RESUMEN
The physiological role of the renal ClC-Ka/ClC-K1 channels is to confer a high Cl- permeability to the thin Ascending Limb of Henle (tAL), which in turn is essential for establishing the high osmolarity of the renal medulla that drives water reabsorption from collecting ducts. Here, we investigated by whole-cell patch-clamp measurements on HEK293 cells co-expressing ClC-Ka (tagged with GFP) and the accessory subunit barttin (tagged with m-Cherry) the effect of a natural diuretic extract from roots of Dandelion (DRE), and other compounds activating PKC, such as ATP, on ClC-Ka activity and its membrane localization. Treatment with 400 µg/ml DRE significantly inhibited Cl- currents time-dependently within several minutes. Of note, the same effect on Cl- currents was obtained upon treatment with 100 µM ATP. Pretreatment of cells with either the intracellular Ca2+ chelator BAPTA-AM (30 µM) or the PKC inhibitor Calphostin C (100 nM) reduced the inhibitory effect of DRE. Conversely, 1 µM of phorbol meristate acetate (PMA), a specific PKC activator, mimicked the inhibitory effect of DRE on ClC-Ka. Finally, we found that pretreatment with 30 µM Heclin, an E3 ubiquitin ligase inhibitor, did not revert DRE-induced Cl- current inhibition. In agreement with this, live-cell confocal analysis showed that DRE treatment did not induce ClC-Ka internalization. In conclusion, we demonstrate for the first time that the activity of ClC-Ka in renal cells could be significantly inhibited by the activation of PKC elicited by classical maneuvers, such as activation of purinergic receptors, or by exposure to herbal extracts that activates a PKC-dependent pathway. Overall, we provide both new information regarding the regulation of ClC-Ka and a proof-of-concept study for the use of DRE as new diuretic.
Asunto(s)
Canales de Cloruro/metabolismo , Diuréticos/farmacología , Asa de la Nefrona/metabolismo , Proteína Quinasa C/metabolismo , Adenosina Trifosfato/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células HEK293 , Humanos , Microscopía Intravital , Asa de la Nefrona/citología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Microscopía Confocal , Naftalenos/farmacología , Técnicas de Placa-Clamp , Extractos Vegetales/farmacología , Raíces de Plantas/química , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Taraxacum/química , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Calcium phosphate (CaP) crystals, which begin to form in the early segments of the loop of Henle (LOH), are known to act as precursors for calcium stone formation. The proximal tubule (PT), which is just upstream of the LOH and is a major site for Ca2+ reabsorption, could be a regulator of such CaP crystal formation. However, PT Ca2+ reabsorption is mostly described as being paracellular. Here, we show the existence of a regulated transcellular Ca2+ entry pathway in luminal membrane PT cells induced by Ca2+-sensing receptor (CSR, also known as CASR)-mediated activation of transient receptor potential canonical 3 (TRPC3) channels. In support of this idea, we found that both CSR and TRPC3 are physically and functionally coupled at the luminal membrane of PT cells. More importantly, TRPC3-deficient mice presented with a deficiency in PT Ca2+ entry/transport, elevated urinary [Ca2+], microcalcifications in LOH and urine microcrystals formations. Taken together, these data suggest that a signaling complex comprising CSR and TRPC3 exists in the PT and can mediate transcellular Ca2+ transport, which could be critical in maintaining the PT luminal [Ca2+] to mitigate formation of the CaP crystals in LOH and subsequent formation of calcium stones.
