RESUMO
Hypercalciuria is the most common metabolic risk factor in people with kidney stone disease. Its etiology is mostly multifactorial, although monogenetic causes of hypercalciuria have also been described. Despite the increased availability of genetic diagnostic tests, the vast majority of individuals with familial hypercalciuria remain unsolved. In this study, we investigated a consanguineous pedigree with idiopathic hypercalciuria. The proband additionally exhibited severe skeletal deformities and hyperparathyroidism. Whole-exome sequencing of the proband revealed a homozygous ultra-rare variant in TRPV5 (NM_019841.7:c.1792G>A; p.(Val598Met)), which encodes for a renal Ca2+-selective ion channel. The variant segregates with the three individuals with hypercalciuria. The skeletal phenotype unique to the proband was due to an additional pathogenic somatic mutation in GNAS (NM_000516.7:c.601C>T; p.(Arg201Cys)), which leads to polyostotic fibrous dysplasia. The variant in TRPV5 is located in the TRP helix, a characteristic amphipathic helix that is indispensable for the gating movements of TRP channels. Biochemical characterization of the TRPV5 p.(Val598Met) channel revealed a complete loss of Ca2+ transport capability. This defect is caused by reduced expression of the mutant channel, due to misfolding and preferential targeting to the proteasome for degradation. Based on these findings, we conclude that biallelic loss of TRPV5 function causes a novel form of monogenic autosomal recessive hypercalciuria, which we name renal Ca2+-wasting hypercalciuria (RCWH). The recessive inheritance pattern explains the rarity of RCWH and underscores the potential prevalence of RCWH in highly consanguineous populations, emphasizing the importance of exploration of this disorder within such communities.
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In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.
Assuntos
Túbulos Renais Coletores , Rim Policístico Autossômico Dominante , Humanos , Rim Policístico Autossômico Dominante/metabolismo , Rim/metabolismo , Expressão Gênica , Trifosfato de Adenosina/metabolismo , Túbulos Renais Coletores/metabolismoRESUMO
Fanconi anemia (FA) is a rare inherited disorder that mainly affects the bone marrow. This condition causes decreased production of all types of blood cells. FA is caused by a defective repair of DNA interstrand crosslinks and to date, mutations in over 20 genes have been linked to the disease. Advances in science and molecular biology have provided new insight between FA gene mutations and the severity of clinical manifestations. Here, we will highlight the current and promising therapeutic options for this rare disease. The current standard treatment for FA patients is hematopoietic stem cell transplantation, a treatment associated to exposure to radiation or chemotherapy, immunological complications, plus opportunistic infections from prolonged immune incompetence or increased risk of morbidity. New arising treatments include gene addition therapy, genome editing using CRISPR-Cas9 nuclease, and hematopoietic stem cell generation from induced pluripotent stem cells. Finally, we will also discuss the revolutionary developments in mRNA therapeutics as an opportunity for this disease.
Assuntos
Anemia de Fanconi , Humanos , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Medula Óssea/metabolismo , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Dano ao DNARESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1-/- mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1-/- mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1-/- iMCD3 cells to EVs derived from Pkd1-/- mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1-/- cells to mDCT15 Pkd1-/- EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth.
Assuntos
Cistos , Vesículas Extracelulares , Rim Policístico Autossômico Dominante , Camundongos , Animais , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Rim/metabolismo , Comunicação Celular , Vesículas Extracelulares/metabolismo , Trifosfato de Adenosina/metabolismo , Cistos/metabolismo , Canais de Cátion TRPP/metabolismoRESUMO
Within the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a highly Ca2+ -selective channel important for active reabsorption of Ca2+ in the kidney. Its channel activity is controlled by a negative feedback mechanism involving calmodulin (CaM) binding. Combining advanced microscopy techniques and biochemical assays, this study characterized the dynamic lobe-specific CaM regulation. We demonstrate for the first time that functional (full-length) TRPV5 interacts with CaM in the absence of Ca2+ , and this interaction is intensified at increasing Ca2+ concentrations sensed by the CaM C-lobe that achieves channel pore blocking. Channel inactivation occurs without requiring CaM N-lobe calcification. Moreover, we show a Ca2+ -dependent binding stoichiometry at the single channel level. In conclusion, our study proposes a new model for CaM-dependent regulation - calmodulation - of this uniquely Ca2+ -selective TRP channel TRPV5 that involves apoCaM interaction and lobe-specific actions, which may be of significant physiological relevance given its role as gatekeeper of Ca2+ transport in the kidney. KEY POINTS: The renal Ca2+ channel TRPV5 is an important player in maintenance of the body's Ca2+ homeostasis. Activity of TRPV5 is controlled by a negative feedback loop that involves calmodulin (CaM), a protein with two Ca2+ -binding lobes. We investigated the dynamics of the interaction between TRPV5 and CaM with advanced fluorescence microscopy techniques. Our data support a new model for CaM-dependent regulation of TRPV5 channel activity with CaM lobe-specific actions and demonstrates Ca2+ -dependent binding stoichiometries. This study improves our understanding of the mechanism underlying fast channel inactivation, which is physiologically relevant given the gatekeeper function of TRPV5 in Ca2+ reabsorption in the kidney.
