RESUMEN
BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.
Asunto(s)
Ceramidasa Alcalina/genética , Diabetes Insípida Nefrogénica/genética , Cloruro de Litio/toxicidad , Equilibrio Ácido-Base/fisiología , Acidosis/inducido químicamente , Acidosis/genética , Animales , Diabetes Insípida Nefrogénica/inducido químicamente , Dinoprostona/orina , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Hematócrito , Hipercalcemia/inducido químicamente , Hipercalcemia/genética , Túbulos Renales Colectores/metabolismo , Ratones , Ratones Endogámicos , Ratones Noqueados , Nefronas/metabolismo , ARN Mensajero/biosíntesis , Sodio/sangre , Especificidad de la EspecieRESUMEN
The renal aldosterone-sensitive distal tubule (ASDT) is crucial for sodium reabsorption and blood pressure regulation. The ASDT consists of the late distal convoluted tubule (DCT2), connecting tubule (CNT), and collecting duct. Due to difficulties in isolating epithelial cells from the ASDT in large quantities, few transcriptome studies have been performed on this segment. Moreover, no studies exist on isolated DCT2 and CNT cells (excluding intercalated cells), and the role of aldosterone for regulating the transcriptome of these specific cell types is largely unknown. A mouse model expressing eGFP in DCT2/CNT/initial cortical collecting duct (iCCD) principal cells was exploited to facilitate the isolation of these cells in high number and purity. Combined with deep RNA sequencing technology, a comprehensive catalog of chronic aldosterone-regulated transcripts from enriched DCT2/CNT/iCCD principal cells was generated. There were 257 significantly downregulated and 290 upregulated transcripts in response to aldosterone ( P < 0.05). The RNA sequencing confirmed aldosterone regulation of well-described aldosterone targets including Sgk1 and Tsc22d3. Changes in selected transcripts such as S100a1 and Cldn4 were confirmed by RT-qPCR. The RNA sequencing showed downregulation of Nr3c2 encoding the mineralocorticoid receptor (MR), and cell line experiments showed a parallel decrease in MR protein. Furthermore, a large number of transcripts encoding transcription factors were downregulated. An extensive mRNA transcriptome reconstruction of an enriched CNT/iCCD principal cell population was also generated. The results provided a comprehensive database of aldosterone-regulated transcripts in the ASDT, allowing development of novel hypotheses for the action of aldosterone.
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Aldosterona/farmacología , Túbulos Renales Colectores/metabolismo , Túbulos Renales Distales/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/efectos de los fármacos , Aldosterona/administración & dosificación , Aldosterona/sangre , Animales , Células Cultivadas , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Túbulos Renales Colectores/citología , Túbulos Renales Distales/citología , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Renal Na+-Cl- cotransporter (NCC) is expressed in early distal convoluted tubule (DCT) 1 and late DCT (DCT2). NCC activity can be stimulated by aldosterone administration, and the mechanism is assumed to depend on the enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), which inactivates glucocorticoids that would otherwise occupy aldosterone receptors. Because 11ß-HSD2 in rat may only be abundantly expressed in DCT2 cells and not in DCT1 cells, it has been speculated that aldosterone specifically stimulates NCC activity in DCT2 cells. In mice, however, it is debated if 11ß-HSD2 is expressed in DCT2 cells. The present study examined whether aldosterone administration in mice stimulates NCC abundance and phosphorylation in DCT2 cells but not in DCT1 cells. B6/C57 male mice were administered 100 µg aldosterone·kg body weight-1·24 h-1 for 6 days and euthanized during isoflurane inhalation. Western blotting of whole kidney homogenate showed that aldosterone administration stimulated NCC and pT58-NCC abundances (P < 0.001). In DCT1 cells, confocal microscopy detected no effect of the aldosterone administration on NCC and pT58-NCC abundances. By contrast, NCC and pT58-NCC abundances were stimulated by aldosterone administration in the middle of DCT2 (P < 0.001 and <0.01, respectively) and at the junction between DCT2 and CNT (P < 0.001 and <0.05, respectively). In contrast to rat, immunohistochemistry in mouse showed no/very weak 11ß-HSD2 expression in DCT2 cells. Collectively, long-term aldosterone administration stimulates mouse NCC and pT58-NCC abundances in DCT2 cells and presumably not in DCT1 cells.
