RESUMO
Autosomal dominant mutations in cullin-3 ( Cul3) cause the most severe form of familial hyperkalemic hypertension (FHHt). Cul3 mutations cause skipping of exon 9, which results in an internal deletion of 57 amino acids from the CUL3 protein (CUL3-∆9). The precise mechanism by which this altered form of CUL3 causes FHHt is controversial. CUL3 is a member of the cullin-RING ubiquitin ligase family that mediates ubiquitination and thus degradation of cellular proteins, including with-no-lysine [K] kinases (WNKs). In CUL3-∆9-mediated FHHt, proteasomal degradation of WNKs is abrogated, leading to overactivation of the WNK targets sterile 20/SPS-1 related proline/alanine-rich kinase and oxidative stress-response kinase-1, which directly phosphorylate and activate the thiazide-sensitive Na+-Cl- cotransporter. Several groups have suggested different mechanisms by which CUL3-∆9 causes FHHt. The majority of these are derived from in vitro data, but recently the Kurz group (Schumacher FR, Siew K, Zhang J, Johnson C, Wood N, Cleary SE, Al Maskari RS, Ferryman JT, Hardege I, Figg NL, Enchev R, Knebel A, O'Shaughnessy KM, Kurz T. EMBO Mol Med 7: 1285-1306, 2015) described the first mouse model of CUL3-∆9-mediated FHHt. Analysis of this model suggested that CUL3-∆9 is degraded in vivo, and thus Cul3 mutations cause FHHt by inducing haploinsufficiency. We recently directly tested this model but found that other dominant effects of CUL3-∆9 must contribute to the development of FHHt. In this review, we focus on our current knowledge of CUL3-∆9 action gained from in vitro and in vivo models that may help unravel this complex problem.
Assuntos
Pressão Sanguínea , Proteínas Culina/genética , Mutação , Néfrons/enzimologia , Pseudo-Hipoaldosteronismo/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Pressão Sanguínea/genética , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Modelos Animais de Doenças , Estabilidade Enzimática , Predisposição Genética para Doença , Haploinsuficiência , Humanos , Proteínas dos Microfilamentos , Néfrons/fisiopatologia , Fenótipo , Pseudo-Hipoaldosteronismo/diagnóstico , Pseudo-Hipoaldosteronismo/enzimologia , Pseudo-Hipoaldosteronismo/fisiopatologiaRESUMO
Reversible posttranslational modification of proteins is a critically important process in physiological regulation in all tissues, including the kidney. Lysine acetylation occurs in all organisms, including prokaryotes, and is regulated by a balance between the lysine acetyltransferases (adding an acetyl group to the ε-amino group of a lysine) and deacetylases (removing it). The kidney is an organ rich with acetylated lysines, which map to >2,000 unique histone and nonhistone proteins. However, the functional significance of these modifications remains to be discovered. Here, we have compiled gene lists of the acetyltransferases and deacetylases in the mammalian genomes and mapped their mRNA expression along the renal tubule. These lists will be useful for generating targeted approaches to test the physiological or pathophysiological significance of lysine acetylation changes in the kidney.
Assuntos
Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Néfrons/enzimologia , Acetilação , Acetiltransferases/genética , Animais , Histona Desacetilases/genética , HumanosRESUMO
Deletion of cyclooxygenase-2 (COX-2) causes impairment of postnatal kidney development. Here we tested whether the renin angiotensin system contributes to COX-2-dependent nephrogenesis in mice after birth and whether a rescue of impaired renal development and function in COX-2-/- mice was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P3 to P8, the critical time frame of renal COX-2 expression, led to hypoplastic glomeruli, a thinned subcapsular cortex and maturational arrest of superficial glomeruli quite similar to that observed in COX-2-/- mice. In contrast, AT2 receptor antagonist PD123319 was without any effect on renal development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L162313 rescued impaired renal function by reducing serum urea and creatinine and mitigated pathologic albumin excretion. Moreover, glomerulosclerosis in the kidneys of COX-2-/- mice was reduced. Thus, angiotensin II-AT1-receptor signaling is necessary for COX-2-dependent normal postnatal nephrogenesis and maturation.
