RESUMO
BACKGROUND: Urinary extracellular vesicles (uEVs) are a promising source for biomarker discovery, but optimal approaches for normalization, quantification, and characterization in spot urines are unclear. METHODS: Urine samples were analyzed in a water-loading study, from healthy subjects and patients with kidney disease. Urine particles were quantified in whole urine using nanoparticle tracking analysis (NTA), time-resolved fluorescence immunoassay (TR-FIA), and EVQuant, a novel method quantifying particles via gel immobilization. RESULTS: Urine particle and creatinine concentrations were highly correlated in the water-loading study (R2 0.96) and in random spot urines from healthy subjects (R2 0.47-0.95) and patients (R2 0.41-0.81). Water loading reduced aquaporin-2 but increased Tamm-Horsfall protein (THP) and particle detection by NTA. This finding was attributed to hypotonicity increasing uEV size (more EVs reach the NTA size detection limit) and reducing THP polymerization. Adding THP to urine also significantly increased particle count by NTA. In both fluorescence NTA and EVQuant, adding 0.01% SDS maintained uEV integrity and increased aquaporin-2 detection. Comparison of intracellular- and extracellular-epitope antibodies suggested the presence of reverse topology uEVs. The exosome markers CD9 and CD63 colocalized and immunoprecipitated selectively with distal nephron markers. Conclusions uEV concentration is highly correlated with urine creatinine, potentially replacing the need for uEV quantification to normalize spot urines. Additional findings relevant for future uEV studies in whole urine include the interference of THP with NTA, excretion of larger uEVs in dilute urine, the ability to use detergent to increase intracellular-epitope recognition in uEVs, and CD9 or CD63 capture of nephron segment-specific EVs.
Assuntos
Vesículas Extracelulares/metabolismo , Nefropatias/diagnóstico , Nefropatias/urina , Adulto , Biomarcadores/urina , Estudos de Casos e Controles , Creatinina/urina , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , UrináliseRESUMO
COX16 is involved in the biogenesis of cytochrome-c-oxidase (complex IV), the terminal complex of the mitochondrial respiratory chain. We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. The absence of COX16 protein expression leads to a complete loss of the holo-complex IV, as detected by Western blot in patient fibroblasts. Lentiviral transduction of patient fibroblasts with wild-type COX16 complementary DNA rescued complex IV biosynthesis. We hypothesize that COX16 could play a role in the copper delivery route of the COX2 module as part of the complex IV assembly. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease.
Assuntos
Acidose Láctica , Encefalopatias , Cardiomiopatias , Deficiência de Citocromo-c Oxidase , Hepatopatias , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Acidose Láctica/genética , Cardiomiopatias/genética , Deficiência de Citocromo-c Oxidase/genética , Humanos , Recém-Nascido , Proteínas Mitocondriais/metabolismoRESUMO
The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC3)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCCSV)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCCSV and NCC3. NCCSV S811A impairs NCC3 activity in a dominant-negative fashion, although it does not affect plasma membrane abundance. This effect may be explained by the heterodimerization of NCCSV with NCC3. Taken together, our study highlights the dominant-negative effect of NCCSV on T55 and T60 phosphorylation and NCC activity. Here, we reveal a new function of NCCSV in humans that broadens the understanding on NCC regulation in blood pressure control.-Tutakhel, O. A. Z., Bianchi, F., Smits, D. A., Bindels, R. J. M., Hoenderop, J. G. J., van der Wijst, J. Dominant functional role of the novel phosphorylation site S811 in the human renal NaCl cotransporter.
Assuntos
Rim/metabolismo , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Pressão Sanguínea/fisiologia , Linhagem Celular , Células HEK293 , Humanos , Isoformas de Proteínas/metabolismoRESUMO
The distal convoluted tubule (DCT) of the kidney plays an important role in blood pressure regulation by modulating Na+ reabsorption via the Na+-Cl- cotransporter (NCC). A diet containing high salt (NaCl) and low K+ activates NCC, thereby causing Na+ retention and a rise in blood pressure. Since high blood pressure, hypertension, is associated with changes in serum calcium (Ca2+) and magnesium (Mg2+) levels, we hypothesized that dietary Na+ and K+ intake affects Ca2+ and Mg2+ transport in the DCT. Therefore, the present study aimed to investigate the effect of a high-Na+/low-K+ diet on renal Ca2+ and Mg2+ handling. Mice were divided in four groups and fed a normal-Na+/normal-K+, normal-Na+/low-K+, high-Na+/normal-K+, or high-Na+/low-K+ diet for 4 days. Serum and urine were collected for electrolyte and hormone analysis. Gene and protein expression of electrolyte transporters were assessed in kidney and intestine by qPCR and immunoblotting. Whereas Mg2+ homeostasis was not affected, the mice had elevated urinary Ca2+ and phosphate (Pi) excretion upon high Na+ intake, as well as significantly lower serum Ca2+ levels in the high-Na+/low-K+ group. Alterations in the gene and protein expression of players involved in Ca2+ and Pi transport indicate that reabsorption in the proximal tubular and TAL is affected, while inducing a compensatory response in the DCT. These effects may contribute to the negative health impact of a high-salt diet, including kidney stone formation, chronic kidney disease, and loss of bone mineral density.