Assuntos
Calmodulina , Canais de Cátion TRPV , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Ligação Proteica , Canais de Cátion TRPV/metabolismoRESUMO
BACKGROUND: Hypomagnesaemia with secondary hypocal-caemia (HSH) is a rare autosomal recessive disorder caused by pathogenic variants in TRPM6, encoding the channel-kinase transient receptor potential melastatin type 6. Patients have very low serum magnesium (Mg2+) levels and suffer from muscle cramps and seizures. Despite genetic testing, a subgroup of HSH patients remains without a diagnosis. METHODS: In this study, two families with an HSH phenotype but negative for TRPM6 pathogenic variants were subjected to whole exome sequencing. Using a complementary combination of biochemical and functional analyses in overexpression systems and patient-derived fibroblasts, the effect of the TRPM7-identified variants on Mg2+ transport was examined. RESULTS: For the first time, variants in TRPM7 were identified in two families as a potential cause for hereditary HSH. Patients suffer from seizures and muscle cramps due to magnesium deficiency and episodes of hypocalcaemia. In the first family, a splice site variant caused the incorporation of intron 1 sequences into the TRPM7 messenger RNA and generated a premature stop codon. As a consequence, patient-derived fibroblasts exhibit decreased cell growth. In the second family, a heterozygous missense variant in the pore domain resulted in decreased TRPM7 channel activity. CONCLUSIONS: We establish TRPM7 as a prime candidate gene for autosomal dominant hypomagnesaemia and secondary hypocalcaemia. Screening of unresolved patients with hypocalcaemia and secondary hypocalcaemia may further establish TRPM7 pathogenic variants as a novel Mendelian disorder.
Assuntos
Hipocalcemia , Canais de Cátion TRPM , Humanos , Magnésio , Canais de Cátion TRPM/metabolismo , Cãibra Muscular/complicações , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Digestion of dietary fibers by gut bacteria has been shown to stimulate intestinal mineral absorption [e.g., calcium (Ca2+) and magnesium (Mg2+)]. Although it has been suggested that local pH and short-chain fatty acid (SCFA) concentrations determine divalent cation absorption, the exact molecular mechanisms are still unknown. Therefore, this study aimed to determine the effects of SCFAs on intestinal Mg2+ absorption. We show that the butyrate concentration in the colon negatively correlates with serum Mg2+ levels in wildtype mice. Moreover, Na-butyrate significantly inhibited Mg2+ uptake in Caco-2 cells, while Ca2+ uptake was unaffected. Although Na-butyrate significantly lowered total ATP production rate, and resulted in increased phosphorylation of AMP-activated protein kinase (AMPK), inhibition of Mg2+ uptake by butyrate preceded these consequences. Importantly, electrophysiological examinations demonstrated that intracellular butyrate directly reduced the activity of the heteromeric Mg2+ channel complex, transient receptor potential melastatin (TRPM)6/7. Blocking cellular butyrate uptake prevented its inhibitory effect on Mg2+ uptake, demonstrating that butyrate acts intracellularly. Our work identified butyrate as novel regulator of intestinal Mg2+ uptake that works independently from metabolic regulation. This finding further highlights the role of microbial fermentation in the regulation of mineral absorption.