Assuntos
Angiotensina II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Néfrons/enzimologia , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina , Transdução de Sinais , Fatores Etários , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Animais Recém-Nascidos , Creatinina/sangue , Ciclo-Oxigenase 2/deficiência , Ciclo-Oxigenase 2/genética , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Feminino , Genótipo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Néfrons/efeitos dos fármacos , Néfrons/crescimento & desenvolvimento , Néfrons/patologia , Fenótipo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/metabolismo , Renina/sangue , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ureia/sangueRESUMO
Adenylyl cyclases (AC) catalyze formation of cAMP, a critical component of G protein-coupled receptor signaling. So far, nine distinct membrane-bound AC isoforms (AC1-9) and one soluble AC (sAC) have been identified and, except for AC8, all of them are expressed in the kidney. While the role of ACs in renal cAMP formation is well established, we are just beginning to understand the function of individual AC isoforms, particularly with regard to hormonal regulation of transporter and channel phosphorylation, membrane abundance, and trafficking. This review focuses on the role of different AC isoforms in regulating renal water and electrolyte transport in health as well as potential pathological implications of disordered AC isoform function. In particular, we focus on modulation of transporter and channel abundance, activity, and phosphorylation, with an emphasis on studies employing genetically modified animals. As will be described, it is now evident that specific AC isoforms can exert unique effects in the kidney that may have important implications in our understanding of normal physiology as well as disease pathogenesis.
Assuntos
Adenilil Ciclases/metabolismo , Néfrons/enzimologia , Equilíbrio Hidroeletrolítico , Animais , Transporte Biológico , AMP Cíclico/metabolismo , Ativação Enzimática , Humanos , Isoenzimas , Nefropatias/enzimologia , Nefropatias/fisiopatologia , Néfrons/fisiopatologia , Fosforilação , Transdução de SinaisRESUMO
MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.
Assuntos
RNA Helicases DEAD-box/metabolismo , Células-Tronco Embrionárias/enzimologia , Rim/enzimologia , Mesoderma/enzimologia , Ribonuclease III/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Rim/anormalidades , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mesoderma/anormalidades , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Néfrons/anormalidades , Néfrons/enzimologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogênese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Transativadores/genética , Transativadores/metabolismo , Ureter/anormalidades , Ureter/enzimologiaRESUMO
A serine-threonine protein kinase, WNK4, reduces Na⺠reabsorption and K⺠secretion in the distal convoluted tubule by reducing trafficking of the thiazide-sensitive Na-Cl cotransporter to and enhancing renal outer medullary potassium channel retrieval from the apical membrane. Epithelial sodium channels (ENaC) in the distal nephron also play a role in regulating Na⺠reabsorption and are also regulated by WNK4, but the mechanism is unclear. In A6 distal nephron cells, transepithelial current measurement and single channel recording show that WNK4 inhibits ENaC activity. Analysis of the number of channel per patch shows that WNK4 reduces channel number but has no effect on channel open probability. Western blots of apical and total ENaC provide additional evidence that WNK4 reduces apical as well as total ENaC expression. WNK4 enhances ENaC internalization independent of Nedd4-2-mediated ENaC ubiquitination. WNK4 also reduced the amount of ENaC available for recycling but has no effect on the rate of transepithelial current increase to forskolin. In contrast, Nedd4-2 not only reduced ENaC in the recycling pool but also decreased the rate of increase of current after forskolin. WNK4 associates with wild-type as well as Liddle's mutated ENaC, and WNK4 reduces both wild-type and mutated ENaC expressed in HEK293 cells.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais Epiteliais de Sódio/metabolismo , Néfrons/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Colforsina/farmacologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Células HEK293 , Humanos , Potenciais da Membrana , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Néfrons/efeitos dos fármacos , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Fatores de Tempo , Transfecção , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Matrix metalloproteinase 9 (MMP9) is a conditionally expressed enzyme and is upregulated in glomerulonephritis. Its function in these diseases, however, remains to be fully elucidated. The induction of nephrotoxic serum nephritis (NTN) in wild-type mice resulted in an upregulation of MMP9, followed by leukocyte infiltration, albuminuria, and subsequent renal failure. MMP9 deficiency ameliorated the course of NTN as indicated by reduced histological injury and reduced infiltration of proinflammatory macrophages. The chemotaxis of MMP9-deficient macrophages in vitro was impaired. Intrarenal macrophages isolated from the kidneys of nephritic MMP9 knockout mice still displayed the typical features of a proinflammatory phenotype and were indistinguishable from wild type-derived cells. Bone marrow transplantation restored renal tissue injury and macrophage recruitment when wild type-derived donor cells were transplanted onto MMP9-deficient mice prior to the induction of NTN. Thus, leukocyte-derived MMP9 mediates the recruitment of proinflammatory macrophages into kidneys during experimental crescentic glomerulonephritis.
