RESUMEN
The Goldblatt model of hypertension (2K-1C) in rats is characterized by renal sympathetic nerve activity (rSNA). We investigated the effects of unilateral renal denervation of the clipped kidney (DNX) on sodium transporters of the unclipped kidneys and the cardiovascular, autonomic, and renal functions in 2K-1C and control (CTR) rats. The mean arterial pressure (MAP) and rSNA were evaluated in experimental groups. Kidney function and NHE3, NCC, ENaCß, and ENaCγ protein expressions were assessed. The glomerular filtration rate (GRF) and renal plasma flow were not changed by DNX, but the urinary (CTR: 0.0042 ± 0.001; 2K-1C: 0.014 ± 0.003; DNX: 0.005 ± 0.0013 mL/min/g renal tissue) and filtration fractions (CTR: 0.29 ± 0.02; 2K-1C: 0.51 ± 0.06; DNX: 0.28 ± 0.04 mL/min/g renal tissue) were normalized. The Na+/H+ exchanger (NHE3) was reduced in 2K-1C, and DNX normalized NHE3 (CTR: 100 ± 6; 2K-1C: 44 ± 14, DNX: 84 ± 13%). Conversely, the Na+/Cl- cotransporter (NCC) was increased in 2K-1C and was reduced by DNX (CTR: 94 ± 6; 2K-1C: 144 ± 8; DNX: 60 ± 15%). In conclusion, DNX in Goldblatt rats reduced blood pressure and proteinuria independently of GRF with a distinct regulation of NHE3 and NCC in unclipped kidneys.
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Riñón , Intercambiador 3 de Sodio-Hidrógeno , Animales , Riñón/inervación , Riñón/metabolismo , Ratas , Masculino , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Tasa de Filtración Glomerular , Desnervación , Isquemia/metabolismo , Presión Sanguínea , Ratas Wistar , Hipertensión/metabolismo , Canales Epiteliales de Sodio/metabolismo , Modelos Animales de Enfermedad , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Aquatic animals residing in saline habitats either allow extracellular sodium concentration to conform to environmental values or regulate sodium to lower levels. The latter strategy requires an energy-driven process to move sodium against a large concentration gradient to eliminate excess sodium that diffuses into the animal. Previous studies of invertebrate and vertebrate species indicate a sodium pump, Na+/K+ ATPase, powers sodium secretion. We provide the first functional evidence of a saline-water animal, Aedes taeniorhynchus mosquito larva, utilizing a proton pump to power this process. Vacuolar-type H+ ATPase (VHA) protein is highly expressed on the apical membrane of the posterior rectal cells, and in situ sodium flux across this epithelium increases significantly in larvae held in higher salinity and is sensitive to Bafilomycin A1, an inhibitor of VHA. We also report the first evidence of splice variants of the sodium/proton exchanger, NHE3, with both high and low molecular weight variants highly expressed on the apical membrane of the posterior rectal cells. Evidence of NHE3 function was indicated with in situ sodium transport significantly inhibited by a NHE3 antagonist, S3226. We propose that the outward proton pumping by VHA establishes a favourable electromotive gradient to drive sodium secretion via NHE3 thus producing a hyperosmotic, sodium-rich urine. This H+- driven Na+ secretion process is the primary mechanism of ion regulation in salt-tolerant culicine mosquito species and was first investigated over 80 years ago.