Assuntos
Butiratos , Magnésio , Humanos , Camundongos , Animais , Butiratos/farmacologia , Butiratos/metabolismo , Células CACO-2 , Magnésio/metabolismo , Colo/metabolismo , Ácidos Graxos Voláteis/metabolismoRESUMO
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited disorder characterized by the development of renal cysts, which frequently leads to renal failure. Hypertension and other cardiovascular symptoms contribute to the high morbidity and mortality of the disease. ADPKD is caused by mutations in the PKD1 gene or, less frequently, in the PKD2 gene. The disease onset and progression are highly variable between patients, whereby the underlying mechanisms are not fully elucidated. Recently, a role of extracellular vesicles (EVs) in the progression of ADPKD has been postulated. However, the mechanisms stimulating EV release in ADPKD have not been addressed and the participation of the distal nephron segments is still uninvestigated. Here, we studied the effect of Pkd1 deficiency on EV release in wild type and Pkd1-/- mDCT15 and mIMCD3 cells as models of the distal convoluted tubule (DCT) and inner medullary collecting duct (IMCD), respectively. By using nanoparticle tracking analysis, we observed a significant increase in EV release in Pkd1-/- mDCT15 and mIMCD3 cells, with respect to the wild type cells. The molecular mechanisms leading to the changes in EV release were further investigated in mDCT15 cells through RNA sequencing and qPCR studies. Specifically, we assessed the relevance of purinergic signaling and ceramide biosynthesis enzymes. Pkd1-/- mDCT15 cells showed a clear upregulation of P2rx7 expression compared to wild type cells. Depletion of extracellular ATP by apyrase (ecto-nucleotidase) inhibited EV release only in wild type cells, suggesting an exacerbated signaling of the extracellular ATP/P2X7 pathway in Pkd1-/- cells. In addition, we identified a significant up-regulation of the ceramide biosynthesis enzymes CerS6 and Smpd3 in Pkd1-/- cells. Altogether, our findings suggest the involvement of the DCT in the EV-mediated ADPKD progression and points to the induction of ceramide biosynthesis as an underlying molecular mechanism. Further studies should be performed to investigate whether CerS6 and Smpd3 can be used as biomarkers of ADPKD onset, progression or severity.
Assuntos
Ceramidas , Vesículas Extracelulares , Rim Policístico Autossômico Dominante , Humanos , Trifosfato de Adenosina , Apirase/metabolismo , Ceramidas/biossíntese , Ceramidas/genética , Vesículas Extracelulares/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/genéticaRESUMO
Purinergic signalling is a receptor-mediated process characterized by the binding of extracellular nucleotides and nucleosides to purinergic receptors, which results in the activation intracellular signalling pathways, and, ultimately, leads to changes in cell physiology. Purinergic signalling has been related to the regulation of important physiological processes (e.g., renal electrolyte reabsorption; platelet aggregation; immune response). In addition, it has been associated with pathophysiological situations such as cancer and inflammation. Extracellular vesicles (EVs) are nanoparticles released by all cells of the organism, which play a key role in cell-cell communication. In this regard, EVs can mediate effects on target cells located at distant locations. Within their cargo, EVs contain molecules with the potential to affect purinergic signalling at the target cells and tissues. Here, we review the studies addressing the regulation of purinergic signalling by EVs based on the cell type or tissue where the regulation takes place. In this regard, EVs are found to play a major role in modulating the extracellular ATP levels and, specially, adenosine. This has a clear impact on, for instance, the inflammatory and immune response against cancer cells. Furthermore, we discuss the data available on the regulation of EV secretion and its cargo by purinergic signalling. Here, a major role of the purinergic receptor P2X7 and again, an impact on processes such as inflammation, immune response and cancer pathogenesis has been established. Finally, we highlight uninvestigated aspects of these two regulatory networks and address their potential as therapeutic targets.
Assuntos
Vesículas Extracelulares , Neoplasias , Comunicação Celular , Vesículas Extracelulares/metabolismo , Humanos , Inflamação/metabolismo , Neoplasias/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais/fisiologiaRESUMO
TRPV5 is a highly selective calcium channel that finetunes urinary calcium excretion by reabsorbing calcium from the pro-urine. New structural findings show how PTH and pH control TRPV5 activity by altering the binding of endogenous ligands calmodulin and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).