Assuntos
Quimiotaxia , Glomerulonefrite/enzimologia , Leucócitos/enzimologia , Macrófagos Peritoneais/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Néfrons/enzimologia , Animais , Transplante de Medula Óssea , Células Cultivadas , Quimiocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Glomerulonefrite/prevenção & controle , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Macrófagos Peritoneais/imunologia , Masculino , Metaloproteinase 9 da Matriz/deficiência , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Néfrons/imunologia , Néfrons/patologia , Fenótipo , Fatores de TempoRESUMO
The study of experimental hypertension and the development of drugs with selective inhibitory effects on the enzymes and receptors constituting the components of the circulating and tissue renin-angiotensin systems have led to newer concepts of how this system participates in both physiology and pathology. Over the last decade, a renewed emphasis on understanding the role of angiotensin-(1-7) and angiotensin-converting enzyme 2 in the regulation of blood pressure and renal function has shed new light on the complexity of the mechanisms by which these components of the renin angiotensin system act in the heart and in the kidneys to exert a negative regulatory influence on angiotensin converting enzyme and angiotensin II. The vasodepressor axis composed of angiotensin-(1-7)/angiotensin-converting enzyme 2/mas receptor emerges as a site for therapeutic interventions within the renin-angiotensin system. This review summarizes the evolving knowledge of the counterregulatory arm of the renin-angiotensin system in the control of nephron function and renal disease.
Assuntos
Angiotensina II/metabolismo , Angiotensina I/metabolismo , Hipertensão/enzimologia , Néfrons/enzimologia , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Enzima de Conversão de Angiotensina 2 , Animais , Anti-Hipertensivos/uso terapêutico , Pressão Sanguínea , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Néfrons/efeitos dos fármacos , Néfrons/fisiopatologia , Proto-Oncogene Mas , Transdução de Sinais/efeitos dos fármacosRESUMO
Cation transport in the distal mammalian nephron relies on the SLC12 family of membrane cotransporters that include the thiazide-sensitive Na(+)-Clâ» cotransporter (NCC). NCC is regulated through a scaffold of interacting proteins, including the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive Gordon's syndrome. Dynamic regulation of NCC function by kinases must involve dephosphorylation by phosphatases, as illustrated by the role of PP1 and PP2B in the regulation of KCC members of the SLC12 family. There are 2 phosphorylation-controlled regulatory pathways for NCC: type 1, mediated by WNK4 and affecting trafficking to the surface membrane, and type 2, affecting intrinsic transporter kinetics by phosphorylation of conserved N-terminal S/T amino acids. Using the Xenopus oocyte expression system, we show that PP4 inhibits NCC activity - but not trafficking to the surface membrane - by a mechanism that requires phosphatase activity and a conserved N-terminal amino acid of NCC, threonine 58. This action is distinct from WNK4 regulation of membrane trafficking. In the mouse kidney, PP4 is selectively expressed in the distal nephron, including cells of the distal convoluted tubule cells, suggesting that PP4 may have a physiological role in regulating NCC and hence NaCl reabsorption in vivo.
Assuntos
Rim/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptores de Droga/metabolismo , Simportadores de Cloreto de Sódio/metabolismo , Animais , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Células Cultivadas , Imuno-Histoquímica , Rim/enzimologia , Túbulos Renais Distais/enzimologia , Túbulos Renais Distais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Néfrons/enzimologia , Néfrons/metabolismo , Oócitos , Fosfoproteínas Fosfatases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Receptores de Droga/genética , Simportadores de Cloreto de Sódio/genética , Transfecção , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl- cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.