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Protones , Sodio , Animales , Sodio/metabolismo , Larva/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Aguas Salinas , Intercambiadores de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Macrólidos/farmacología , Bombas de Protones/metabolismo , SalinidadRESUMEN
Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
Asunto(s)
Bicarbonatos , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Duodeno , Ratones Noqueados , Péptidos , Transportadores de Sulfato , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Animales , Ratones , Bicarbonatos/metabolismo , Humanos , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/genética , Péptidos/farmacología , Fibrosis Quística/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/patología , Duodeno/metabolismo , Duodeno/efectos de los fármacos , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Masculino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Antiportadores , Antiportadores de Cloruro-BicarbonatoRESUMEN
Sodium-glucose cotransporter 2 inhibitors (SGLT2is), initially developed for type 2 diabetes (T2D) treatment, have demonstrated significant cardiovascular and renal benefits in heart failure (HF) and chronic kidney disease (CKD), irrespective of T2D. This review provides an analysis of the multifaceted mechanisms underlying the cardiorenal benefits of SGLT2i in HF and CKD outside of the T2D context. Eight major aspects of the protective effects of SGLT2i beyond glycemic control are explored: 1) the impact on renal hemodynamics and tubuloglomerular feedback; 2) the natriuretic effects via proximal tubule Na+/H+ exchanger NHE3 inhibition; 3) the modulation of neurohumoral pathways with evidence of attenuated sympathetic activity; 4) the impact on erythropoiesis, not only in the context of local hypoxia but also systemic inflammation and iron regulation; 5) the uricosuria and mitigation of the hyperuricemic environment in cardiorenal syndromes; 6) the multiorgan metabolic reprogramming including the potential induction of a fasting-like state, improvement in glucose and insulin tolerance, and stimulation of lipolysis and ketogenesis; 7) the vascular endothelial growth factor A (VEGF-A) upregulation and angiogenesis, and 8) the direct cardiac effects. The intricate interplay between renal, neurohumoral, metabolic, and cardiac effects underscores the complexity of SGLT2i actions and provides valuable insights into their therapeutic implications for HF and CKD. Furthermore, this review sets the stage for future research to evaluate the individual contributions of these mechanisms in diverse clinical settings.
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Insuficiencia Cardíaca , Insuficiencia Renal Crónica , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/fisiopatología , Animales , Riñón/efectos de los fármacos , Riñón/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/antagonistas & inhibidoresRESUMEN
Lack of understanding of the pathophysiology of gastrointestinal (GI) complications in type 1 diabetes (T1D), including altered intestinal transcriptomes and protein expression represents a major gap in the management of these patients. Human enteroids have emerged as a physiologically relevant model of the intestinal epithelium but establishing enteroids from individuals with long-standing T1D has proven difficult. We successfully established duodenal enteroids using endoscopic biopsies from pediatric T1D patients and compared them with aged-matched enteroids from healthy subjects (HS) using bulk RNA sequencing (RNA-seq), and functional analyses of ion transport processes. RNA-seq analysis showed significant differences in genes and pathways associated with cell differentiation and proliferation, cell fate commitment, and brush border membrane. Further validation of these results showed higher expression of enteroendocrine cells, and the proliferating cell marker Ki-67, significantly lower expression of NHE3, lower epithelial barrier integrity, and higher fluid secretion in response to cAMP and elevated calcium in T1D enteroids. Enteroids established from pediatric T1D duodenum identify characteristics of an abnormal intestinal epithelium and are distinct from HS. Our data supports the use of pediatric enteroids as an ex-vivo model to advance studies of GI complications and drug discovery in T1D patients.
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Diabetes Mellitus Tipo 1 , Duodeno , Mucosa Intestinal , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Niño , Duodeno/metabolismo , Duodeno/patología , Femenino , Masculino , Proliferación Celular , Adolescente , Células Enteroendocrinas/metabolismo , Células Enteroendocrinas/patología , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Diferenciación Celular , Organoides/metabolismo , Organoides/patología , Antígeno Ki-67/metabolismoRESUMEN
OBJECTIVES: Potassium supplementation reduces blood pressure and the occurrence of cardiovascular diseases, with K + -induced natriuresis playing a potential key role in this process. However, whether these beneficial effects occur in diabetes remains unknown. METHODS: In this study, we examined the impact of high-K + intake on renal Na + /K + transport by determining the expression of major apical Na + transporters, diuretics responses (as a proxy for specific Na + transporter function), urinary Na + /K + excretion, and plasma Na + /K + concentrations in db/db mice, a model of type 2 diabetes mellitus. RESULTS: Although db/m mice exhibited increased fractional excretion of sodium (FE Na ) and fractional excretion of potassium (FE K ) under high-K + intake, these responses were largely blunted in db/db mice, suggesting impaired K + -induced natriuresis and kaliuresis in diabetes. Consequently, high-K + intake increased plasma K + levels in db/db mice, which could be attributed to the abnormal activity of sodium-hydrogen exchanger 3 (NHE3), sodium-chloride cotransporter (NCC), and epithelial Na + channel (ENaC), as high-K + intake could not effectively decrease NHE3 and NCC and increase ENaC expression and activity in the diabetic group. Inhibition of NCC by hydrochlorothiazide could correct the hyperkalemia in db/db mice fed a high-K + diet, indicating a key role for NCC in K + -loaded diabetic mice. Treatment with metformin enhanced urinary Na + /K + excretion and normalized plasma K + levels in db/db mice with a high-K + diet, at least partially, by suppressing NCC activity. CONCLUSION: Collectively, the impaired K + -induced natriuresis in diabetic mice under high-K + intake may be primarily attributed to impaired NCC-mediated renal K + excretion, despite the role of NHE3.
