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2.
J Am Soc Nephrol ; 30(8): 1454-1470, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31253651

RESUMEN

BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.


Asunto(s)
Aldosterona/farmacología , Túbulos Renales Distales/metabolismo , Transducción de Señal , Tiazidas/farmacología , Aldosterona/metabolismo , Animales , Presión Sanguínea , Calcio/metabolismo , Línea Celular , Membrana Celular/metabolismo , Biología Computacional , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Síndrome de Gitelman/metabolismo , Riñón/metabolismo , Masculino , Ratones , Mineralocorticoides/metabolismo , Fosforilación , Proteómica , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cloruro de Sodio/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo
3.
Clin Sci (Lond) ; 132(16): 1779-1796, 2018 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-29941522

RESUMEN

Adenylyl cyclase (AC) isoform 6 (AC6) is highly expressed throughout the renal tubule and collecting duct (CD), catalyzes the synthesis of cAMP and contributes to various aspects of renal transport. Several proteins involved in acid-base homeostasis are regulated by cAMP. In the present study, we assess the relative contribution of AC6 to overall acid-base regulation using mice with global deletion of AC6 (AC6-/-) or newly generated mice lacking AC6 in the renal tubule and CD (AC6loxloxPax8Cre). Higher energy expenditure in AC6-/- relative to wild-type (WT) mice, was associated with lower urinary pH, mild alkalosis in conjunction with elevated blood HCO3- concentrations, and significantly higher renal abundance of the H+-ATPase B1 subunit. In contrast with WT mice, AC6-/- mice have a less pronounced increase in urinary pH after 8 days of HCO3- challenge, which is associated with increased blood pH and HCO3- concentrations. Immunohistochemistry demonstrated that AC6 was expressed in intercalated cells (IC), but subcellular distribution of the H+-ATPase B1 subunit, pendrin, and the anion exchangers 1 and 2 in AC6-/- mice was normal. In the AC6-/- mice, H+-ATPase B1 subunit levels after HCO3- challenge were greater, which correlated with a higher number of type A IC. In contrast with the AC6-/- mice, AC6loxloxPax8Cre mice had normal urinary pH under baseline conditions but higher blood HCO3- than controls after HCO3- challenge. In conclusion, AC6 is required for maintaining normal acid-base homeostasis and energy expenditure. Under baseline conditions, renal AC6 is redundant for acid-base balance but becomes important under alkaline conditions.


Asunto(s)
Equilibrio Ácido-Base/fisiología , Adenilil Ciclasas/metabolismo , Homeostasis/fisiología , Riñón/metabolismo , Adenilil Ciclasas/genética , Animales , Análisis Químico de la Sangre , Metabolismo Energético , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Orina/química , ATPasas de Translocación de Protón Vacuolares/metabolismo
4.
Physiol Genomics ; 50(5): 343-354, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29521601

RESUMEN

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.


Asunto(s)
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 Transgénicos
5.
JCI Insight ; 2(7): e91042, 2017 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-28405619

RESUMEN

Psychiatric patients treated with lithium (Li+) may develop nephrogenic diabetes insipidus (NDI). Although the etiology of Li+-induced NDI (Li-NDI) is poorly understood, it occurs partially due to reduced aquaporin-2 (AQP2) expression in the kidney collecting ducts. A mechanism postulated for this is that Li+ inhibits adenylyl cyclase (AC) activity, leading to decreased cAMP, reduced AQP2 abundance, and less membrane targeting. We hypothesized that Li-NDI would not develop in mice lacking AC6. Whole-body AC6 knockout (AC6-/-) mice and potentially novel connecting tubule/principal cell-specific AC6 knockout (AC6loxloxCre) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li+ administration increased water intake and reduced urine osmolality in control, AC6-/-, and AC6loxloxCre mice. Consistent with AC6-/- mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6loxloxCre mice compared with controls under standard conditions, and levels were further reduced after Li+ administration. AC6loxloxCre and control mice had a similar increase in the numbers of proliferating cell nuclear antigen-positive cells in response to Li+. However, AC6loxloxCre mice had a higher number of H+-ATPase B1 subunit-positive cells under standard conditions and after Li+ administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell-to-principal cell ratio.


Asunto(s)
Adenilil Ciclasas/metabolismo , Acuaporina 2/metabolismo , Diabetes Insípida Nefrogénica/enzimología , Litio/toxicidad , Adenilil Ciclasas/genética , Animales , AMP Cíclico/metabolismo , Diabetes Insípida Nefrogénica/inducido químicamente , Modelos Animales de Enfermedad , Femenino , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Masculino , Ratones , Ratones Noqueados
6.
Am J Physiol Renal Physiol ; 313(3): F756-F766, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27733368

RESUMEN

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.


Asunto(s)
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 Arriba
7.
Am J Physiol Renal Physiol ; 310(4): F300-10, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26582762

RESUMEN

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/genética
8.
Curr Opin Nephrol Hypertens ; 24(5): 463-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26125647

RESUMEN

PURPOSE OF REVIEW: Sodium-glucose cotransporters (SGLTs) are important mediators of glucose uptake across apical cell membranes. SGLT1 mediates almost all sodium-dependent glucose uptake in the small intestine, while in the kidney SGLT2, and to a lesser extent SGLT1, account for more than 90% and nearly 3%, respectively, of glucose reabsorption from the glomerular ultrafiltrate. Although the recent availability of SGLT2 inhibitors for the treatment of diabetes mellitus has increased the number of clinical studies, this review has a focus on mechanisms contributing to the cellular regulation of SGLTs. RECENT FINDINGS: Studies have focused on the regulation of SGLT expression under different physiological/pathophysiological conditions, for example diet, age or diabetes mellitus. Several studies provide evidence of SGLT regulation via cyclic adenosine monophosphate/protein kinase A, protein kinase C, glucagon-like peptide 2, insulin, leptin, signal transducer and activator of transcription-3 (STAT3), phosphoinositide-3 kinase (PI3K)/Akt, mitogen-activated protein kinases (MAPKs), nuclear factor-kappaB (NF-kappaB), with-no-K[Lys] kinases/STE20/SPS1-related proline/alanine-rich kinase (Wnk/SPAK) and regulatory solute carrier protein 1 (RS1) pathways. SUMMARY: SGLT inhibitors are important drugs for glycemic control in diabetes mellitus. Although the contribution of SGLT1 for absorption of glucose from the intestine as well as SGLT2/SGLT1 for renal glucose reabsorption has been comprehensively defined, this review provides an up-to-date outline for the mechanistic regulation of SGLT1/SGLT2.


Asunto(s)
Glucemia/metabolismo , Glucosa/metabolismo , Hipoglucemiantes/uso terapéutico , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Sodio/metabolismo , Animales , Humanos , Riñón/metabolismo
9.
Nephrol Dial Transplant ; 28(8): 2058-65, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23615760

RESUMEN

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 Tiempo
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