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Cálcio , Prótons , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Casamento , Canais de Cátion TRPV/metabolismoRESUMO
Dietary fibers have been shown to increase the intestinal absorption of calcium (Ca2+) and magnesium (Mg2+). However, the mechanisms that explain the enhanced electrolyte absorption remain unknown. Therefore, this study aims to investigate the short-term and long-term effects of 5% (w/w) sodium butyrate (Na-butyrate), an important end-metabolite of bacterial fermentation of dietary fibers, on Ca2+ and Mg2+ homeostasis in mice. Serum Ca2+ levels were only significantly increased in mice treated with Na-butyrate for 1 day. This was associated with a twofold increase in the mRNA expression levels of Trpv6 in the proximal and distal colon. Contrary, Na-butyrate did not affect serum Mg2+ concentrations at either of the intervention periods. However, we observed a reduction in urinary Mg2+ excretion, although not significantly, after 1 day of treatment. A significant reduction of 2.5-fold in urinary Mg2+ excretion was observed after 14 days of treatment. Indeed, 14-day Na-butyrate supplementation increased colonic Trpm7 expression by 1.2-fold compared to control mice. In conclusion, short-term Na-butyrate supplementation increases serum Ca2+ levels in mice. This was associated with increased mRNA expression levels of Trpv6 in the colon, suggesting that Na-butyrate regulates the expression of genes involved in active intestinal Ca2+ absorption.
Assuntos
Sódio na Dieta , Canais de Cátion TRPM , Animais , Ácido Butírico/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Colo , Fibras na Dieta/metabolismo , Fibras na Dieta/farmacologia , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sódio/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Sódio na Dieta/metabolismo , Sódio na Dieta/farmacologia , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
The kidneys are essential for maintaining electrolyte homeostasis. Blood electrolyte composition is controlled by active reabsorption and secretion processes in dedicated segments of the kidney tubule. Specifically, the distal convoluted tubule (DCT) and connecting tubule are important for regulating the final excretion of sodium, magnesium, and calcium. Studies unravelling the specific function of these segments have greatly improved our understanding of DCT (patho)physiology. Over the years, experimental models used to study the DCT have changed and the field has advanced from early dissection studies with rats and rabbits to the use of various transgenic mouse models. Developments in dissection techniques and cell culture methods have resulted in immortalized mouse DCT cell lines and made it possible to specifically obtain DCT fragments for ex vivo studies. However, we still do not fully understand the complex (patho)physiology of this segment and there is need for advanced human DCT models. Recently, kidney organoids and tubuloids have emerged as new complex cell models that provide excellent opportunities for physiological studies, disease modeling, drug discovery, and even personalized medicine in the future. This review presents an overview of cell models used to study the DCT and provides an outlook on kidney organoids and tubuloids as model for DCT (patho)physiology. Impact statement This study provides a detailed overview of past and future developments on cell models used to study kidney (patho)physiology and specifically the distal convoluted tubule (DCT) segment. Hereby, we highlight the need for an advanced human cell model of this segment and summarize recent advances in the field of kidney organoids and tubuloids with a focus on DCT properties. The findings reported in this review are significant for future developments toward an advanced human model of the DCT that will help to increase our understanding of DCT (patho)physiology.
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Túbulos Renais Distais , Magnésio , Animais , Cálcio , Camundongos , Camundongos Transgênicos , Organoides , Coelhos , RatosRESUMO
Transient receptor potential vanilloid (TRPV) channels are part of the superfamily of TRP ion channels and play important roles in widespread physiological processes including both neuronal and non-neuronal pathways. Various diseases such as skeletal abnormalities, chronic pain, and cancer are associated with dysfunction of a TRPV channel. In order to obtain full understanding of disease pathogenesis and create opportunities for therapeutic intervention, it is essential to unravel how these channels function at a molecular level. In the past decade, incredible progress has been made in biochemical sample preparation of large membrane proteins and structural biology techniques, including cryo-electron microscopy. This has resulted in high resolution structures of all TRPV channels, which has provided novel insights into the molecular mechanisms of channel gating and regulation that will be summarized in this review.