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Hiperpotasemia , Natriuresis , Potasio , Intercambiador 3 de Sodio-Hidrógeno , Animales , Natriuresis/efectos de los fármacos , Ratones , Potasio/orina , Potasio/sangre , Potasio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Masculino , Diabetes Mellitus Tipo 2/complicaciones , Suplementos DietéticosRESUMEN
To explore the mechanism of the hypertension in dopamine receptor-4 (Drd4) null mice, we determined the salt sensitivity and renal sodium transport proteins in Drd4-/- and Drd4+/+ mice with varied salt diets. On normal NaCl diet (NS), mean arterial pressures (MAP, telemetry) were higher in Drd4-/- than Drd4+/+; Low NaCl diet (LS) tended to decrease MAP in both strains; high NaCl diet (HS) elevated MAP with sodium excretion decreased and pressure-natriuresis curve shifted to right in Drd4-/- relative to Drd4+/+ mice. Drd4-/- mice exhibited increased renal sodium-hydrogen exchanger 3 (NHE3), sodium-potassium-2-chloride cotransporter (NKCC2), sodium-chloride cotransporter (NCC), and outer medullary α-epithelial sodium channel (αENaC) on NS, decreased NKCC2, NCC, αENaC, and αNa+-K+-ATPase on LS, and increased αENaC on HS. NKCC2, NCC, αENaC, and αNa+-K+-ATPase in plasma membrane were greater in Drd4-/- than in Drd4+/+ mice with HS. D4R was expressed in proximal and distal convoluted tubules, thick ascending limbs, and outer medullary collecting ducts and colocalized with NKCC2 and NCC. The phosphorylation of NKCC2 was enhanced but ubiquitination was reduced in the KO mice. There were no differences between the mouse strains in serum aldosterone concentrations and urinary dopamine excretions despite their changes with diets. The mRNA expressions of renal NHE3, NKCC2, NCC, and αENaC on NS were not altered in Drd4-/- mice. Thus, increased protein expressions of NHE3, NKCC2, NCC and αENaC are associated with hypertension in Drd4-/- mice; increased plasma membrane protein expression of NKCC2, NCC, αENaC, and αNa+-K+-ATPase may mediate the salt sensitivity of Drd4-/- mice.
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Riñón , Ratones Noqueados , Receptores de Dopamina D4 , Animales , Riñón/metabolismo , Ratones , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/metabolismo , Regulación hacia Arriba , Cloruro de Sodio Dietético , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Masculino , Hipertensión/metabolismo , Hipertensión/genética , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Presión Sanguínea/fisiología , Canales Epiteliales de Sodio/metabolismo , Canales Epiteliales de Sodio/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: The renal proximal tubule (RPT) plays a pivotal role in regulating sodium reabsorption and thus blood pressure (BP). Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigate high-salt intake-induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function. METHODS: Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group], or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for 6 months. Urinary sodium excretion, reactive oxygen species (ROS) production, mitochondrial function, and the expression of sodium hydrogen exchanger isoform 3 (NHE3) and Na+/K+-ATPase of RPTs were determined. RESULTS: Chronic dietary cinnamaldehyde supplementation reduced tail systolic BP and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production, and alleviated mitochondrial dysfunction of RPTs in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice. CONCLUSIONS: The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension.
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Acroleína , Hipertensión , Ratones Noqueados , Mitocondrias , Cloruro de Sodio Dietético , Canal Catiónico TRPA1 , Animales , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Acroleína/análogos & derivados , Acroleína/farmacología , Hipertensión/metabolismo , Hipertensión/fisiopatología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Modelos Animales de Enfermedad , Especies Reactivas de Oxígeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Ratones , Presión Sanguínea/efectos de los fármacosRESUMEN
Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.