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Aldosterona/administración & dosificación , Túbulos Renales Distales/efectos de los fármacos , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/metabolismo , Animales , Bombas de Infusión Implantables , Infusiones Subcutáneas , Túbulos Renales Distales/metabolismo , Masculino , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Fluorescente , Fosforilación , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Genetic inactivation of the epithelial Na(+) channel α-subunit (αENaC) in the renal collecting duct (CD) does not interfere with Na(+) and K(+) homeostasis in mice. However, inactivation in the CD and a part of the connecting tubule (CNT) induces autosomal recessive pseudohypoaldosteronism type 1 (PHA-1) symptoms in subjects already on a standard diet. In the present study, we further examined the importance of αENaC in the CNT. Knockout mice with αENaC deleted primarily in a part of the CNT (CNT-KO) were generated using Scnn1a(lox/lox) mice and Atp6v1b1::Cre mice. With a standard diet, plasma Na(+) concentration ([Na(+)]) and [K(+)], and urine Na(+) and K(+) output were unaffected. Seven days of Na(+) restriction (0.01% Na(+)) led to a higher urine Na(+) output only on days 3-5, and after 7 days plasma [Na(+)] and [K(+)] were unaffected. In contrast, the CNT-KO mice were highly susceptible to a 2-day 5% K(+) diet and showed lower food intake and relative body weight, lower plasma [Na(+)], higher fractional excretion (FE) of Na(+), higher plasma [K(+)], and lower FE of K(+). The higher FE of Na(+) coincided with lower abundance and phosphorylation of the Na(+)-Cl(-) cotransporter. In conclusion, reducing ENaC expression in the CNT induces clear PHA-1 symptoms during high dietary K(+) loading.
Asunto(s)
Canales Epiteliales de Sodio/biosíntesis , Túbulos Renales Colectores/metabolismo , Potasio/metabolismo , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/metabolismo , Aldosterona/metabolismo , Animales , Peso Corporal , Colon/metabolismo , Dieta , Ingestión de Alimentos , Canales Epiteliales de Sodio/genética , Femenino , Túbulos Renales Colectores/patología , Masculino , Ratones , Ratones Noqueados , Fosforilación , Potasio/sangre , Seudohipoaldosteronismo/patología , Sodio/sangre , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/biosíntesis , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/biosíntesis , Miembro 3 de la Familia de Transportadores de Soluto 12/genéticaRESUMEN
Aldosterone promotes electrogenic sodium reabsorption through the amiloride-sensitive epithelial sodium channel (ENaC). Here, we investigated the importance of ENaC and its positive regulator channel-activating protease 1 (CAP1/Prss8) in colon. Mice lacking the αENaC subunit in colonic superficial cells (Scnn1a(KO)) were viable, without fetal or perinatal lethality. Control mice fed a regular or low-salt diet had a significantly higher amiloride-sensitive rectal potential difference (∆PDamil) than control mice fed a high-salt diet. In Scnn1a(KO) mice, however, this salt restriction-induced increase in ∆PDamil did not occur, and the circadian rhythm of ∆PDamil was blunted. Plasma and urinary sodium and potassium did not change with regular or high-salt diets or potassium loading in control or Scnn1a(KO) mice. However, Scnn1a(KO) mice fed a low-salt diet lost significant amounts of sodium in their feces and exhibited high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAP1/Prss8 in colonic superficial cells (Prss8(KO)) were viable, without fetal or perinatal lethality. Compared with controls, Prss8(KO) mice fed regular or low-salt diets exhibited significantly reduced ∆PDamil in the afternoon, but the circadian rhythm was maintained. Prss8(KO) mice fed a low-salt diet also exhibited sodium loss through feces and higher plasma aldosterone levels. Thus, we identified CAP1/Prss8 as an in vivo regulator of ENaC in colon. We conclude that, under salt restriction, activation of the renin-angiotensin-aldosterone system in the kidney compensated for the absence of ENaC in colonic surface epithelium, leading to colon-specific pseudohypoaldosteronism type 1 with mineralocorticoid resistance without evidence of impaired potassium balance.