Assuntos
Rim/enzimologia , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Humanos , Rim/citologia , Néfrons/citologia , Néfrons/enzimologia , Néfrons/metabolismo , Proteínas Serina-Treonina Quinases/genética , Receptores de Detecção de Cálcio/genética , Transdução de Sinais/fisiologiaRESUMO
Calcineurin inhibitors like FK506 (tacrolimus) are routinely used for immunosuppression following transplantation. Its use is limited by many side effects, including renal tubular acidosis (RTA), mainly of the distal type. In this study, rats were treated with FK506 and at baseline (after 9 days) systemic acid-base status was similar to that in control animals. However, FK506-treated rats given NH(4)Cl in the drinking water for 2 days developed a more severe metabolic acidosis than control animals. Urine pH was more alkaline, but net acid excretion was normal. After 7 days of acid load, all differences related to acid-base homeostasis were equalized in both groups. Protein abundance of type IIa Na-P(i) cotransporter, type 3 Na(+)/H(+) exchanger, and electrogenic Na(+)-bicarbonate cotransporter, and both a4 and B2 subunits of the vacuolar H(+)-ATPase were reduced under baseline conditions, while induction of metabolic acidosis enhanced protein abundance of these transporters in FK506-treated animals. In parallel, protein expression of AE1 was reduced at baseline and increased together with pendrin during NH(4)Cl loading in FK506 rats. Protein abundance of the Na(+)-bicarbonate cotransporter NBCn1 was reduced under baseline conditions but remained downregulated during metabolic acidosis. Morphological analysis revealed an increase in the relative number of non-type A intercalated cells in the connecting tubule and cortical collecting duct at the expense of principal cells. Additionally, subcellular distribution of the a4 subunit of the vacuolar H(+)-ATPase was affected by FK506 with less luminal localization in the connecting tubule and outer medullary collecting duct. These data suggest that FK506 impacts on several major acid-base transport proteins in the kidney, and its use is associated with transient metabolic acidosis and altered expression of key renal acid-base transport proteins.
Assuntos
Equilíbrio Ácido-Base/efeitos dos fármacos , Acidose Tubular Renal/induzido quimicamente , Inibidores de Calcineurina , Inibidores Enzimáticos/toxicidade , Proteínas de Membrana Transportadoras/metabolismo , Néfrons/efeitos dos fármacos , Tacrolimo/toxicidade , Acidose Tubular Renal/enzimologia , Acidose Tubular Renal/patologia , Cloreto de Amônio , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Biomarcadores/sangue , Biomarcadores/urina , Calcineurina/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/administração & dosagem , Injeções Subcutâneas , Masculino , Néfrons/enzimologia , Néfrons/patologia , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Transportadores de Sulfato , Tacrolimo/administração & dosagem , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Stimulation of the release of nitric oxide (NO) in the kidney has been shown to result in renal hemodynamic changes and natriuresis. NO is a potent stimulator of soluble guanylate cyclase, leading to an increase of cyclic GMP. The precise localization of NO synthase and soluble guanylate cyclase in the renal structure is not known. In this study, the microlocalization of mRNAs coding for constitutive NO synthase and soluble guanylate cyclase was carried out in the rat kidney, using an assay of reverse transcription and polymerase chain reaction in individual microdissected renal tubule segments along the nephron, glomeruli, vasa recta bundle, and arcuate arteries. A large signal for constitutive NO synthase was detected in inner medullary collecting duct. Small signals were detected in inner medullary thin limb, cortical collecting duct, outer medullary collecting duct, glomerulus, vasa recta, and arcuate artery. Soluble guanylate cyclase mRNA is expressed largely in glomerulus, proximal convoluted tubule, proximal straight tubule, and cortical collecting duct, and in small amounts in medullary thick ascending limb, inner medullary thin limb, outer medullary collecting duct, inner medullary collecting duct, and the vascular system. Our data demonstrate that NO can be produced locally in the kidney, and that soluble guanylate cyclase is widely distributed in glomerulus, renal tubules, and the vascular system.