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Compuestos de Bencidrilo , Glucósidos , Hipertensión , Ratas Endogámicas SHR , Ratas Wistar , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Intercambiador 3 de Sodio-Hidrógeno , Regulación hacia Arriba , Animales , Masculino , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/genética , Intercambiador 3 de Sodio-Hidrógeno/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Hipertensión/fisiopatología , Glucósidos/farmacología , Compuestos de Bencidrilo/farmacología , Regulación hacia Arriba/efectos de los fármacos , Ratas , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Presión Sanguínea/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Riñón/metabolismo , Riñón/efectos de los fármacosRESUMEN
Beyond glycemic control, SGLT2 inhibitors (SGLT2is) have protective effects on cardiorenal function. Renoprotection has been suggested to involve inhibition of NHE3 leading to reduced ATP-dependent tubular workload and mitochondrial oxygen consumption. NHE3 activity is also important for regulation of endosomal pH, but the effects of SGLT2i on endocytosis are unknown. We used a highly differentiated cell culture model of proximal tubule (PT) cells to determine the direct effects of SGLT2i on Na+-dependent fluid transport and endocytic uptake in this nephron segment. Strikingly, canagliflozin but not empagliflozin reduced fluid transport across cell monolayers and dramatically inhibited endocytic uptake of albumin. These effects were independent of glucose and occurred at clinically relevant concentrations of drug. Canagliflozin acutely inhibited surface NHE3 activity, consistent with a direct effect, but did not affect endosomal pH or NHE3 phosphorylation. In addition, canagliflozin rapidly and selectively inhibited mitochondrial complex I activity. Inhibition of mitochondrial complex I by metformin recapitulated the effects of canagliflozin on endocytosis and fluid transport, whereas modulation of downstream effectors AMPK and mTOR did not. Mice given a single dose of canagliflozin excreted twice as much urine over 24 h compared with empagliflozin-treated mice despite similar water intake. We conclude that canagliflozin selectively suppresses Na+-dependent fluid transport and albumin uptake in PT cells via direct inhibition of NHE3 and of mitochondrial function upstream of the AMPK/mTOR axis. These additional targets of canagliflozin contribute significantly to reduced PT Na+-dependent fluid transport in vivo.NEW & NOTEWORTHY Reduced NHE3-mediated Na+ transport has been suggested to underlie the cardiorenal protection provided by SGLT2 inhibitors. We found that canagliflozin, but not empagliflozin, reduced NHE3-dependent fluid transport and endocytic uptake in cultured proximal tubule cells. These effects were independent of SGLT2 activity and resulted from inhibition of mitochondrial complex I and NHE3. Studies in mice are consistent with greater effects of canagliflozin versus empagliflozin on fluid transport. Our data suggest that these selective effects of canagliflozin contribute to reduced Na+-dependent transport in proximal tubule cells.
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Canagliflozina , Túbulos Renales Proximales , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Intercambiador 3 de Sodio-Hidrógeno , Animales , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/enzimología , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Canagliflozina/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Ratones , Masculino , Transportador 2 de Sodio-Glucosa/metabolismo , Endocitosis/efectos de los fármacos , Ratones Endogámicos C57BL , Albúminas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Compuestos de Bencidrilo , GlucósidosRESUMEN
BACKGROUND: Hypertension is one of the major etiologies that cause chronic kidney disease (CKD) and can exacerbate kidney dysfunction. Zinc is an essential trace element playing a role in blood pressure regulation, and zinc deficiency, a common comorbidity in patients with CKD, can cause hypertension. However, the precise mechanism underlying zinc deficiency-induced hypertension is unknown. Sodium (Na+) retention due to inappropriate Na+ reabsorption in the renal tubule is the principal pathophysiology of hypertension. Therefore, this study aimed to investigate the association between zinc deficiency and salt sensitivity. METHODS: Adult mice were fed a zinc-adequate (ZnA) or zinc-deficient (ZnD) diet combined with/without high salt in drinking water (HS) for 4 weeks (n = 6 each). Changes in blood pressure, urinary sodium excretion, and the expressions of the proximal tubular Na+ transporter, Na+/H+ exchanger 3 (NHE3), which mostly contributes to filtered Na+ reabsorption and the downstream Na+-Cl- transporter (NCC) were analyzed. RESULTS: Urinary Na+ excretion significantly increased in ZnD mice, indicating that zinc deficiency causes natriuresis. NHE3 expressions were significantly suppressed, whereas NCC was upregulated in ZnD mice. Interestingly, the combination of high salt and ZnD diet (HS-ZnD) reversed the urinary Na+ loss. The NCC remained activated and NHE3 expressions paradoxically increased in HS-ZnD mice compared with those fed the combination of high salt and ZnA diet. In addition, blood pressure significantly increased only in HS-ZnD mice. CONCLUSION: The combination of zinc deficiency and high salt causes hypertension. Zinc is associated with salt-sensitivity, potentially through NHE3 and NCC regulation.