Assuntos
Canais de Cátion TRPV/química , Canais de Cátion TRPV/ultraestrutura , Animais , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico , Conformação Proteica , Relação Estrutura-Atividade , Canais de Cátion TRPV/metabolismoRESUMO
Nephrolithiasis or renal stone disease is an increasingly common problem, and its relatively high recurrence rate demands better treatment options. The majority of patients with nephrolithiasis have stones that contain calcium (Ca2+), which develop upon "supersaturation" of the urine with insoluble Ca2+ salts; hence processes that influence the delivery and renal handling of Ca2+ may influence stone formation. Idiopathic hypercalciuria is indeed frequently observed in patients with kidney stones that contain Ca2+. Genetic screens of nephrolithiasis determinants have identified an increasing number of gene candidates, most of which are involved in renal Ca2+ handling. This review provides an outline of the current knowledge regarding genetics of nephrolithiasis and will mainly focus on the epithelial Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), an important player in Ca2+ homeostasis. Being a member of the TRP family of ion channels, TRPV5 is currently part of a revolution in structural biology. Recent technological breakthroughs in the cryo-electron microscopy field, combined with improvements in biochemical sample preparation, have resulted in high-resolution 3-dimensional structural models of integral membrane proteins, including TRPV5. These models currently are being used to explore the proteins' structure-function relationship, elucidate the molecular mechanisms of channel regulation, and study the putative effects of disease variants. Combined with other multidisciplinary approaches, this approach may open an avenue toward better understanding of the pathophysiological mechanisms involved in hypercalciuria and stone formation, and ultimately it may facilitate prevention of stone recurrence through the development of effective drugs.
Assuntos
Cálcio/metabolismo , Túbulos Renais/metabolismo , Nefrolitíase/genética , Canais de Cátion TRPV/genética , Humanos , Nefrolitíase/metabolismo , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismoRESUMO
Intestinal enterocytes are key players in the absorption of magnesium (Mg2+) and calcium (Ca2+). Understanding the exact molecular mechanisms by which their absorption behavior is regulated could greatly improve treatment strategies for stimulating intestinal absorption in diseases with Mg2+ and/or Ca2+ deficiency. However, such studies are hampered by the lack of in vitro intestinal cell models mimicking the mechanical and physiological properties of the gut. In this study we develop an in vitro gut model based on porous micropatterned membranes with villi-like surface topography and mechanical properties closely mimicking that of intestinal tissue. These membranes are prepared via phase separation micromolding using poly-ε-caprolactone/poly-lactic-glycolic acid (PCL/PLGA) polymer blend and can facilitate cellular differentiation of Caco-2 cells similar to native enterocytes. In fact, cells cultured on these micropatterned membranes form a brush border of microvilli with spatial differences in morphology and tight junction formation along the villous-base axis. Moreover, cells cultured on our membranes show a 2-fold increased alkaline phosphatase activity at the end of differentiation. Finally, we demonstrate that cells cultured on our micropatterned membranes have a 4- and 1.5-fold increased uptake of 25Mg and 45Ca, respectively, compared to non-patterned membranes. These results indicate that the new membranes can mimic the intestinal environment and therefore can have a great impact on mineral uptake in vitro. STATEMENT OF SIGNIFICANCE: This study presents the development of an in vitro gut model consisting of villi-like PCL/PLGA micropatterned membranes. These membranes are prepared via phase separation micromolding (PSµM), a technique which allows tailoring of the membrane surface topography combined with membrane porosity and interconnectivity which are important parameters for membranes used for in vitro transport studies. The culture of Caco-2 cells on these micropatterned membranes shows that they facilitate cellular differentiation similar to gut enterocytes. Our data indicate that mimicking the 3D geometry of the gut is very important for improving the physiological relevance of in vitro gut models. In the future, our micropatterned membranes with segment-specific geometries, in combination with isotopic measurements, would be applied to perform detailed ion uptake and transport studies.