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Aldosterona/metabolismo , Colon/metabolismo , Canales Epiteliales de Sodio/fisiología , Sodio/metabolismo , Animales , Canales Epiteliales de Sodio/deficiencia , Femenino , Masculino , Ratones , Serina Endopeptidasas/fisiologíaRESUMEN
The cellular morphology of the collecting duct is altered by chronic lithium treatment. We have previously shown that lithium increases the fraction of type-A intercalated cells and lowers the fraction of principal cells along the collecting duct. Moreover, type-A intercalated cells acquire a long-row distribution pattern along the tubules. In the present study, we show that these morphological changes reverse progressively after discontinuation of lithium and finally disappear after 19 days from lithium suspension. In this time frame we have identified for the first time, in vivo, a novel cellular type positive for both intercalated and principal cells functional markers, as recognized by colabeling with H(+)-ATPase/aquaporin-4 (AQP4) and anion exchanger-1 (AE-1)/AQP2 and Foxi1/AQP4. This cell type is mainly present after 6 days of lithium washout, and it disappears in parallel with the long-row pattern of the type-A intercalated cells. It usually localizes either in the middle or at the edge of the long-row pattern. Its ultrastructure resembles the intercalated cells as shown both by differential interference contrast and by electron microscopy. The time course of appearance, the localization along the collecting duct, and the ultrastructure suggest that the cells double labeled for principal and intercalated cells markers could represent a transition element driving the conversion of intercalated cells into principal cells.
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Diabetes Insípida Nefrogénica/inducido químicamente , Diabetes Insípida Nefrogénica/patología , Túbulos Renales Colectores/citología , Litio/efectos adversos , Síndrome de Abstinencia a Sustancias , Animales , Acuaporina 2/biosíntesis , Acuaporina 4/biosíntesis , Proliferación Celular , Diabetes Insípida Nefrogénica/metabolismo , Factores de Transcripción Forkhead/biosíntesis , Masculino , ATPasas de Translocación de Protón/biosíntesis , Ratas , Síndrome de Abstinencia a Sustancias/patologíaRESUMEN
Aquaporins may facilitate transepithelial water absorption in the intestine of seawater (SW)-acclimated fish. Here we have characterized three full-length aqp8 paralogs from Atlantic salmon (Salmo salar). Bayesian inference revealed that each paralog is a representative of the three major classes of aqp8aa, aqp8ab and aqp8b genes found in other teleosts. The permeability properties were studied by heterologous expression in Xenopus laevis oocytes, and the expression levels examined by qPCR, immunofluorescence and immunoelectron microscopy, and immunoblotting of membrane fractions from intestines of SW-challenged smolts. All three Aqp8 paralogs were permeable to water and urea, whereas Aqp8ab and -8b were, surprisingly, also permeable to glycerol. The mRNA tissue distribution of each paralog was distinct, although some tissues such as the intestine showed redundant expression of more than one paralog. Immunofluorescence microscopy localized Aqp8aa(1+2) to intracellular compartments of the liver and intestine, and Aqp8ab and Aqp8b to apical plasma membrane domains of the intestinal epithelium, with Aqp8b also in goblet cells. In a control experiment with rainbow trout, immunoelectron microscopy confirmed abundant labeling of Aqp8ab and -8b at apical plasma membranes of enterocytes in the middle intestine and also in subapical vesicular structures. During SW challenge, Aqp8ab showed significantly increased levels of protein expression in plasma-membrane-enriched fractions of the intestine. These data indicate that the Atlantic salmon Aqp8 paralogs have neofunctionalized on a transcriptional as well as a functional level, and that Aqp8ab may play a central role in the intestinal transcellular uptake of water during SW acclimation.