Asunto(s)
Calcio/metabolismo , Cálculos Renales/etiología , Túbulos Renales Proximales/metabolismo , Receptores Sensibles al Calcio/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Células Epiteliales/metabolismo , Túbulos Renales Proximales/citología , Células LLC-PK1 , Asa de la Nefrona/citología , Asa de la Nefrona/metabolismo , Ratones , Transducción de Señal , PorcinosRESUMEN
In this study, we examined an ammonium conductance in the mouse thick ascending limb cell line ST-1. Whole cell patch clamp was performed to measure currents evoked by NH4Cl in the presence of BaCl2, tetraethylammonium, and BAPTA Application of 20 mmol/L NH4Cl induced an inward current (-272 ± 79 pA, n = 9). In current-voltage (I-V) relationships, NH4Cl application caused the I-V curve to shift down in an inward direction. The difference in current before and after NH4Cl application, which corresponds to the current evoked by NH4Cl, was progressively larger at more negative potentials. The reversal potential for NH4Cl was +15 mV, higher than the equilibrium potential for chloride, indicating that the current should be due to NH4+ We then injected ST-1 poly(A) RNA into Xenopus oocytes and performed two-electrode voltage clamp. NH4Cl application in the presence of BaCl2 caused the I-V curve to be steeper. The NH4+ current was retained at pH 6.4, where endogenous oocyte current was abolished. The NH4+ current was unaffected by 10 µmol/L amiloride but abolished after incubation in Na+-free media. These results demonstrate that the renal cell line ST-1 produces an NH4+ conductance.
Asunto(s)
Potenciales de Acción , Compuestos de Amonio/metabolismo , Asa de la Nefrona/citología , Amilorida/farmacología , Animales , Bario/farmacología , Línea Celular , Cloruros/metabolismo , Diuréticos/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Transporte Iónico , Asa de la Nefrona/metabolismo , Ratones , XenopusRESUMEN
MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this, we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle's loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal, whereas peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation, and morphogenic functions including α-smooth muscle actin, integrin-α8, -ß1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p, and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration, and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis.
Asunto(s)
Diferenciación Celular/genética , ARN Helicasas DEAD-box/fisiología , Neovascularización Fisiológica/genética , Nefronas/embriología , Ribonucleasa III/fisiología , Actinas/metabolismo , Animales , Capilares/embriología , Movimiento Celular/genética , Proliferación Celular/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Femenino , Expresión Génica , Cadenas alfa de Integrinas/metabolismo , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/citología , Glomérulos Renales/embriología , Túbulos Renales/irrigación sanguínea , Túbulos Renales/citología , Túbulos Renales/embriología , Túbulos Renales Distales/irrigación sanguínea , Túbulos Renales Distales/citología , Túbulos Renales Distales/embriología , Túbulos Renales Proximales/irrigación sanguínea , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/embriología , Asa de la Nefrona/irrigación sanguínea , Asa de la Nefrona/citología , Asa de la Nefrona/embriología , Ratones , MicroARNs/genética , Nefronas/anomalías , Nefronas/citología , Organogénesis/genética , Podocitos/fisiología , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Células del Estroma/fisiología , Transcriptoma , Uréter/anomalías , Vía de Señalización Wnt/genéticaRESUMEN
We previously characterized a H(+) transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13(BN)) rats, and superoxide production was enhanced in low Na(+) media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na(+). We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H(+) currents. To test this hypothesis, we developed HV1(-/-) null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH4Cl prepulse in 0 sodium/BaCl2 containing media resulted in superoxide production in thick limb from wild-type but not HV1(-/-) rats (P<0.05) and more rapid recovery of intracellular pH in wild-type rats (ΔpHI 0.005 versus 0.002 U/s, P=0.046, respectively). Superoxide production was enhanced by low intracellular sodium (<10 mmol/L) in both thick limb and peritoneal macrophages only when HV1 was present. When fed a high-salt diet, blood pressure, outer medullary renal injury (tubular casts), and oxidative stress (4-hydroxynonenal staining) were significantly reduced in HV1(-/-) rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na(+) is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.