Assuntos
Cálcio/metabolismo , Túbulos Renais Distais/metabolismo , Magnésio/metabolismo , Fosfatos/metabolismo , Potássio na Dieta/administração & dosagem , Reabsorção Renal , Cloreto de Sódio na Dieta/administração & dosagem , Ração Animal , Animais , Cálcio/sangue , Cálcio/urina , Regulação da Expressão Gênica , Homeostase , Magnésio/sangue , Magnésio/urina , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Fosfatos/sangue , Fosfatos/urina , Potássio na Dieta/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Fatores de TempoRESUMO
The thiazide-sensitive NaCl cotransporter (NCC), located apically in distal convoluted tubule epithelia, regulates the fine-tuning of renal sodium excretion. Three isoforms of NCC are generated through alternative splicing of the transcript, of which the third isoform has been the most extensively investigated in pathophysiological conditions. The aim of this study was to investigate the effect of different anti-hypertensive treatments on the abundance and phosphorylation of all three NCC isoforms in urinary extracellular vesicles (uEVs) of essential hypertensive patients. In uEVs isolated from patients (n = 23) before and after hydrochlorothiazide or valsartan treatment, the abundance and phosphorylation of the NCC isoforms was determined. Additionally, clinical biochemistry and blood pressure of the patients was assessed. Our results show that NCC detected in human uEVs has a glycosylated and oligomeric structure, comparable to NCC present in human kidney membrane fractions. Despite the inhibitory action of hydrochlorothiazide on NCC activity, immunoblot analysis of uEVs showed significantly increased abundance of NCC isoforms 1 and 2 (NCC1/2), total NCC (NCC1-3), and the phosphorylated form of total NCC (pNCC1-3-T55/T60) in essential hypertensive patients treated with hydrochlorothiazide but not with valsartan. This study highlights that NCC1/2, NCC1-3, and pNCC1-3-T55/T60 are upregulated by hydrochlorothiazide, and the increase in NCC abundance in uEVs of essential hypertensive patients correlates with the blood pressure response to hydrochlorothiazide.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Vesículas Extracelulares/efeitos dos fármacos , Hidroclorotiazida/uso terapêutico , Hipertensão/tratamento farmacológico , Rim/efeitos dos fármacos , Inibidores de Simportadores de Cloreto de Sódio/uso terapêutico , Valsartana/uso terapêutico , Adolescente , Adulto , Idoso , Biomarcadores/urina , Pressão Sanguínea/efeitos dos fármacos , Estudos Cross-Over , Vesículas Extracelulares/metabolismo , Feminino , Glicosilação , Humanos , Hipertensão/fisiopatologia , Hipertensão/urina , Rim/metabolismo , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Países Baixos , Fosforilação , Estudos Prospectivos , Isoformas de Proteínas , Membro 3 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/urina , Resultado do Tratamento , Regulação para Cima , Adulto JovemRESUMO
The thiazide-sensitive NaCl cotransporter (NCC) is an important pharmacological target in the treatment of hypertension. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms. Only the third isoform has been extensively investigated. The aim of the present study was, therefore, to establish the abundance and localization of the almost identical isoforms 1 and 2 (NCC1/2) in the human kidney and to determine their functional properties and regulation in physiological conditions. Immunohistochemical analysis of NCC1/2 in the human kidney revealed that NCC1/2 localizes to the apical plasma membrane of the distal convoluted tubule. Importantly, NCC1/2 mRNA constitutes â¼ 44% of all NCC isoforms in the human kidney. Functional analysis performed in the Xenopus laevis oocyte revealed that thiazide-sensitive (22)Na(+) transport of NCC1 was significantly increased compared with NCC3. Mimicking a constitutively active phosphorylation site at residue 811 (S811D) in NCC1 further augmented Na(+) transport, while a nonphosphorylatable variant (S811A) of NCC1 prevented this enhanced response. Analysis of human urinary exosomes demonstrated that water loading in human subjects significantly reduces the abundance of NCC1/2 in urinary exosomes. The present study highlights that previously underrepresented NCC1/2 is a fully functional thiazide-sensitive NaCl-transporting protein. Being significantly expressed in the kidney, it may constitute a unique route of renal NaCl reabsorption and could, therefore, play an important role in blood pressure regulation.