Asunto(s)
Acuaporinas/metabolismo , Permeabilidad de la Membrana Celular , Salmo salar/metabolismo , Agua de Mar , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Acuaporinas/química , Acuaporinas/genética , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/ultraestructura , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Filogenia , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Xenopus laevisRESUMEN
BACKGROUND: Desmopressin (dDAVP) induces a decrease in immunolabelling of aquaporin (AQP) 4 protein in the terminal inner medulla (IM) of the Brattleboro (BB) rat kidney. It is disputed, however, whether the decreased labelling reflects real down-regulation of protein abundance, or whether it is a result of epitope shielding in the AQP4 protein, preventing binding of the antibody as previously suggested. Furthermore, it is unknown if vasopressin regulates AQP4 in the connecting tubule (CNT) and in the cortical collecting duct (CCD). Using BB rats, we aimed to determine (i) whether the dDAVP-induced decrease in AQP4 labelling in the terminal IM reflects down-regulation in protein abundance and (ii) whether dDAVP increases the AQP4 protein abundance in the CNT and the CCD. METHODS: BB rats received dDAVP or saline (control) via osmotic minipumps pumps for 6 days. RESULTS: Immunolight microscopy revealed strong AQP4 labelling in the initial inner medullary collecting duct (IMCD1), weak labelling in the middle IMCD (IMCD2) and weak/absent labelling in the terminal IMCD (IMCD3) after 6 days of dDAVP administration. AQP3 labelling was similar to that of AQP4. Two-hour administration with dDAVP (previously described BB rats) did not change the labelling pattern of AQP4, suggesting that potential acute phosphorylation did not induce epitope shielding, thereby preventing the binding of the antibody. CONCLUSIONS: In BB rats, long-term administration with dDAVP (i) increased the AQP4 protein abundance in the IMCD1 and decreased the abundance in the IMCD2 and the IMCD3, and (ii) increased the AQP4 protein abundance in the CNT and the CCD.
Asunto(s)
Acuaporina 4/metabolismo , Regulación de la Expresión Génica , Corteza Renal/metabolismo , Médula Renal/metabolismo , Túbulos Renales/metabolismo , Receptores de Vasopresinas/fisiología , Animales , Fármacos Antidiuréticos/farmacología , Desamino Arginina Vasopresina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Corteza Renal/citología , Corteza Renal/efectos de los fármacos , Médula Renal/citología , Médula Renal/efectos de los fármacos , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Masculino , Ratas , Ratas Brattleboro , Factores de TiempoRESUMEN
Steroid hormones such as 17ß-estradiol (E2) are known to modulate ion transporter expression in the kidney through classic intracellular receptors. Steroid hormones are also known to cause rapid nongenomic responses in a variety of nonrenal tissues. However, little is known about renal short-term effects of steroid hormones. Here, we studied the acute actions of E2 on intracellular Ca(2+) signaling in isolated distal convoluted tubules (DCT2), connecting tubules (CNT), and initial cortical collecting ducts (iCCD) by fluo 4 fluorometry. Physiological concentrations of E2 induced transient increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in a subpopulation of cells. The [Ca(2+)](i) increases required extracellular Ca(2+) and were inhibited by Gd(3+). Strikingly, the classic E2 receptor antagonist ICI 182,780 also increased [Ca(2+)](i), which is inconsistent with the activation of classic E2 receptors. G protein-coupled estrogen receptor 1 (GPER1 or GPR30) was detected in microdissected DCT2/CNT/iCCD by RT-PCR. Stimulation with the specific GPER1 agonist G-1 induced similar [Ca(2+)](i) increases as E2, and in tubules from GPER1 knockout mice, E2, G-1, and ICI 182,780 failed to induce [Ca(2+)](i) elevations. The intercalated cells showed both E2-induced concanamycin-sensitive H(+)-ATPase activity by BCECF fluorometry and the E2-mediated [Ca(2+)](i) increment. We propose that E2 via GPER1 evokes [Ca(2+)](i) transients and increases H(+)-ATPase activity in intercalated cells in mouse DCT2/CNT/iCCD.