Asunto(s)
Hipertensión/metabolismo , Canales Iónicos/fisiología , Enfermedades Renales/metabolismo , Asa de la Nefrona/metabolismo , Sodio/metabolismo , Superóxidos/metabolismo , Animales , Modelos Animales de Enfermedad , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Hipertensión/fisiopatología , Canales Iónicos/deficiencia , Canales Iónicos/genética , Enfermedades Renales/fisiopatología , Asa de la Nefrona/citología , Masculino , NADP/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Endogámicas Dahl , Ratas Mutantes , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Prior studies have linked renoprotective effects of estrogens to G-protein-coupled estrogen receptor-1 (GPER-1) and suggest that aldosterone may also activate GPER-1. Here, the role of GPER-1 in murine renal tissue was further evaluated by examining its anatomical distribution, subcellular distribution and steroid binding specificity. Dual immunofluorescent staining using position-specific markers showed that GPER-1 immunoreactivity primarily resides in distal convoluted tubules and the Loop of Henle (stained with Tamm-Horsfall Protein-1). Lower GPER-1 expression was observed in proximal convoluted tubules marked with megalin, and GPER-1 was not detected in collecting ducts. Plasma membrane fractions prepared from whole kidney tissue or HEK293 cells expressing recombinant human GPER-1 (HEK-GPER-1) displayed high-affinity, specific [(3)H]-17ß-estradiol ([(3)H]-E2) binding, but no specific [(3)H]-aldosterone binding. In contrast, cytosolic preparations exhibited specific binding to [(3)H]-aldosterone but not to [(3)H]-E2, consistent with the subcellular distribution of GPER-1 and mineralocorticoid receptor (MR) in these preparations. Aldosterone and MR antagonists, spironolactone and eplerenone, failed to compete for specific [(3)H]-E2 binding to membranes of HEK-GPER-1 cells. Furthermore, aldosterone did not increase [(35)S]-GTP-γS binding to membranes of HEK-GPER-1 cells, indicating that it is not involved in G protein signaling mediated through GPER-1. During the secretory phases of the estrus cycle, GPER-1 is upregulated on cortical epithelia and localized to the basolateral surface during proestrus and redistributed intracellularly during estrus. GPER-1 is down-modulated during luteal phases of the estrus cycle with significantly less receptor on the surface of renal epithelia. Our results demonstrate that GPER-1 is associated with specific estrogen binding and not aldosterone binding and that GPER-1 expression is modulated during the estrus cycle which may suggest a physiological role for GPER-1 in the kidney during reproduction.
Asunto(s)
Estradiol/metabolismo , Estro/fisiología , Túbulos Renales Distales/metabolismo , Asa de la Nefrona/metabolismo , Receptores Acoplados a Proteínas G/genética , Reproducción/fisiología , Aldosterona/metabolismo , Animales , Eplerenona , Femenino , Regulación de la Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HEK293 , Humanos , Túbulos Renales Distales/citología , Asa de la Nefrona/citología , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Antagonistas de Receptores de Mineralocorticoides/farmacología , Unión Proteica , Receptores de Estrógenos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Espironolactona/análogos & derivados , Espironolactona/farmacologíaRESUMEN
The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in â¼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.
Asunto(s)
Células Cultivadas , Asa de la Nefrona/citología , Uromodulina/biosíntesis , Animales , Ratones , Ratones Noqueados , Canales de Potasio de Rectificación Interna/biosíntesis , Miembro 1 de la Familia de Transportadores de Soluto 12/biosíntesisRESUMEN
Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteoma/metabolismo , Shigella flexneri/crecimiento & desarrollo , Shigella flexneri/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Carbono/metabolismo , Línea Celular , Ciclo del Ácido Cítrico/fisiología , Disentería Bacilar/patología , Fermentación/fisiología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Humanos , Hierro/metabolismo , Asa de la Nefrona/citología , Asa de la Nefrona/microbiología , Proteínas de Transporte de Membrana/biosíntesis , NADP Transhidrogenasas/biosíntesis , Shigella flexneri/patogenicidadRESUMEN
Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidney's thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross talk between the two functionally distinct tubular segments. However, the extent, timing, specificity, and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is downregulated at the peak of injury, it is significantly upregulated 48 h after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium, and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of Na(+)-K(+)-2Cl cotransporter (NKCC2) and basolateral targeting of Na(+)-K(+)-ATPase. Compared with the wild-type, THP(-/-) mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of proinflammatory cytokines and chemokines such as monocyte chemoattractant protein-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.