Assuntos
Processamento Alternativo , Rim/metabolismo , Cloreto de Sódio/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico , Western Blotting , Membrana Celular/metabolismo , Ingestão de Líquidos , Exossomos/metabolismo , Imunofluorescência , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Fosforilação , Isoformas de Proteínas , Proteômica/métodos , RNA Mensageiro/metabolismo , Reabsorção Renal , Membro 3 da Família 12 de Carreador de Soluto/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/urina , Espectrometria de Massas em Tandem , Fatores de Tempo , Transfecção , Xenopus laevisRESUMO
Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations.
Assuntos
Síndrome de Gitelman/genética , Membro 3 da Família 12 de Carreador de Soluto/genética , Bioensaio/métodos , Síndrome de Gitelman/metabolismo , Glicosilação , Células HEK293 , Humanos , Mutação , Fosforilação , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Ionic calcium (Ca2+) is a key messenger in signal transduction and its mitochondrial uptake plays an important role in cell physiology. This uptake is mediated by the mitochondrial Ca2+ uniporter (MCU), which is regulated by EMRE (essential MCU regulator) encoded by the SMDT1 (single-pass membrane protein with aspartate rich tail 1) gene. This work presents the genetic, clinical and cellular characterization of two patients harbouring SMDT1 variants and presenting with muscle problems. Analysis of patient fibroblasts and complementation experiments demonstrated that these variants lead to absence of EMRE protein, induce MCU subcomplex formation and impair mitochondrial Ca2+ uptake. However, the activity of oxidative phosphorylation enzymes, mitochondrial morphology and membrane potential, as well as routine/ATP-linked respiration were not affected. We hypothesize that the muscle-related symptoms in the SMDT1 patients result from aberrant mitochondrial Ca2+ uptake.
Assuntos
Canais de Cálcio , Cálcio , Humanos , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Transporte de Íons , Mitocôndrias/genética , Mitocôndrias/metabolismo , Músculos/metabolismoRESUMO
The renal thiazide-sensitive sodium-chloride cotransporter (NCC), located in the distal convoluted tubule (DCT) of the kidney, plays an important role in blood pressure regulation by fine-tuning sodium excretion. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms, of which only the third isoform (NCC3) has been extensively investigated so far. However, recent studies unraveled the importance of the isoforms 1 and 2, collectively referred to as NCC splice variant (NCCSV), in several (patho)physiological conditions. In the human kidney, NCCSV localizes to the apical membrane of the DCT and could constitute a functional route for renal sodium-chloride reabsorption. Analysis of urinary extracellular vesicles (uEVs), a non-invasive method for measuring renal responses, demonstrated that NCCSV abundance changes in response to acute water loading and correlates with patients' thiazide responsiveness. Furthermore, a novel phosphorylation site at serine 811 (S811), exclusively present in NCCSV, was shown to play an instrumental role in NCCSV as well as NCC3 function. This review aims to summarize these new insights of NCCSV function in humans that broadens the understanding on NCC regulation in blood pressure control.
Assuntos
Processamento Alternativo/genética , Pressão Sanguínea/genética , Túbulos Renais Distais/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/genética , Animais , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Animal studies have shown that the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus can activate the thiazide-sensitive NaCl cotransporter (NCC). A common side effect of CNIs is hypertension. Renal salt transporters such as NCC are excreted in urinary extracellular vesicles (uEVs) after internalization into multivesicular bodies. Human studies indicate that CNIs also increase NCC abundance in uEVs, but results are conflicting and no relationship with NCC function has been shown. Therefore, we investigated the effects of CsA and Tac on the abundance of both total NCC (tNCC) and phosphorylated NCC at Thr60 phosphorylation site (pNCC) in uEVs, and assessed whether NCC abundance in uEVs predicts the blood pressure response to thiazide diuretics. Our results show that in kidney transplant recipients treated with cyclosporine (n = 9) or tacrolimus (n = 23), the abundance of both tNCC and pNCC in uEVs is 4-5 fold higher than in CNI-free kidney transplant recipients (n = 13) or healthy volunteers (n = 6). In hypertensive kidney transplant recipients, higher abundances of tNCC and pNCC prior to treatment with thiazides predicted the blood pressure response to thiazides. During thiazide treatment, the abundance of pNCC in uEVs increased in responders (n = 10), but markedly decreased in non-responders (n = 8). Thus, our results show that CNIs increase the abundance of both tNCC and pNCC in uEVs, and these increases correlate with the blood pressure response to thiazides. This implies that assessment of NCC in uEVs could represent an alternate method to guide anti-hypertensive therapy in kidney transplant recipients.