Asunto(s)
Señalización del Calcio/fisiología , Estradiol/metabolismo , Receptor alfa de Estrógeno/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales Distales/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Aldosterona/metabolismo , Aldosterona/farmacología , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Estrógenos/metabolismo , Estrógenos/farmacología , Espacio Extracelular/metabolismo , Femenino , Inmunohistoquímica , Túbulos Renales Colectores/ultraestructura , Túbulos Renales Distales/ultraestructura , Masculino , Ratones , Ratones Noqueados , Microscopía Inmunoelectrónica , ATPasas de Translocación de Protón/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Lithium-induced nephrogenic diabetes insipidus (NDI) is accompanied by polyuria, downregulation of aquaporin 2 (AQP2), and cellular remodeling of the collecting duct (CD). The amiloride-sensitive epithelial sodium channel (ENaC) is a likely candidate for lithium entry. Here, we subjected transgenic mice lacking αENaC specifically in the CD (knockout [KO] mice) and littermate controls to chronic lithium treatment. In contrast to control mice, KO mice did not markedly increase their water intake. Furthermore, KO mice did not demonstrate the polyuria and reduction in urine osmolality induced by lithium treatment in the control mice. Lithium treatment reduced AQP2 protein levels in the cortex/outer medulla and inner medulla (IM) of control mice but only partially reduced AQP2 levels in the IM of KO mice. Furthermore, lithium induced expression of H(+)-ATPase in the IM of control mice but not KO mice. In conclusion, the absence of functional ENaC in the CD protects mice from lithium-induced NDI. These data support the hypothesis that ENaC-mediated lithium entry into the CD principal cells contributes to the pathogenesis of lithium-induced NDI.
Asunto(s)
Diabetes Insípida Nefrogénica/inducido químicamente , Canales Epiteliales de Sodio/fisiología , Cloruro de Litio/toxicidad , Absorción , Animales , Acuaporina 2/análisis , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/patología , Cloruro de Litio/farmacocinética , Ratones , Ratones Noqueados , ATPasas de Translocación de Protón/análisisRESUMEN
Mutations in α, ß, or γ subunits of the epithelial sodium channel (ENaC) can downregulate ENaC activity and cause a severe salt-losing syndrome with hyperkalemia and metabolic acidosis, designated pseudohypoaldosteronism type 1 in humans. In contrast, mice with selective inactivation of αENaC in the collecting duct (CD) maintain sodium and potassium balance, suggesting that the late distal convoluted tubule (DCT2) and/or the connecting tubule (CNT) participates in sodium homeostasis. To investigate the relative importance of ENaC-mediated sodium absorption in the CNT, we used Cre-lox technology to generate mice lacking αENaC in the aquaporin 2-expressing CNT and CD. Western blot analysis of microdissected cortical CD (CCD) and CNT revealed absence of αENaC in the CCD and weak αENaC expression in the CNT. These mice exhibited a significantly higher urinary sodium excretion, a lower urine osmolality, and an increased urine volume compared with control mice. Furthermore, serum sodium was lower and potassium levels were higher in the genetically modified mice. With dietary sodium restriction, these mice experienced significant weight loss, increased urinary sodium excretion, and hyperkalemia. Plasma aldosterone levels were significantly elevated under both standard and sodium-restricted diets. In summary, αENaC expression within the CNT/CD is crucial for sodium and potassium homeostasis and causes signs and symptoms of pseudohypoaldosteronism type 1 if missing.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Túbulos Renales Colectores/metabolismo , Túbulos Renales/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Aldosterona/sangre , Animales , Acuaporina 2/metabolismo , Canales Epiteliales de Sodio/genética , Femenino , Homeostasis/fisiología , Corteza Renal/citología , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Sodio en la Dieta/farmacologíaRESUMEN