Asunto(s)
Lesión Renal Aguda/metabolismo , Lesión Renal Aguda/patología , Asa de la Nefrona/metabolismo , Nefritis/metabolismo , Circulación Renal/fisiología , Uromodulina/metabolismo , Animales , Biomarcadores/sangre , Polaridad Celular/fisiología , Modelos Animales de Enfermedad , Asa de la Nefrona/citología , Asa de la Nefrona/ultraestructura , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Microscopía Inmunoelectrónica , Nefritis/patología , Pronóstico , Recuperación de la Función/fisiología , Daño por Reperfusión/metabolismo , Daño por Reperfusión/patología , Transducción de Señal/fisiología , Uromodulina/sangre , Uromodulina/orinaRESUMEN
In the inner medulla, radial organization of nephrons and blood vessels around collecting duct (CD) clusters leads to two lateral interstitial regions and preferential intersegmental fluid and solute flows. As the descending (DTLs) and ascending thin limbs (ATLs) pass through these regions, their transepithelial fluid and solute flows are influenced by variable transepithelial solute gradients and structure-to-structure interactions. The goal of this study was to quantify structure-to-structure interactions, so as to better understand compartmentation and flows of transepithelial water, NaCl, and urea and generation of the axial osmotic gradient. To accomplish this, we determined lateral distances of AQP1-positive and AQP1-negative DTLs and ATLs from their nearest CDs, so as to gauge interactions with intercluster and intracluster lateral regions and interactions with interstitial nodal spaces (INSs). DTLs express reduced AQP1 and low transepithelial water permeability along their deepest segments. Deep AQP1-null segments, prebend segments, and ATLs lie equally near to CDs. Prebend segments and ATLs abut CDs and INSs throughout much of their descent and ascent, respectively; however, the distal 30% of ATLs of the longest loops lie distant from CDs as they approach the outer medullary boundary and have minimal interaction with INSs. These relationships occur regardless of loop length. Finally, we show that ascending vasa recta separate intercluster AQP1-positive DTLs from descending vasa recta, thereby minimizing dilution of gradients that drive solute secretion. We hypothesize that DTLs and ATLs enter and exit CD clusters in an orchestrated fashion that is important for generation of the corticopapillary solute gradient by minimizing NaCl and urea loss.
Asunto(s)
Capacidad de Concentración Renal/fisiología , Asa de la Nefrona/citología , Asa de la Nefrona/metabolismo , Animales , Acuaporina 1/metabolismo , Transporte Biológico/fisiología , Permeabilidad de la Membrana Celular/fisiología , Masculino , Modelos Animales , Ósmosis/fisiología , Ratas , Ratas Wistar , Cloruro de Sodio/metabolismo , Urea/metabolismoRESUMEN
Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.
Asunto(s)
Riñón/metabolismo , Factores de Transcripción NFATC/metabolismo , Cloruro de Sodio/farmacología , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Riñón/citología , Riñón/efectos de los fármacos , Asa de la Nefrona/citología , Asa de la Nefrona/efectos de los fármacos , Asa de la Nefrona/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Miembro 1 de la Familia de Transportadores de Soluto 12 , Factor de Necrosis Tumoral alfa/orinaRESUMEN
Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.