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Presión Sanguínea , Hipertensión , Cloruro de Sodio Dietético , Intercambiador 3 de Sodio-Hidrógeno , Zinc , Animales , Zinc/deficiencia , Zinc/metabolismo , Hipertensión/etiología , Hipertensión/fisiopatología , Hipertensión/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Sodio/orina , Sodio/metabolismo , NatriuresisRESUMEN
Chronic angiotensin II (ANG II) infusion is an experimental model that induces hypertension in rodents. The natriuresis, diuresis, and blood pressure responses differ between males and females. This is perhaps not unexpected, given the rodent kidney, which plays a key role in blood pressure regulation, exhibits marked sex differences. Under normotensive conditions, compared with males, the female rat nephron exhibits lower Na+/H+ exchanger 3 (NHE3) activity along the proximal tubule but higher Na+ transporter activities along the distal segments. ANG II infusion-induced hypertension induces a pressure natriuretic response that reduces NHE3 activity and shifts Na+ transport capacity downstream. The goals of this study were to apply a computational model of epithelial transport along a rat nephron 1) to understand how a 14-day ANG II infusion impacts segmental electrolyte transport in male and female rat nephrons and 2) to identify and explain any sex differences in the effects of loop diuretics, thiazide diuretics, and K+-sparing diuretics. Model simulations suggest that the NHE3 downregulation in the proximal tubule is a major contributor to natriuresis and diuresis in hypertension, with the effects stronger in males. All three diuretics are predicted to induce stronger natriuretic and diuretic effects under hypertension compared with normotension, with relative increases in sodium excretion higher in hypertensive females than in males. The stronger natriuretic responses can be explained by the downstream shift of Na+ transport load in hypertension and by the larger distal transport load in females, both of which limit the ability of the distal segments to further elevate their Na+ transport.NEW & NOTEWORTHY Sex differences in the prevalence of hypertension are found in human and animal models. The kidney, which regulates blood pressure, exhibits sex differences in morphology, hemodynamics, and membrane transporter distributions. This computational modeling study provides insights into how the sexually dimorphic responses to a 14-day angiotensin II infusion differentially impact segmental electrolyte transport in rats. Simulations of diuretic administration explain how the natriuretic and diuretic effects differ between normotension and hypertension and between the sexes.
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Angiotensina II , Hipertensión , Natriuresis , Intercambiador 3 de Sodio-Hidrógeno , Animales , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Femenino , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Natriuresis/efectos de los fármacos , Diuréticos/farmacología , Presión Sanguínea/efectos de los fármacos , Factores Sexuales , Simulación por Computador , Sodio/metabolismo , Ratas , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Caracteres Sexuales , Modelos Animales de Enfermedad , Diuresis/efectos de los fármacos , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Riñón/metabolismo , Riñón/efectos de los fármacos , Riñón/fisiopatologíaRESUMEN
The sodium/proton exchanger isoform 3 (NHE3) is expressed in the intestine and the kidney, where it contributes to hydrogen secretion and sodium (re)absorption. The roles of this transporter have been studied by the use of the respective knockout mice and by using pharmacological inhibitors. Whole-body NHE3 knockout mice suffer from a high mortality rate (with only â¼30% of mice surviving into adulthood), and based on the expression of NHE3 in both intestine and kidney, some conclusions that were originally derived were based on this rather complex phenotype. In the last decade, more refined models have been developed that added temporal and spatial control of NHE3 expression. For example, novel mouse models have been developed with a knockout of NHE3 in intestinal epithelial cells, tubule/collecting duct of the kidney, proximal tubule of the kidney, and thick ascending limb of the kidney. These refined models have significantly contributed to our understanding of the role of NHE3 in a tissue/cell type-specific manner. In addition, tenapanor was developed, which is a non-absorbable, intestine-specific NHE3 inhibitor. In rat and human studies, tenapanor lowered intestinal Pi uptake and was effective in lowering plasma Pi levels in patients on hemodialysis. Of note, diarrhea is seen as a side effect of tenapanor (with its indication for the treatment of constipation) and in intestine-specific NHE3 knockout mice; however, effects on plasma Pi were not supported by this mouse model which showed enhanced and not reduced intestinal Pi uptake. Further studies indicated that the gut microbiome in mice lacking intestinal NHE3 resembles an intestinal environment favoring the competitive advantage of inflammophilic over anti-inflammatory species, something similar seen in patients with inflammatory bowel disease. This review will highlight recent developments and summarize newly gained insight from these refined models.