Lithium (Li+) salts are widely used to treat bipolar mood disorders. Recent trials suggest a potential efficacy also in the treatment of amyotrophic lateral sclerosis and Alzheimer's disease. Li+ is freely filtered by the glomerulus and mainly reabsorbed in the proximal convoluted tubule. Reabsorption in the distal nephron becomes significant under sodium-restricted conditions. Nevertheless, the distal nephron is greatly affected by Li+ even under normal sodium intake. Polyuria, renal tubular acidosis and finally chronic renal failure are the most frequent adverse effects. The occurrence of an overt nephrogenic diabetes insipidus (NDI) limits Li+ usage and imposes suspension. The molecular mechanisms of Li+-related urinary concentration defect involve a dysregulation of the aquaporin system in principal cells of the collecting duct. ENaC is crucial as the entry route for intracellular Li+ accumulation. The basolateral exit route is not clearly identified, but some evidence suggests Na+/H+ exchanger 1 (NHE1) as a potential candidate. Li+ promotes polyuria mainly counteracting the intracellular vasopressin signaling. An additional role of the inner medullary interstitial cells and PGE-2 pathway has to be considered. The GSK3ß cascade is also regulated by Li+. GSK3ß inhibition could lead not only to the polyuria, but also to the Li+-dependent proliferative effect on principal cells. Cellular reorganization of the collecting duct and microcysts are the main pathological findings during Li+ treatment. Their relationship with the urinary concentration defect and an eventual Li+-induced ciliopathy has to been investigated. Li+-induced NDI has been a matter of investigation since the early 1970s. This manuscript reports the latest clinical and experimental findings in combination with the older fundamental results.
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Diabetes Insípida Nefrogénica/inducido químicamente , Litio/toxicidad , Animales , Glucógeno Sintasa Quinasa 3/fisiología , Glucógeno Sintasa Quinasa 3 beta , Humanos , Riñón/metabolismo , Túbulos Renales Colectores/metabolismo , Litio/metabolismoRESUMEN
Lithium is used commonly to treat bipolar mood disorders. In addition to its primary therapeutic effects in the central nervous system lithium has a number of side effects in the kidney. The side effects include nephrogenic diabetes insipidus with polyuria, mild sodium wasting, and changes in acid/base balance. These functional changes are associated with marked structural changes in collecting duct cell composition and morphology, likely contributing to the functional changes. Over the past few years, investigations of lithium-induced renal changes have provided novel insight into the molecular mechanisms that are responsible for the disturbances in water, sodium, and acid/base metabolism. This includes dysregulation of renal aquaporins, epithelial sodium channel, and acid/base transporters. This review focuses on these issues with the aim to present this in context with clinically relevant features.
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Acuaporinas/fisiología , Diabetes Insípida Nefrogénica/fisiopatología , Canales Epiteliales de Sodio/fisiología , Compuestos de Litio/efectos adversos , Aldosterona/fisiología , Diabetes Insípida Nefrogénica/inducido químicamente , Diabetes Insípida Nefrogénica/patología , Humanos , Túbulos Renales Colectores/fisiología , Proteómica , Sodio/metabolismoRESUMEN
Previous reports identify a voltage dependent distal renal tubular acidosis (dRTA) secondary to lithium (Li+) salt administration. This was based on the inability of Li+-treated patients to increase the urine-blood (U-B) pCO2 when challenged with NaHCO3 and, the ability of sodium neutral phosphate or Na2SO4 administration to restore U-B pCO2 in experimental animal models. The underlying mechanisms for the Li+-induced dRTA are still unknown. To address this point, a 7 days time course of the urinary acid-base parameters was investigated in rats challenged with LiCl, LiCitrate, NaCl, or NaCitrate. LiCl induced the largest polyuria and a mild metabolic acidosis. Li+-treatment induced a biphasic response. In the first 2 days, proper urine volume and acidification occurred, while from the 3rd day of treatment, polyuria developed progressively. In this latter phase, the LiCl-treated group progressively excreted more NH4+ and less pCO2, suggesting that NH3/NH4+ became the main urinary buffer. This physiological parameter was corroborated by the upregulation of NBCn1 (a marker of increased ammonium recycling) in the inner stripe of outer medulla of LiCl treated rats. Finally, by investigating NH4+ excretion in ENaC-cKO mice, a model resistant to Li+-induced polyuria, a primary role of the CD was confirmed. By definition, dRTA is characterized by deficient urinary ammonium excretion. Our data question the presence of a voltage-dependent Li+-induced dRTA in rats treated with LiCl for 7 days and the data suggest that the alkaline urine pH induced by NH3/NH4+ as the main buffer has lead to the interpretation dRTA in previous studies.