Asunto(s)
Linaje de la Célula/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Asa de la Nefrona/embriología , Receptores Acoplados a Proteínas G/biosíntesis , Células Madre/metabolismo , Animales , Humanos , Corteza Renal/citología , Corteza Renal/embriología , Asa de la Nefrona/citología , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/genética , Células Madre/citologíaRESUMEN
Inappropriate Na(+) reabsorption by thick ascending limbs (THALs) induces hypertension. NO produced by NO synthase type 3 (NOS3) inhibits NaCl reabsorption by THALs. Tumor necrosis factor α (TNF-α) decreases NOS3 expression in endothelial cells and contributes to increases in blood pressure. However, the effects of TNF-α on THAL NOS3 and the signaling cascade are unknown. TNF-α activates several signaling pathways, including Rho/Rho kinase (ROCK), which is known to reduce NOS3 expression in endothelial cells. Therefore, we hypothesized that TNF-α decreases NOS3 expression via Rho/ROCK in rat THAL primary cultures. THAL cells were incubated with either vehicle or 1 nmol/L of TNF-α for 24 hours, and NOS3 expression was measured by Western blot. TNF-α decreased NOS3 expression by 51 ± 6% (P<0.002) and blunted stimulus-induced NO production. A 10-minute treatment with TNF-α stimulated RhoA activity by 60 ± 23% (P<0.04). Inhibition of Rho GTPase with 0.05 µg/mL of C3 exoenzyme blocked TNF-α-induced reductions in NOS3 expression by 30 ± 8% (P<0.02). Inhibition of ROCK with 10 µmol/L of H-1152 blocked TNF-α-induced decreases in NOS3 expression by 66 ± 15% (P<0.001). Simultaneous inhibition of Rho and ROCK had no additive effect. Myosin light chain kinase, NO, protein kinase C, mitogen-activated kinase kinase, c-Jun amino terminal kinases, and Rac-1 were also not involved in TNF-α-induced decreases in NOS3 expression. We conclude that TNF-α decreases NOS3 expression primarily via Rho/ROCK in rat THALs. These data suggest that some of the beneficial effects of ROCK inhibitors in hypertension could be attributed to the mitigation of TNF-α-induced reduction in NOS3 expression.
Asunto(s)
Asa de la Nefrona/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Western Blotting , Células Cultivadas , Asa de la Nefrona/citología , Asa de la Nefrona/metabolismo , Masculino , Microscopía Fluorescente , Óxido Nítrico/metabolismo , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Quinasas Asociadas a rho/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Low-oxygen tension is an important component of the stem cell microenvironment. In rodents, renal resident stem cells have been described in the papilla, a relatively hypoxic region of the kidney. In the present study, we found that CD133(+) cells, previously described as renal progenitors in the human cortex, were enriched in the renal inner medulla and localized within the Henle's loop and thin limb segments. Once isolated, the CD133(+) cell population expressed renal embryonic and stem-related transcription factors and was able to differentiate into mature renal epithelial cells. When injected subcutaneously in immunodeficient mice within Matrigel, CD133(+) cells generated canalized structures positive for renal specific markers of different nephron segments. Oct4A levels and differentiation potential of papillary CD133(+) cells were higher than those of CD133(+) cells from cortical tubuli. Hypoxia was able to promote the undifferentiated phenotype of CD133(+) progenitors from papilla. Hypoxia stimulated clonogenicity, proliferation, vascular endothelial growth factor synthesis, and expression of CD133 that were in turn reduced by epithelial differentiation with parallel HIF-1α downregulation. In addition, hypoxia downregulated microRNA-145 and promoted the synthesis of Oct4A. Epithelial differentiation increased microRNA-145 and reduced Oct4 level, suggesting a balance between Oct4 and microRNA-145. MicroRNA-145 overexpression in CD133(+) cells induced downrelation of Oct4A at the protein level, inhibited cell proliferation, and stimulated terminal differentiation. This study underlines the role of the hypoxic microenvironment in controlling the proliferation and maintaining a progenitor phenotype and stem/progenitor properties of CD133(+) cells of the nephron. This mechanism may be at the basis of the maintenance of a CD133(+) population in the papillary region and may be involved in renal regeneration after injury.
