04). The high-K diet also reduced SK activity in ROMK(+/-) mice by approximately 40% (from 0.95 to 0.55) but increased NP(o) of the 70-pS K channel from 0 to 0.09 in ROMK(+/-) mice. This corresponds to approximately 30% of channel activity (NP(o) = 0.27) observed in ROMK(+/+) mice. Neither the 70-pS nor the 30-pS K channels were observed in TAL cells from ROMK(-/-) mice on either the normal or high-K diets. Thus functional expression of the 70-pS K channel is enhanced by increasing dietary K and requires expression of ROMK. It is likely that ROMK forms a critical subunit of the 70-pS K channel, accounting for the loss of apical K secretory channel activity in ROMK Bartter syndrome.