Asunto(s)
Acidosis Tubular Renal/inducido químicamente , Acidosis Tubular Renal/orina , Compuestos de Amonio/orina , Dióxido de Carbono/orina , Túbulos Renales Distales , Poliuria/orina , Animales , Tampones (Química) , Dióxido de Carbono/sangre , Citratos/efectos adversos , Canales Epiteliales de Sodio/genética , Concentración de Iones de Hidrógeno , Médula Renal/metabolismo , Túbulos Renales Colectores/fisiopatología , Cloruro de Litio/efectos adversos , Masculino , Ratones , Ratones Noqueados , Presión Parcial , Poliuria/inducido químicamente , Poliuria/genética , Ratas , Cloruro de Sodio/efectos adversos , Citrato de Sodio/efectos adversos , Simportadores de Sodio-Bicarbonato/metabolismo , Factores de Tiempo , UrinálisisRESUMEN
Lithium (Li) treatment for 4 wk has previously been shown to increase the fraction of intercalated cells in parallel with a decrease in the fraction of principal cells in the kidney collecting duct (Christensen BM, Marples D, Kim YH, Wang W, Frøkiaer J, and Nielsen S. Am J Physiol Cell Physiol 286: C952-C964, 2004; Kim YH, Kwon TH, Christensen BM, Nielsen J, Wall SM, Madsen KM, Frøkiaer J, and Nielsen S. Am J Physiol Renal Physiol 285: F1244-F1257, 2003). To study how early this fractional change starts, the origin of the cells and the possible mechanism behind the changes, we did time course studies in rats subjected to different durations of Li treatment (i.e., for 4, 10, and 15 days). Increased urine output was already observed at day 4 of Li treatment with decreased AQP2 levels although not statistically significant. At days 10 and 15, both a significant polyuria and downregulation in AQP2 expression were observed. At day 10, the density of H+-ATPase-positive cells was increased in the IMCD of Li-treated rats and this was further pronounced at day 15. Some of the H+-ATPase-positive cells did not costain with Cl-/HCO3- exchanger AE1, indicating that they were not fully differentiated to type A IC. By double labeling for either H+-ATPase and proliferating-cell nuclear antigen (PCNA) or for AQP4 and PCNA, we found that proliferation mainly occurred in proximal IMCD cells at day 4 and it increased toward the middle part of the IMCD in response to prolonged Li treatment. Most cells expressing PCNA were stained with AQP4 but not with H+-ATPase. Triple-labeling for H+-ATPase, AQP4, and PCNA showed a subset of cells negative for all three proteins or only positive for PCNA. In contrast, a 4-wk recovery period after 4 wk of Li treatment reversed the enhanced proliferative rate to the control levels. In conclusion, the Li-induced increase in the density of intercalated cells is associated with a high proliferative rate of principal cells in the IM-1 and IM-2 rather than a selective proliferation of intercalated cells as expected. This is likely to contribute to the remodeling of the collecting duct after Li treatment.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Médula Renal/citología , Médula Renal/efectos de los fármacos , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/efectos de los fármacos , Litio/farmacología , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Apoptosis/efectos de los fármacos , Factor Inductor de la Apoptosis , Acuaporina 2/metabolismo , Acuaporina 4/metabolismo , Diferenciación Celular/efectos de los fármacos , Diabetes Insípida/inducido químicamente , Diabetes Insípida/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Médula Renal/química , Túbulos Renales Colectores/química , Litio/efectos adversos , Masculino , Mitosis/efectos de los fármacos , Poliuria/inducido químicamente , Antígeno Nuclear de Célula en Proliferación/metabolismo , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The purpose of the present study was to examine whether there is axial heterogeneity in the basolateral plasma membrane (BLM) localization of AQP2 and whether altered vasopressin action or medullary tonicity affects the BLM localization of AQP2. Immunocytochemistry and immunoelectron microscopy revealed AQP2 labeling of the BLM in connecting