Assuntos
Cálcio/metabolismo , Mucosa Intestinal/metabolismo , Magnésio/metabolismo , Alicerces Teciduais , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis , Células CACO-2 , Diferenciação Celular , Proliferação de Células , Enterócitos/metabolismo , Humanos , Microvilosidades/metabolismo , Permeabilidade , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Porosidade , Propriedades de Superfície , Junções Íntimas , Engenharia Tecidual/métodosRESUMO
Proton pump inhibitors (PPIs) are used by millions of patients for the treatment of stomach acid-reflux diseases. Although PPIs are generally considered safe, about 13% of the users develop hypomagnesemia. Despite rising attention for this issue, the underlying mechanism is still unknown. Here, we examine whether the gut microbiome is involved in the development of PPI-induced hypomagnesemia in wild-type C57BL/6J mice. After 4 wk of treatment under normal or low dietary Mg2+ availability, omeprazole significantly reduced serum Mg2+ levels only in mice on a low-Mg2+ diet without affecting the mRNA expression of colonic or renal Mg2+ transporters. Overall, 16S rRNA gene sequencing revealed a lower gut microbial diversity in omeprazole-treated mice. Omeprazole induced a shift in microbial composition, which was associated with a 3- and 2-fold increase in the abundance of Lactobacillus and Bifidobacterium, respectively. To examine the metabolic consequences of these microbial alterations, the colonic composition of organic acids was evaluated. Low dietary Mg2+ intake, independent of omeprazole treatment, resulted in a 10-fold increase in formate levels. Together, these results imply that both omeprazole treatment and low dietary Mg2+ intake disturb the gut internal milieu and may pose a risk for the malabsorption of Mg2+ in the colon.-Gommers, L. M. M., Ederveen, T. H. A., van der Wijst, J., Overmars-Bos, C., Kortman, G. A. M., Boekhorst, J., Bindels, R. J. M., de Baaij, J. H. F., Hoenderop, J. G. J. Low gut microbiota diversity and dietary magnesium intake are associated with the development of PPI-induced hypomagnesemia.
Assuntos
Microbioma Gastrointestinal/fisiologia , Magnésio/metabolismo , Inibidores da Bomba de Prótons/efeitos adversos , Animais , Bifidobacterium/fisiologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/microbiologia , Dieta , Lactobacillus/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Omeprazol/efeitos adversos , RNA Ribossômico 16S/metabolismoRESUMO
The identification of genes causing inherited kidney diseases yielded crucial insights in the molecular basis of disease and improved our understanding of physiological processes that operate in the kidney. Monogenic kidney disorders are caused by mutations in genes coding for a large variety of proteins including receptors, channels and transporters, enzymes, transcription factors, and structural components, operating in specialized cell types that perform highly regulated homeostatic functions. Common variants in some of these genes are also associated with complex traits, as evidenced by genome-wide association studies in the general population. In this review, we discuss how the molecular genetics of inherited disorders affecting different tubular segments of the nephron improved our understanding of various transport processes and of their involvement in homeostasis, while providing novel therapeutic targets. These include inherited disorders causing a dysfunction of the proximal tubule (renal Fanconi syndrome), with emphasis on epithelial differentiation and receptor-mediated endocytosis, or affecting the reabsorption of glucose, the handling of uric acid, and the reabsorption of sodium, calcium, and magnesium along the kidney tubule.
Assuntos
Nefropatias/genética , Nefropatias/fisiopatologia , Rim/fisiologia , Rim/fisiopatologia , Animais , Humanos , Doenças RarasRESUMO
TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.
Assuntos
Canais de Cátion TRPV/fisiologia , Canais de Cátion TRPV/ultraestrutura , Animais , Cálcio/metabolismo , Radioisótopos de Cálcio , Clonagem Molecular , Microscopia Crioeletrônica , Modelos Químicos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Coelhos , Canais de Cátion TRPV/classificação , Canais de Cátion TRPV/genéticaRESUMO
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
Active reabsorption of magnesium (Mg2+ ) in the distal convoluted tubule (DCT) of the kidney is crucial for maintaining Mg2+ homeostasis. Impaired activity of the Na+ -Cl- -cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg2+ balance. To further elucidate the possible interrelationship between NCC activity and renal Mg2+ handling, plasma Mg2+ levels and urinary excretion of sodium (Na+ ) and Mg2+ were measured in a mouse model of Gordon syndrome. In this model, DCT1-specific expression of a constitutively active mutant form of the NCC-phosphorylating kinase, SPAK (CA-SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)-sensitive Na+ reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg2+ levels in CA-SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA-SPAK mice exhibited greater NCC-dependent Na+ reabsorption together with decreased Mg2+ reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA-SPAK mice exhibited higher urinary Mg2+ excretion, while urinary Na+ excretion decreased to levels observed in control animals. Surprisingly, CA-SPAK mice had unaltered renal expression of Trpm6, encoding the Mg2+ -permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA-SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na+ reabsorption. Thus, Mg2+ reabsorption is not coupled to increased thiazide-sensitive Na+ reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg2+ excretion after prolonged HCTZ administration.