Asunto(s)
Antígenos CD/inmunología , Hipoxia de la Célula/fisiología , Glicoproteínas/inmunología , Médula Renal/citología , Asa de la Nefrona/citología , MicroARNs/fisiología , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Péptidos/inmunología , Células Madre/metabolismo , Antígeno AC133 , Animales , Diferenciación Celular/genética , Microambiente Celular , Humanos , Médula Renal/metabolismo , Ratones , Ratones SCID , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre/efectos de los fármacosRESUMEN
Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Túbulos Renales Distales/metabolismo , Asa de la Nefrona/metabolismo , Receptores de Ghrelina/biosíntesis , Animales , Transporte Iónico/fisiología , Túbulos Renales Distales/citología , Asa de la Nefrona/citología , Ratones , Ratones Transgénicos , Especificidad de Órganos/fisiología , Receptores de Ghrelina/genética , Sodio/metabolismoRESUMEN
The renal thick ascending limb (TAL) and distal convoluted tubule (DCT) play central roles in salt homeostasis and blood pressure regulation. An emerging model suggests that bumetanide- and thiazide-sensitive NaCl transporters (NKCC2 and NCC) along these segments are phosphorylated and activated by WNK kinases, via SPAK and OSR1. Here, we show that a kidney-specific SPAK isoform, which lacks the kinase domain, inhibits phosphorylation of NCC and NKCC2 by full-length SPAK in vitro. Kidney-specific SPAK is highly expressed along the TAL, whereas full-length SPAK is more highly expressed along the DCT. As predicted from the differential expression, SPAK knockout in animals has divergent effects along TAL and DCT, with increased phosphorylated NKCC2 along TAL and decreased phosphorylated NCC along DCT. In mice, extracellular fluid volume depletion shifts SPAK isoform abundance to favor NaCl retention along both segments, indicating that a SPAK isoform switch modulates sodium avidity along the distal nephron.
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Presión Sanguínea/fisiología , Líquido Extracelular/metabolismo , Isoenzimas , Túbulos Renales Distales/metabolismo , Asa de la Nefrona/metabolismo , Proteínas Serina-Treonina Quinasas , Cloruro de Sodio/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Regulación de la Expresión Génica , Homeostasis , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/farmacología , Túbulos Renales Distales/citología , Asa de la Nefrona/citología , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Estructura Terciaria de Proteína , Receptores de Droga/genética , Receptores de Droga/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales (Química)/metabolismo , Transducción de Señal , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12 , Miembro 3 de la Familia de Transportadores de Soluto 12 , Simportadores/genética , Simportadores/metabolismoRESUMEN
The thick ascending limb of Henle's loop (TALH) is normally exposed to variable and often very high osmotic stress and involves different mechanisms to counteract this stress. ER resident calcium binding proteins especially calreticulin (CALR) play an important role in different stress balance mechanisms. To investigate the role of CALR in renal epithelial cells adaptation and survival under osmotic stress, two-dimensional fluorescence difference gel electrophoresis combined with mass spectrometry and functional proteomics were performed. CALR expression was significantly altered in TALH cells exposed to osmotic stress, whereas renal inner medullary collecting duct cells and interstitial cells exposed to hyperosmotic stress showed no significant changes in CALR expression. Moreover, a time dependent downregulation of CALR was accompanied with continuous change in the level of free intracellular calcium. Inhibition of the calcium release, through IP3R antagonist, prevented CALR expression alteration under hyperosmotic stress, whereas the cell viability was significantly impaired. Overexpression of wild type CALR in TALH cells resulted in significant decrease in cell viability under hyperosmotic stress. In contrast, the hyperosmotic stress did not have any effect on cells overexpressing the CALR mutant, lacking the calcium-binding domain. Silencing CALR with siRNA significantly improved the cell survival under osmotic stress conditions. Taken together, our data clearly highlight the crucial role of CALR and its calcium-binding role in TALH adaptation and survival under osmotic stress.
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Calcio/metabolismo , Calreticulina/metabolismo , Asa de la Nefrona/metabolismo , Animales , Señalización del Calcio , Calreticulina/deficiencia , Calreticulina/genética , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Supervivencia Celular/fisiología , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Homeostasis , Humanos , Médula Renal/citología , Asa de la Nefrona/citología , Presión Osmótica , Proteoma/metabolismo , Proteómica , Conejos , TransfecciónRESUMEN
The effects of Na(+)-K(+)-2Cl(-) cotransporter type 2 (NKCC2) isoforms on the regulation of nuclear factor of activated T cells isoform 5 (NFAT5) were determined in mouse medullary thick ascending limb (mTAL) cells exposed to high NaCl concentration. Primary cultures of mTAL cells and freshly isolated mTAL tubules, both derived from the outer medulla (outer stripe>inner stripe), express NKCC2 isoforms A and F. The relative expression of NKCC2A mRNA was approximately twofold greater than NKCC2F in these preparations. The abundance of NKCC2A mRNA, but not NKCC2F mRNA, increased approximately twofold when mTAL cells were exposed for 2 h to a change in osmolality from 300 to 500 mosmol/kgH2O, produced with NaCl. Total NKCC2 protein expression also increased. Moreover, a 2.5-fold increase in NFAT5 mRNA accumulation was observed after cells were exposed to 500 mosmol/kgH2O for 4 h. Laser-scanning cytometry detected a twofold increase in endogenous NFAT5 protein expression in response to high NaCl concentration. Pretreatment with the loop diuretic bumetanide dramatically reduced transcriptional activity of the NFAT5-specific reporter construct TonE-Luc in mTAL cells exposed to high NaCl. Transient transfection of mTAL cells with shRNA vectors targeting NKCC2A prevented increases in NFAT5 mRNA abundance and protein expression and inhibited NFAT5 transcriptional activity in response to hypertonic stress. Silencing of NKCC2F mRNA did not affect NFAT5 mRNA accumulation but partially inhibited NFAT5 transcriptional activity. These findings suggest that NKCC2A and NKCC2F exhibit differential effects on NFAT5 expression and transcriptional activity in response to hypertonicity produced by high NaCl concentration.
Asunto(s)
Médula Renal/metabolismo , Asa de la Nefrona/metabolismo , Factores de Transcripción NFATC/metabolismo , Isoformas de Proteínas/metabolismo , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Análisis de Varianza , Animales , Western Blotting , Células Cultivadas , Regulación de la Expresión Génica , Médula Renal/citología , Médula Renal/efectos de los fármacos , Asa de la Nefrona/citología , Asa de la Nefrona/efectos de los fármacos , Masculino , Ratones , Factores de Transcripción NFATC/genética , Isoformas de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Simportadores de Cloruro de Sodio-Potasio/genética , Miembro 1 de la Familia de Transportadores de Soluto 12RESUMEN
Connexins are the main components of gap junction channels, which are important for intercellular communication. In the kidney, several members of the connexin (Cx) family have been identified. Renal vascular expression and hemodynamic impacts have so far been shown for Cx37, Cx40, and Cx43. Additionally, Cx30, Cx30.3, and Cx43 have been identified to be part of tubular epithelial gap junctions and/or hemichannels. However, the localization and role of other Cx family members in renal epithelial structures remain undetermined. We aimed to localize Cx37 in the kidney to obtain information on its epithelial expression and potential functions. Immunohistochemistry in rodent kidney showed characteristic punctate patterns in the vasculature and along the nephron. Strong basolateral expression was found in the thick ascending limb and distal convoluted tubule. Weaker abundances were found in the proximal tubule and the collecting duct also at the basolateral side. In situ hybridization and real-time PCR of isolated nephron segments confirmed this distribution at the mRNA level. Ultrastructurally, Cx37 immunostaining was confined to basolateral cell interdigitations and infoldings. As a functional approach, rats were fed low- or high-salt diets. Compared with control and high-salt diets, rats treated with low-salt diet showed significantly increased Cx37 mRNA and protein levels. This may be indicative of an adaptive tubular response to changes in sodium reabsorption. In summary, renal epithelia express Cx37 in their basolateral membranes. Here, the formation of Cx37 gap junctions may be involved in cellular communication and adjustments of vectorial epithelial transport.