Assuntos
Aminoácido Oxirredutases/genética , Guanilato Ciclase/genética , Néfrons/enzimologia , Animais , Sequência de Bases , Expressão Gênica , Dados de Sequência Molecular , Óxido Nítrico Sintase , Oligodesoxirribonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/metabolismo , RatosRESUMO
Observations in vivo suggest that catecholamines modulate reabsorptive functions of proximal tubules by acting on beta-adrenoceptors. However, beta-catecholamine binding sites or beta-adrenoceptor-sensitive adenylate cyclase (AdC) has not been found in segments of proximal tubules of rat, rabbit, or mouse kidney. In the present study, we investigated the responsiveness of AdC to catecholamines, [8-Arg]vasopressin (AVP), and to parathyroid hormone (PTH) in proximal convoluted tubules (PCT), proximal straight tubules (PST), and in late distal convoluted tubules (LDCT) microdissected from canine kidney. Isoproterenol (ISO) caused a marked and dose-dependent stimulation of AdC in PST (maximum: delta + 850%; half maximum stimulation at 10(-7) M ISO), but ISO had no effect on AdC in PCT. The AdC in both PCT and PST was markedly stimulated by PTH; AVP stimulated the AdC in LDCT but not in PST or in PCT. The stimulatory effect of 10(-5) M ISO in PST (delta + 725%) was significantly greater than in LDCT (delta + 307%); norepinephrine and epinephrine had stimulatory effects in PST similar to ISO. The stimulation of AdC in PST by ISO was blocked by propranolol and by beta 2-blocker ICI-118551. On the other hand, alpha-blocker phentolamine and beta 1-blocker metoprolol did not abolish the stimulation of AdC in PST by ISO. The accumulation of cAMP in intact PCT and PST incubated in vitro was stimulated by PTH both in PST and in PCT, but ISO elevated cAMP (delta + 683%) only in PST. Our results show that proximal tubules of canine nephron, PST but not PCT, contain beta-adrenoceptors of beta 2 subtype coupled to AdC. These observations provide direct evidence that the effects of catecholamines, either released from renal nerve endings or arriving from blood supply, can act directly on beta 2-adrenoceptors located in proximal tubules, and also suggest that at least some of the catecholamine effects in proximal tubules are mediated via cAMP generation.
Assuntos
Adenilil Ciclases/metabolismo , Isoproterenol/farmacologia , Túbulos Renais Proximais/metabolismo , Néfrons/enzimologia , Receptores Adrenérgicos beta/metabolismo , Animais , AMP Cíclico/metabolismo , Cães , Metoprolol/farmacologia , Néfrons/efeitos dos fármacos , Norepinefrina/farmacologia , Propanolaminas/farmacologia , Propranolol/farmacologiaRESUMO
The action sites for parathyroid hormone (PTH), salmon calcitonin (SCT), and arginine-vasopressin (AVP) were investigated along the human nephron by measuring adenylate cyclase activity, using a single tubule in vitro microassay. Well-localized segments of tubule were isolated by microdissection from five human kidneys unsuitable for transplantation. PTH (10 IU/ml) increased adenylate cyclase activity in the convoluted and the straight proximal tubule, in the medullary and cortical portions of the thick ascending limb, and in the early portion of the distal convoluted tubule (corresponding stimulated:basal activity ratios were 64, 19, 10, 18, and 22, respectively). SCT (10 ng/ml) increased adenylate cyclase activity in the medullary and cortical portions of the thick ascending limb, in the early portion of the distal convoluted tubule, and, to a lesser extent, in the cortical and the medullay collecting tubule (activity ratios were 7, 14, 15, 3, and 3, respectively). AVP (1 microM) stimulated adenylate cyclase activity in the terminal nephron segments only, i.e., the late portion of the distal convoluted tubule, the cortical and medullary portions of the collecting tubule (activity ratios 81, 51, and 97, respectively). As measured in one experiment, nearly one-half maximal responses were obtained with 0.1 IU/ml PTH or 0.3 ng/ml SCT in thick ascending limbs and with 1 nM AVP in collecting tubules, suggesting that enzyme sensitivity to hormones as well preserved under the conditions used in this study.