tubule (CNT) cells and inner medullary collecting duct (IMCD) principal cells in normal rats and vasopressin-deficient Brattleboro rats. In contrast there was little basolateral AQP2 labeling in cortical (CCD) and outer medullary collecting duct principal cells. Short-term desamino-Cys(1), (D)-Arg(8) vasopressin (dDAVP) treatment (2 h) of Brattleboro rats caused no increase in AQP2 labeling of the BLM. In contrast, long-term dDAVP treatment (6 days) of Brattleboro rats caused an increased BLM labeling in CNT, CCD, and IMCD. Treatment of normal rats with V(2)-receptor antagonist for 60 min caused retrieval of AQP2 from the apical plasma membrane. Moreover, AQP2 labeling of the BLM was unchanged in CNT and IMCD but increased in CCD. In conclusion, there is an axial heterogeneity in the subcellular localization of AQP2 with prominent AQP2 labeling of the BLM in CNT and IMCD. There was no increase in AQP2 labeling of the BLM in response to short-term dDAVP. Moreover, acute V(2)-receptor antagonist treatment did not cause retrieval of AQP2 from the BLM. In contrast, long-term dDAVP treatment caused a major increase in AQP2 expression in the BLM in CCD.
Asunto(s)
Acuaporinas/metabolismo , Corteza Renal/efectos de los fármacos , Corteza Renal/metabolismo , Vasopresinas/deficiencia , Vasopresinas/farmacología , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Acuaporina 2 , Acuaporina 6 , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Desamino Arginina Vasopresina/farmacología , Inmunohistoquímica , Corteza Renal/ultraestructura , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Túbulos Renales/ultraestructura , Masculino , Ratas , Ratas Brattleboro , Ratas Wistar , Fármacos Renales/farmacologíaRESUMEN
Lithium treatment for 4 wk caused severe polyuria, dramatic downregulation in aquaporin-2 (AQP-2) expression, and marked decrease in AQP-2 immunoreactivity with the appearance of a large number of cells without AQP-2 labeling in the collecting ducts after lithium treatment. Surprisingly, this was not all due to an increase in AQP-2-negative principal cells, because double immunolabeling revealed that the majority of the AQP-2-negative cells displayed [H(+)]ATPase labeling, which identified them as intercalated cells. Moreover, multiple [H(+)]ATPase-labeled cells were adjacent, which was never seen in control rats. Quantitation confirmed a significant decrease in the fraction of collecting duct cells that exhibited detectable AQP-2 labeling compared with control rats: in cortical collecting ducts, 40 +/- 3.4 vs. 62 +/- 1.8% of controls (P < 0.05; n = 4) and in inner medullary collecting ducts, 58 +/- 1.6 vs. 81 +/- 1.3% of controls (P < 0.05; n = 4). In parallel, a significant increase in the fraction of intercalated ([H(+)]ATPase-positive) cells was shown. Urine output, whole kidney AQP-2 expression, cellular organization, and the fractions of principal and intercalated cells in cortex and inner medulla returned to control levels after 4 wk on a lithium-free diet following 4 wk on a lithium-containing diet. In conclusion, lithium treatment not only decreased AQP-2 expression, but dramatically and reversibly reduced the fraction of principal cells and altered the cellular organization in collecting ducts. These effects are likely to be important in lithium-induced nephrogenic diabetes insipidus.
Asunto(s)
Diabetes Insípida Nefrogénica/inducido químicamente , Diabetes Insípida Nefrogénica/metabolismo , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Litio/farmacología , Proteínas de Transporte de Membrana , Animales , Anticuerpos , Acuaporina 2 , Acuaporina 4 , Acuaporina 6 , Acuaporinas/inmunología , Acuaporinas/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Túbulos Renales Colectores/ultraestructura , Microscopía Inmunoelectrónica , ATPasas de Translocación de Protón/inmunología , ATPasas de Translocación de Protón/metabolismo , Ratas , Ratas Wistar , Recuperación de la Función , Transportadores de Sulfato , OrinaRESUMEN
Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart.