RESUMO
The amiloride-sensitive epithelial sodium channel (ENaC) and the thiazide-sensitive sodium chloride cotransporter (NCC) are key regulators of sodium and potassium and colocalize in the late distal convoluted tubule of the kidney. Loss of the αENaC subunit leads to a perinatal lethal phenotype characterized by sodium loss and hyperkalemia resembling the human syndrome pseudohypoaldosteronism type 1 (PHA-I). In adulthood, inducible nephron-specific deletion of αENaC in mice mimics the lethal phenotype observed in neonates, and as in humans, this phenotype is prevented by a high sodium (HNa+)/low potassium (LK+) rescue diet. Rescue reflects activation of NCC, which is suppressed at baseline by elevated plasma potassium concentration. In this study, we investigated the role of the γENaC subunit in the PHA-I phenotype. Nephron-specific γENaC knockout mice also presented with salt-wasting syndrome and severe hyperkalemia. Unlike mice lacking αENaC or ßΕΝaC, an HNa+/LK+ diet did not normalize plasma potassium (K+) concentration or increase NCC activation. However, when K+ was eliminated from the diet at the time that γENaC was deleted, plasma K+ concentration and NCC activity remained normal, and progressive weight loss was prevented. Loss of the late distal convoluted tubule, as well as overall reduced ßENaC subunit expression, may be responsible for the more severe hyperkalemia. We conclude that plasma K+ concentration becomes the determining and limiting factor in regulating NCC activity, regardless of Na+ balance in γENaC-deficient mice.
Assuntos
Canais Epiteliais de Sódio/genética , Hiperpotassemia/genética , Potássio/sangue , Pseudo-Hipoaldosteronismo/sangue , Pseudo-Hipoaldosteronismo/genética , Animais , Quelantes/uso terapêutico , Suplementos Nutricionais , Hiperpotassemia/sangue , Hiperpotassemia/tratamento farmacológico , Camundongos , Camundongos Knockout , Néfrons , Poliestirenos/uso terapêutico , Potássio na Dieta/administração & dosagem , Sódio na Dieta/administração & dosagem , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Uric acid (UA) is a metabolite of purine degradation and is involved in gout flairs and kidney stones formation. GLUT9 (SLC2A9) was previously shown to be a urate transporter in vitro. In vivo, humans carrying GLUT9 loss-of-function mutations have familial renal hypouricemia type 2, a condition characterized by hypouricemia, UA renal wasting associated with kidney stones, and an increased propensity to acute renal failure during strenuous exercise. Mice carrying a deletion of GLUT9 in the whole body are hyperuricemic and display a severe nephropathy due to intratubular uric acid precipitation. However, the precise role of GLUT9 in the kidney remains poorly characterized. We developed a mouse model in which GLUT9 was deleted specifically along the whole nephron in a tetracycline-inducible manner (subsequently called kidney-inducible KO or kiKO). The urate/creatinine ratio was increased as early as 4 days after induction of the KO and no GLUT9 protein was visible on kidney extracts. kiKO mice are morphologically identical to their wild-type littermates and had no spontaneous kidney stones. Twenty-four-hour urine collection revealed a major increase of urate urinary excretion rate and of the fractional excretion of urate, with no difference in urate concentration in the plasma. Polyuria was observed, but kiKO mice were still able to concentrate urine after water restriction. KiKO mice displayed lower blood pressure accompanied by an increased heart rate. Overall, these results indicate that GLUT9 is a crucial player in renal handling of urate in vivo and a putative target for uricosuric drugs.
Assuntos
Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Rim/metabolismo , Reabsorção Renal , Ácido Úrico/metabolismo , Animais , Pressão Sanguínea , Feminino , Proteínas Facilitadoras de Transporte de Glucose/genética , Frequência Cardíaca , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Úrico/sangue , Ácido Úrico/urinaRESUMO
Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is critical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the molecular identity of the protein(s) participating in the basolateral Pi efflux remains unknown. Evidence has suggested that xenotropic and polytropic retroviral receptor 1 (XPR1) might be involved in this process. Here, we show that conditional inactivation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dysfunction. These results identify XPR1 as a major regulator of Pi homeostasis and as a potential therapeutic target in bone and kidney disorders.
Assuntos
Síndrome de Fanconi/etiologia , Néfrons , Receptores Acoplados a Proteínas G/fisiologia , Receptores Virais/fisiologia , Raquitismo Hipofosfatêmico/etiologia , Animais , Feminino , Masculino , Camundongos , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
In adulthood, an induced nephron-specific deficiency of αENaC (Scnn1a) resulted in pseudohypoaldosteronism type 1 (PHA-1) with sodium loss, hyperkalemia, and metabolic acidosis that is rescued through high-sodium/low-potassium (HNa+/LK+) diet. In the present study, we addressed whether renal ßENaC expression is required for sodium and potassium balance or can be compensated by remaining (α and γ) ENaC subunits using adult nephron-specific knockout (Scnn1bPax8/LC1) mice. Upon induction, these mice present a severe PHA-1 phenotype with weight loss, hyperkalemia, and dehydration, but unlike the Scnn1aPax8/LC1 mice without persistent salt wasting. This is followed by a marked downregulation of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and Na+/Cl- co-transporter (NCC) protein expression and activity. Most of the experimental Scnn1bPax8/LC1 mice survived with a HNa+/LK+ diet that partly normalized NCC phosphorylation, but not total NCC expression. Since salt loss was minor, we applied a standard-sodium/LK+ diet that efficiently rescued these mice resulting in normokalemia and normalization of NCC phosphorylation, but not total NCC expression. A further switch to LNa+/standard-K+ diet induced again a severe PHA-1-like phenotype, but with only transient salt wasting indicating that low-K+ intake is critical to decrease hyperkalemia in a NCC-dependent manner. In conclusion, while the ßENaC subunit plays only a minor role in sodium balance, severe hyperkalemia results in downregulation of NCC expression and activity. Our data demonstrate the importance to primarily correct the hyperkalemia with a low-potassium diet that normalizes NCC activity.
Assuntos
Dieta Hipossódica , Canais Epiteliais de Sódio/metabolismo , Hiperpotassemia/metabolismo , Potássio/metabolismo , Animais , Rim/metabolismo , Camundongos Transgênicos , Néfrons/metabolismo , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Sódio/metabolismoRESUMO
The circadian clock controls a wide variety of metabolic and homeostatic processes in a number of tissues, including the kidney. However, the role of the renal circadian clocks remains largely unknown. To address this question, we performed a combined functional, transcriptomic, and metabolomic analysis in mice with inducible conditional knockout (cKO) of BMAL1, which is critically involved in the circadian clock system, in renal tubular cells (Bmal1lox/lox/Pax8-rtTA/LC1 mice). Induction of cKO in adult mice did not produce obvious abnormalities in renal sodium, potassium, or water handling. Deep sequencing of the renal transcriptome revealed significant changes in the expression of genes related to metabolic pathways and organic anion transport in cKO mice compared with control littermates. Furthermore, kidneys from cKO mice exhibited a significant decrease in the NAD+-to-NADH ratio, which reflects the oxidative phosphorylation-to-glycolysis ratio and/or the status of mitochondrial function. Metabolome profiling showed significant changes in plasma levels of amino acids, biogenic amines, acylcarnitines, and lipids. In-depth analysis of two selected pathways revealed a significant increase in plasma urea level correlating with increased renal Arginase II activity, hyperargininemia, and increased kidney arginine content as well as a significant increase in plasma creatinine concentration and a reduced capacity of the kidney to secrete anionic drugs (furosemide) paralleled by an approximate 80% decrease in the expression level of organic anion transporter 3 (SLC22a8). Collectively, these results indicate that the renal circadian clocks control a variety of metabolic/homeostatic processes at the intrarenal and systemic levels and are involved in drug disposition.
Assuntos
Fatores de Transcrição ARNTL/genética , Relógios Circadianos/genética , Diuréticos/metabolismo , Furosemida/metabolismo , Rim/metabolismo , Metaboloma/genética , Animais , Diuréticos/sangue , Furosemida/sangue , Camundongos , NéfronsRESUMO
Systemic pseudohypoaldosteronism type 1 (PHA-1) is a severe salt-losing syndrome caused by loss-of-function mutations of the amiloride-sensitive epithelial sodium channel (ENaC) and characterized by neonatal life-threatening hypovolemia and hyperkalemia. The very high plasma aldosterone levels detected under hypovolemic or hyperkalemic challenge can lead to increased or decreased sodium reabsorption, respectively, through the Na(+)/Cl(-) cotransporter (NCC). However, the role of ENaC deficiency remains incompletely defined, because constitutive inactivation of individual ENaC subunits is neonatally lethal in mice. We generated adult inducible nephron-specific αENaC-knockout mice (Scnn1a(Pax8/LC1)) that exhibit hyperkalemia and body weight loss when kept on a regular-salt diet, thus mimicking PHA-1. Compared with control mice fed a regular-salt diet, knockout mice fed a regular-salt diet exhibited downregulated expression and phosphorylation of NCC protein, despite high plasma aldosterone levels. In knockout mice fed a high-sodium and reduced-potassium diet (rescue diet), although plasma aldosterone levels remained significantly increased, NCC expression returned to control levels, and body weight, plasma and urinary electrolyte concentrations, and excretion normalized. Finally, shift to a regular diet after the rescue diet reinstated the symptoms of severe PHA-1 syndrome and significantly reduced NCC phosphorylation. In conclusion, lack of ENaC-mediated sodium transport along the nephron cannot be compensated for by other sodium channels and/or transporters, only by a high-sodium and reduced-potassium diet. We further conclude that hyperkalemia becomes the determining factor in regulating NCC activity, regardless of sodium loss, in the ENaC-mediated salt-losing PHA-1 phenotype.
Assuntos
Canais Epiteliais de Sódio/genética , Hiperpotassemia/genética , Pseudo-Hipoaldosteronismo/genética , Animais , Camundongos , Camundongos Knockout , Néfrons , Índice de Gravidade de DoençaRESUMO
Aldosterone is the main mineralocorticoid hormone controlling sodium balance, fluid homeostasis, and blood pressure by regulating sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). Germline loss-of-function mutations of the mineralocorticoid receptor (MR) in humans and in mice lead to the "renal" form of type 1 pseudohypoaldosteronism (PHA-1), a case of aldosterone resistance characterized by salt wasting, dehydration, failure to thrive, hyperkalemia, and metabolic acidosis. To investigate the importance of MR in adult epithelial cells, we generated nephron-specific MR knockout mice (MR(Pax8/LC1)) using a doxycycline-inducible system. Under standard diet, MR(Pax8/LC1) mice exhibit inability to gain weight and significant weight loss compared to control mice. Interestingly, despite failure to thrive, MR(Pax8/LC1) mice survive but develop a severe PHA-1 phenotype with higher urinary Na(+) levels, decreased plasma Na(+), hyperkalemia, and higher levels of plasma aldosterone. This phenotype further worsens and becomes lethal under a sodium-deficient diet. Na(+)/Cl(-) co-transporter (NCC) protein expression and its phosphorylated form are downregulated in the MR(Pax8/LC1) knockouts, as well as the αENaC protein expression level, whereas the expression of glucocorticoid receptor (GR) is increased. A diet rich in Na(+) and low in K(+) does not restore plasma aldosterone to control levels but is sufficient to restore body weight, plasma, and urinary electrolytes. In conclusion, MR deletion along the nephron fully recapitulates the features of severe human PHA-1. ENaC protein expression is dependent on MR activity. Suppression of NCC under hyperkalemia predominates in a hypovolemic state.
Assuntos
Néfrons/metabolismo , Fenótipo , Pseudo-Hipoaldosteronismo/metabolismo , Receptores de Mineralocorticoides/deficiência , Aldosterona/sangue , Animais , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Deleção de Genes , Camundongos , Potássio/sangue , Potássio/urina , Pseudo-Hipoaldosteronismo/genética , Pseudo-Hipoaldosteronismo/patologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Sódio/sangue , Sódio/urina , Simportadores de Cloreto de Sódio/genética , Simportadores de Cloreto de Sódio/metabolismo , Redução de PesoRESUMO
Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.
Assuntos
Acidose/metabolismo , Amônia/metabolismo , Rim/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Acidose/patologia , Acidose/urina , Amônia/urina , Animais , Western Blotting , Feminino , Glucosiltransferases/deficiência , Glucosiltransferases/genética , Homeostase , Concentração de Íons de Hidrogênio , Túbulos Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Sulfotransferases/deficiência , Sulfotransferases/genética , Simportadores/genética , Simportadores/metabolismo , Urina/químicaRESUMO
Renal endothelin-converting enzyme (ECE)-1 is induced in experimental diabetes and following radiocontrast administration, conditions characterized by renal hypoxia, hypoxia-inducible factor (HIF) stabilization, and enhanced endothelin synthesis. Here we tested whether ECE-1 might be a HIF-target gene in vitro and in vivo. ECE-1 transcription and expression increased in cultured vascular endothelial and proximal tubular cell lines, subject to hypoxia, to mimosine or cobalt chloride. These interventions are known to stabilize HIF signaling by inhibition of HIF-prolyl hydroxylases. In rats, HIF-prolyl-hydroxylase inhibition by mimosine or FG-4497 increased HIF-1α immunostaining in renal tubules, principally in distal nephron segments. This was associated with markedly enhanced ECE-1 protein expression, predominantly in the renal medulla. A progressive and dramatic increase in ECE-1 immunostaining over time, in parallel with enhanced HIF expression, was also noted in conditional von Hippel-Lindau knockout mice. Since HIF and STAT3 are cross-stimulated, we triggered HIF expression by STAT3 activation in mice, transfected by or injected with a chimeric IL-6/IL-6-receptor protein, and found a similar pattern of enhanced ECE-1 expression. Chromatin immunoprecipitation sequence (ChIP-seq) and PCR analysis in hypoxic endothelial cells identified HIF binding at the ECE-1 promoter and intron regions. Thus, our findings suggest that ECE-1 may be a novel HIF-target gene.
Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Cobalto/farmacologia , Dioxigenases/antagonistas & inibidores , Enzimas Conversoras de Endotelina , Células Endoteliais da Veia Umbilical Humana , Humanos , Íntrons , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mimosina/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Inibidores de Prolil-Hidrolase/farmacologia , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transcrição Gênica , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/metabolismoRESUMO
The proximal renal epithelia express three different Na-dependent inorganic phosphate (Pi) cotransporters: NaPi-IIa/SLC34A1, NaPi-IIc/SLC34A3, and PiT2/SLC20A2. Constitutive mouse knockout models of NaPi-IIa and NaPi-IIc suggested that NaPi-IIa mediates the bulk of renal reabsorption of Pi whereas the contribution of NaPi-IIc to this process is minor and probably restricted to young mice. However, many reports indicate that mutations of NaPi-IIc in humans lead to hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report the generation of a kidney-specific and inducible NaPi-IIc-deficient mouse model based on the loxP-Cre system. We found that the specific removal of the cotransporter from the kidneys of young mice does not impair the capacity of the renal epithelia to transport Pi. Moreover, the levels of Pi in plasma and urine as well as the circulating levels of parathyroid hormone, FGF-23, and vitamin D3 remained unchanged. These findings are in agreement with the data obtained with the constitutive knockout model and suggest that, under steady-state conditions of normal dietary Pi, NaPi-IIc is not an essential Na-Pi cotransporter in murine kidneys. However, and unlike the constitutive mutants, the kidney-specific depletion of NaPi-IIc does not result in alteration of the homeostasis of calcium. This suggests that the calcium-related phenotype observed in constitutive knockout mice may not be related to inactivation of the cotransporter in kidney.
Assuntos
Cálcio/metabolismo , Fosfatos/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/genética , Animais , Calcitriol/metabolismo , Doxiciclina/farmacologia , Raquitismo Hipofosfatêmico Familiar/fisiopatologia , Fator de Crescimento de Fibroblastos 23 , Homeostase/efeitos dos fármacos , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIc/deficiênciaRESUMO
Renal hypoxia occurs in AKI of various etiologies, but adaptation to hypoxia, mediated by hypoxia-inducible factor (HIF), is incomplete in these conditions. Preconditional HIF activation protects against renal ischemia-reperfusion injury, yet the mechanisms involved are largely unknown, and HIF-mediated renoprotection has not been examined in other causes of AKI. Here, we show that selective activation of HIF in renal tubules, through Pax8-rtTA-based inducible knockout of von Hippel-Lindau protein (VHL-KO), protects from rhabdomyolysis-induced AKI. In this model, HIF activation correlated inversely with tubular injury. Specifically, VHL deletion attenuated the increased levels of serum creatinine/urea, caspase-3 protein, and tubular necrosis induced by rhabdomyolysis in wild-type mice. Moreover, HIF activation in nephron segments at risk for injury occurred only in VHL-KO animals. At day 1 after rhabdomyolysis, when tubular injury may be reversible, the HIF-mediated renoprotection in VHL-KO mice was associated with activated glycolysis, cellular glucose uptake and utilization, autophagy, vasodilation, and proton removal, as demonstrated by quantitative PCR, pathway enrichment analysis, and immunohistochemistry. In conclusion, a HIF-mediated shift toward improved energy supply may protect against acute tubular injury in various forms of AKI.
Assuntos
Injúria Renal Aguda/prevenção & controle , Rabdomiólise/complicações , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/análise , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Caspase 3/análise , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Rim/patologia , Masculino , Camundongos , Camundongos KnockoutRESUMO
In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Rim/metabolismo , Fator de Transcrição STAT3/metabolismo , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Animais , Hipóxia/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxigênio/farmacologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
ß-Catenin/Wnt signaling is essential during early inductive stages of kidney development, but its role during postinductive stages of nephron development and maturation is not well understood. In this study, we used Pax8Cre mice to target ß-catenin deficiency to renal epithelial cells at the late S-shaped body stage and the developing collecting ducts. The conditional ß-catenin knockout mice formed abnormal kidneys and had reduced renal function. The kidneys were hypoplastic with a thin cortex; a superficial layer of tubules was missing. A high proportion of glomeruli had small, underdeveloped capillary tufts. In these glomeruli, well differentiated podocytes replaced parietal epithelial cells in Bowman's capsule; capillaries toward the outer aspect of these podocytes mimicked the formation of glomerular capillaries. Tracing nephrogenesis in embryonic conditional ß-catenin knockout mice revealed that these "parietal podocytes" derived from precursor cells in the parietal layer of the S-shaped body by direct lineage switch. Taken together, these findings demonstrate that ß-catenin/Wnt signaling is important during the late stages of nephrogenesis and for the lineage specification of parietal epithelial cells.
Assuntos
Rim/embriologia , Organogênese , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Epiteliais/citologia , Rim/citologia , Rim/metabolismo , Camundongos , Camundongos Knockout , beta Catenina/genéticaRESUMO
We investigated the signaling basis for tubule pathology during fibrosis after renal injury. Numerous signaling pathways are activated physiologically to direct tubule regeneration after acute kidney injury (AKI) but several persist pathologically after repair. Among these, transforming growth factor (TGF)-ß is particularly important because it controls epithelial differentiation and profibrotic cytokine production. We found that increased TGF-ß signaling after AKI is accompanied by PTEN loss from proximal tubules (PT). With time, subpopulations of regenerating PT with persistent loss of PTEN (phosphate and tension homolog) failed to differentiate, became growth arrested, expressed vimentin, displayed profibrotic JNK activation, and produced PDGF-B. These tubules were surrounded by fibrosis. In contrast, PTEN recovery was associated with epithelial differentiation, normal tubule repair, and less fibrosis. This beneficial outcome was promoted by TGF-ß antagonism. Tubule-specific induction of TGF-ß led to PTEN loss, JNK activation, and fibrosis even without prior AKI. In PT culture, high TGF-ß depleted PTEN, inhibited differentiation, and activated JNK. Conversely, TGF-ß antagonism increased PTEN, promoted differentiation, and decreased JNK activity. Cre-Lox PTEN deletion suppressed differentiation, induced growth arrest, and activated JNK. The low-PTEN state with JNK signaling and fibrosis was ameliorated by contralateral nephrectomy done 2 wk after unilateral ischemia, suggesting reversibility of the low-PTEN dysfunctional tubule phenotype. Vimentin-expressing tubules with low-PTEN and JNK activation were associated with fibrosis also after tubule-selective AKI, and with human chronic kidney diseases of diverse etiology. By preventing tubule differentiation, the low-PTEN state may provide a platform for signals initiated physiologically to persist pathologically and cause fibrosis after injury.
Assuntos
Diferenciação Celular , Túbulos Renais Proximais/patologia , MAP Quinase Quinase 4/fisiologia , PTEN Fosfo-Hidrolase/deficiência , Fenótipo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Células Cultivadas , Doença Crônica , Fibrose , Humanos , Nefropatias/patologia , Nefropatias/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Ratos , Ratos Sprague-Dawley , Regeneração/fisiologia , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologiaRESUMO
In many kidney diseases, the original insult primarily involves the glomerulus and may then pass onto the tubulointerstitium. Several hypotheses link glomerular disease to tubular injury; perhaps the foremost hypothesis involves chronic tubular hypoxia. The reported effects of hypoxia and consecutive stabilization of hypoxia-inducible factors (HIFs), however, are controversial. Hypoxia induces interstitial fibrosis but also has beneficial effects on renal disease progression when HIF is activated pharmacologically. To analyze the impact of HIF on tubulointerstitial disease development in primary glomerular disease, transgenic von Hippel Lindau (VHL)-knockout mice were generated and null expression was induced before the onset of autoimmune IgG-mediated anti-glomerular basement membrane glomerulonephritis (GN). Tubular VHL knockout and, thus, local HIF-α stabilization increased renal production of vascular endothelial growth factor, tumor growth factor-ß(1), and platelet-derived growth factor-B, resulting in augmented formation of capillaries and interstitial matrix, and conversion of fibroblasts to myofibroblasts. Within the glomerular disease, VHL knockout reduced the glomerular damage and attenuated tubulointerstitial injury. Likewise, proteinuria, plasma urea concentration, and tubulointerstitial matrix were decreased in VHL knockout with GN. These findings shown that tubular HIF-α stabilization in glomerular disease is beneficial for disease outcome. In comparison with VHL knockout alone, GN is a much stronger activator of fibrosis such that stimuli other than hypoxia may be considered important for renal disease progression.
Assuntos
Doença Antimembrana Basal Glomerular/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Doença de von Hippel-Lindau/fisiopatologia , Animais , Autoanticorpos/administração & dosagem , Autoanticorpos/farmacologia , Capilares/citologia , Proliferação de Células , Progressão da Doença , Imuno-Histoquímica , Glomérulos Renais/irrigação sanguínea , Masculino , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Endothelin, via endothelin A receptors (ETA), exerts multiple pathologic effects that contribute to disease pathogenesis throughout the body. ETA antagonists ameliorate many experimental diseases and have been extensively utilized in clinical trials. The utility of ETA blockers has been greatly limited, however, by fluid retention, sometimes leading to heart failure or death. To begin to examine this issue, the effect of genetic disruption of ETA in the nephron on blood pressure and salt handling was determined. METHODS: Mice were generated with doxycycline-inducible nephron-specific ETA deletion using Pax8-rtTA and LC-1 transgenes on the background of homozygous loxP-flanked ETA alleles. Arterial pressure, Na metabolism and measures of body fluid volume status (hematocrit and impedance plethysmography) were assessed. RESULTS: Absence of nephron ETA did not alter arterial pressure whether mice were ingesting a normal or high Na diet. Nephron ETA disruption did not detectably affect 24 hr Na excretion or urine volume regardless of Na intake. However, mice with nephron ETA knockout that were fed a high Na diet had mild fluid retention as evidenced by an increase in body weight and a fall in hematocrit. CONCLUSIONS: Genetic deletion of nephron ETA causes very modest fluid retention that does not alter arterial pressure. Nephron ETA, under normal conditions, likely do not play a major role in regulation of Na excretion or systemic hemodynamics.
Assuntos
Pressão Sanguínea , Volume Sanguíneo/fisiologia , Líquidos Corporais/fisiologia , Néfrons/fisiologia , Receptor de Endotelina A/deficiência , Animais , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Volume Sanguíneo/genética , Feminino , Hemodinâmica/genética , Hemodinâmica/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Receptor de Endotelina A/genéticaRESUMO
Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.
Assuntos
Aldosterona/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Distais/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Regulação para Baixo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação , Transdução de Sinais , Ubiquitinação , Proteínas de Xenopus , Xenopus laevisRESUMO
The paired-box transcription factor Pax2 plays a major role in early development of the kidney and the central nervous system. It is expressed in the metanephric mesenchyme of the developing kidney, at the midbrain-hindbrain boundary and the anlagen of the inner ear. The early expression of Pax2, especially in the developing kidney, prompted us to use this locus as a novel genetic tool to introduce temporally-controlled expression of transgenes. We generated a transgenic Pax2-rtTA mouse strain through genetic recombineering using a large BAC clone which drives expression of TetO-controlled transgenes upon doxycycline treatment in natively Pax2-expressing tissues. We show that expression of a TetO-responsive lacZ gene is tightly regulated by addition of doxycycline and can be detected in all Pax2-expressing tissues. Our transgenic Pax2-rtTA mouse thus represents a suitable tool to study the cell fates and molecular pathways in Pax2-positive tissues during development, such as the kidney. We further propose that the Pax2-rtTA tool has great potential to induce time-controlled, tissue-specific alterations for tumorigenic transformation of Pax2-expressing cells for generating in vivo tumor models, such as Wilms tumor.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Doxiciclina/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição PAX2/genética , Animais , Cromossomos Artificiais Bacterianos , Proteínas de Ligação a DNA/metabolismo , Rim/embriologia , Rim/metabolismo , Óperon Lac , Camundongos , Camundongos Transgênicos , Fator de Transcrição PAX2/metabolismo , Regiões Promotoras Genéticas , TransgenesRESUMO
We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-beta1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena. The initial response to tubular cell-derived TGF-beta1 consisted of a robust proliferation of peritubular cells and deposition of collagen. On sustained expression, nephrons degenerated in a focal pattern. This process started with tubular dedifferentiation and proceeded to total decomposition of tubular cells by autophagy. The final outcome was empty collapsed remnants of tubular basement membrane embedded into a dense collagenous fibrous tissue. The corresponding glomeruli survived as atubular remnants. Thus, TGF-beta1 driven autophagy may represent a novel mechanism of tubular decomposition. The fibrosis seen in between intact tubules and in areas of tubular decomposition resulted from myofibroblasts that were derived from local fibroblasts. No evidence was found for a transition of tubular cells into myofibroblasts. Neither tracing of injured tubules in electron micrographs nor genetic tagging of tubular epithelial cells revealed cells transgressing the tubular basement membrane. In conclusion, overexpression of TGF-beta1 in renal tubules in vivo induces interstitial proliferation, tubular autophagy, and fibrosis, but not epithelial-to-mesenchymal transition.
Assuntos
Autofagia/fisiologia , Células Epiteliais/fisiologia , Transição Epitelial-Mesenquimal/fisiologia , Túbulos Renais , Rim , Fator de Crescimento Transformador beta1/metabolismo , Animais , Células Epiteliais/ultraestrutura , Fibrose/metabolismo , Fibrose/patologia , Rim/citologia , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Camundongos , Camundongos Endogâmicos , Camundongos TransgênicosRESUMO
The role of vascular endothelial growth factor (VEGF) in renal fibrosis, tubular cyst formation, and glomerular diseases is incompletely understood. We studied a new conditional transgenic mouse system [Pax8-rtTA/(tetO)(7)VEGF], which allows increased tubular VEGF production in adult mice. The following pathology was observed. The interstitial changes consisted of a ubiquitous proliferation of peritubular capillaries and fibroblasts, followed by deposition of matrix leading to a unique kind of fibrosis, ie, healthy tubules amid a capillary-rich dense fibrotic tissue. In tubular segments with high expression of VEGF, cysts developed that were surrounded by a dense network of peritubular capillaries. The glomerular effects consisted of a proliferative enlargement of glomerular capillaries, followed by mesangial proliferation. This resulted in enlarged glomeruli with loss of the characteristic lobular structure. Capillaries became randomly embedded into mesangial nodules, losing their filtration surface. Serum VEGF levels were increased, whereas endogenous VEGF production by podocytes was down-regulated. Taken together, this study shows that systemic VEGF interferes with the intraglomerular cross-talk between podocytes and the endocapillary compartment. It suppresses VEGF secretion by podocytes but cannot compensate for the deficit. VEGF from podocytes induces a directional effect, attracting the capillaries to the lobular surface, a relevant mechanism to optimize filtration surface. Systemic VEGF lacks this effect, leading to severe deterioration in glomerular architecture, similar to that seen in diabetic nephropathy.