Assuntos
Adenilil Ciclases/metabolismo , Arginina Vasopressina/farmacologia , Calcitonina/farmacologia , Néfrons/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Adolescente , Adulto , Humanos , Técnicas In Vitro , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Distais/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Alça do Néfron/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Néfrons/enzimologiaRESUMO
Heat shock proteins (HSP) are components of the steroid receptor complex and are released into the cell cytosol after hormone binding. We tested whether HSPs released from steroid receptors mediate an increase in calcineurin phosphatase activity by steroid hormones. Aldosterone increased calcineurin activity in microdissected rat cortical collecting ducts (CCD) and connecting tubules, but not in proximal tubules, medullary thick ascending limb, or outer medullary collecting ducts. In contrast, 5 microM dexamethasone increased calcineurin activity in both CCD and proximal tubules. Aldosterone increased CCD calcineurin activity after 30 min and this response was blocked by spironolactone, but not by actinomycin D. An antibody recognizing HSP-56 did not change basal calcineurin activity, but completely blocked the stimulation of calcineurin by aldosterone. Rapamycin, an immunosuppressive drug that stabilizes the HSP-steroid receptor complex, also blocked the aldosterone response, whereas HSP-90 or HSP-70 increased calcineurin activity in permeabilized CCD. In summary, (a) aldosterone increases calcineurin activity in CCD through a transcription-independent process; (b) maneuvers inactivating HSP-56 or slowing HSP disassociation from the receptor complex blocks stimulation of calcineurin by steroid hormones; (c) HSP-90 and HSP-70 increase CCD calcineurin activity in the absence of steroid hormone. We conclude that HSPs released from transformed steroid receptors can stimulate calcineurin activity through a transcription-independent pathway.
Assuntos
Aldosterona/farmacologia , Proteínas de Ligação a Calmodulina/metabolismo , Dexametasona/farmacologia , Proteínas de Choque Térmico/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Receptores de Esteroides/fisiologia , Transcrição Gênica , Adrenalectomia , Animais , Calcineurina , Proteínas de Ligação a Calmodulina/efeitos dos fármacos , Proteínas de Ligação a Calmodulina/genética , Ativação Enzimática/efeitos dos fármacos , Isomerismo , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/enzimologia , Masculino , Néfrons/efeitos dos fármacos , Néfrons/enzimologia , Especificidade de Órgãos , Fosfoproteínas Fosfatases/efeitos dos fármacos , Fosfoproteínas Fosfatases/genética , Ratos , Ratos Sprague-DawleyRESUMO
Na-K-ATPase activity was measured with an ultramicromethod in single portions of the proximal and distal convolution and of the thick ascending limb of Henle from adrenalectomized rats and after treatment with 5 mug aldosterone per 100 g body wt. The activity in all tubular structures returned to normal within 1 h after injection. This rapid activation of Na-K-ATPase induced by hormone was completely prevented by actinomycin D and cycloheximide. It appears that this aldosterone effect on Na-K-ATPase requires an intact protein synthetic process.
Assuntos
Adenosina Trifosfatases , Aldosterona/farmacologia , Potássio/farmacologia , Sódio/farmacologia , Adenosina Trifosfatases/metabolismo , Adrenalectomia , Aldosterona/administração & dosagem , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Depressão Química , Ativação Enzimática/efeitos dos fármacos , Túbulos Renais Distais/efeitos dos fármacos , Túbulos Renais Distais/enzimologia , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Alça do Néfron/enzimologia , Alça do Néfron/metabolismo , Masculino , Néfrons/enzimologia , Potássio/metabolismo , Ratos , Sódio/metabolismoRESUMO
The renal epithelial sodium channel (ENaC) is of fundamental importance in the control of sodium reabsorption through the distal nephron. ENaC is an important component in the overall control of sodium balance, blood volume and thereby of blood pressure. This is clearly demonstrated by rare genetic disorders of sodium channel activity (Liddle's Syndrome and Pseudohypoaldosteronism type 1 associated with contrasting effects on blood pressure). Subtle dysregulation of ENaC however may also be important in essential hypertension - a common condition and a major cause of cardiovascular morbidity and mortality. The epithelial sodium channel is formed from three partly homologous subunits. In this review we deals firstly with current views of structural and functional features of the renal epithelial sodium channel with particular emphasis on mechanisms and processes involved in the control of sodium channel activity at the biochemical and cellular levels. We then focus on genetic aspects with reference to the significance of genetic variation in the sodium channel genes in relation to blood pressure. In particular, we review recent investigations on the potential clinical significance of mutations within the genes encoding ENaC subunits in individuals with high blood pressure. Lastly, we also examine the potential value of pharmacological targeting of the renal epithelial sodium channel with the sodium channel inhibitor amiloride for the treatment of hypertension.
Assuntos
Amilorida/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Néfrons/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/efeitos dos fármacos , Aldosterona/metabolismo , Amilorida/uso terapêutico , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte , Canais Epiteliais de Sódio , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Proteínas Imediatamente Precoces/metabolismo , Ativação do Canal Iônico , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Néfrons/enzimologia , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Bloqueadores dos Canais de Sódio/uso terapêutico , Canais de Sódio/química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Prior to this work, we found that adrenal as well as extra-adrenal factors activate the response of renal 11beta-hydroxysteroid dehydrogenase 2 to stressful situations. These results -showing ways through which the organism hinders the pathological occupation of mineralocorticoid receptors by glucocorticoids leading to sodium retention and hypertension- prompted the present study on the nature of the above-mentioned extra-adrenal factors. Serotonin was chosen because of its properties as a widely distributed neurohormone, known to interact with glucocorticoids at many sites, also exhibiting increased levels and effects under stressful situations. We studied serotonin effects on 11beta-hydroxysteroid dehydrogenase 2 activity in a cell line derived from distal nephron polarized-epithelium, employing 3H-corticosterone as substrate. The end-product, 3H- 11 -dehydrocorticosterone was separated from the substrate by HPLC and quantified. Serotonin stimulated 1I beta-hydroxysteroid dehydrogenase 2 activity only at 2nM and 25pM, the magnitude of the response depending also on substrate concentration. The stimulation was blocked by the specific inhibitors methiothepin and ketanserin. We postulate that the organism partially prevents renal mineralocorticoid receptor occupancy by glucocorticoids, circulating at enhanced levels under stressful situations, through serotonin-mediated catabolic regulation of the 11beta-hydroxysteroid dehydrogenase 2 activity. Given many, mostly positive, interactions between both hormones, this might eventually pave the way to studies on a new regulatory axis.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Ativação Enzimática/efeitos dos fármacos , Néfrons/enzimologia , Serotonina/farmacologia , Animais , Linhagem Celular , Corticosterona/análogos & derivados , Corticosterona/metabolismo , Cães , Comunicação ParácrinaRESUMO
We describe studies in whole kidney, cortical and medullary homogenates and, glomerular cells in culture to determine the relative levels of expression of alpha (Ya, Yc, Yk), mu (Yb1/Yb2), pi (Yf) glutathione S-transferases (GST) and CuZn superoxide dismutase (CuZn SOD) in different regions of the nephron. Immunoblotting and immunohistochemistry were used to demonstrate relatively weak expression of alpha, mu GST and, CuZn SOD in the glomerulus compared to that in particularly distal tubules. Whilst expression of Ya was found within glomerular cells, Yc, Yk and Yf were not detected. Immunofluorescence showed that Ya and Yb1/Yb2 but not Yf were expressed in cultured epithelial and mesangial cells studied between passages 1 and 3. While Ya was distributed in cytosol, Yb1/Yb2 was primarily located in nuclei.
Assuntos
Glutationa Transferase/análise , Rim/enzimologia , Superóxido Dismutase/análise , Animais , Células Cultivadas/enzimologia , Imunofluorescência , Glutationa Transferase/classificação , Immunoblotting , Córtex Renal/enzimologia , Medula Renal/enzimologia , Néfrons/enzimologia , Ratos , Ratos Endogâmicos WKYRESUMO
Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies to determine the developmental expression of these isoforms in human kidney. Before 35 weeks of gestation, both isoenzymes were expressed by the collecting tubules and developing nephrons. After this time, expression of the alpha set was restricted to the proximal tubule and that of the pi set to the distal and collecting tubules and the loop of Henle.