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Isoquinolinas , Intercambiadores de Sodio-Hidrógeno , Sodio , Sulfonamidas , Animales , Humanos , Ratones , Ratas , Ratones Noqueados , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.
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Microdominios de Membrana , Intercambiador 3 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno , Animales , Ratones , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Intestino Delgado/metabolismo , Microdominios de Membrana/metabolismo , Microvellosidades/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismoRESUMEN
The pathogenesis of irritable bowel syndrome (IBS) is multifactorial, characterized in part by increased intestinal permeability, and visceral hypersensitivity. Increased permeability is associated with IBS severity and abdominal pain. Tenapanor is FDA-approved for the treatment of IBS with constipation (IBS-C) and has demonstrated improvements in bowel motility and a reduction in IBS-related pain; however, the mechanism by which tenapanor mediates these functions remains unclear. Here, the effects of tenapanor on colonic pain signaling and intestinal permeability were assessed through behavioral, electrophysiological, and cell culture experiments. Intestinal motility studies in rats and humans demonstrated that tenapanor increased luminal sodium and water retention and gastrointestinal transit versus placebo. A significantly reduced visceral motor reflex (VMR) to colonic distension was observed with tenapanor treatment versus vehicle in two rat models of visceral hypersensitivity (neonatal acetic acid sensitization and partial restraint stress; both P < 0.05), returning VMR responses to that of nonsensitized controls. Whole cell voltage patch-clamp recordings of retrogradely labeled colonic dorsal root ganglia (DRG) neurons from sensitized rats found that tenapanor significantly reduced DRG neuron hyperexcitability to capsaicin versus vehicle (P < 0.05), an effect not mediated by epithelial cell secretions. Tenapanor also attenuated increases in intestinal permeability in human colon monolayer cultures caused by incubation with proinflammatory cytokines (P < 0.001) or fecal supernatants from patients with IBS-C (P < 0.005). These results support a model in which tenapanor reduces IBS-related pain by strengthening the intestinal barrier, thereby decreasing permeability to macromolecules and antigens and reducing DRG-mediated pain signaling.NEW & NOTEWORTHY A series of nonclinical experiments support the theory that tenapanor inhibits IBS-C-related pain by strengthening the intestinal barrier. Tenapanor treatment reduced visceral motor responses to nonsensitized levels in two rat models of hypersensitivity and reduced responses to capsaicin in sensitized colonic nociceptive dorsal root ganglia neurons. Intestinal permeability experiments in human colon monolayer cultures found that tenapanor attenuates increases in permeability induced by either inflammatory cytokines or fecal supernatants from patients with IBS-C.
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Síndrome del Colon Irritable , Isoquinolinas , Sulfonamidas , Humanos , Ratas , Animales , Síndrome del Colon Irritable/tratamiento farmacológico , Colon/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Funcion de la Barrera Intestinal , Capsaicina/farmacología , Células Receptoras Sensoriales/metabolismo , Dolor Abdominal/metabolismo , Citocinas/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
BACKGROUND: The use of cyclosporin A (CsA) is hampered by the development of nephrotoxicity including hypertension, which is partially dependent on renal sodium retention. To address this issue, we have investigated in vivo sodium reabsorption in different nephron segments of CsA-treated rats through micropuncture study coupled to expression analyses of sodium transporters. To translate the findings in rats to human, kidney-transplanted patients having CsA treatment were enrolled in the study. METHODS: Adult male Sprague-Dawley rats were treated with CsA (15 mg/kg/day) for 21 days, followed by micropuncture study and expression analyses of sodium transporters. CsA-treated kidney-transplanted patients with resistant hypertension were challenged with 50 mg furosemide. RESULTS: CsA-treated rats developed hypertension associated with reduced glomerular filtration rate. In vivo microperfusion study demonstrated a significant decrease in rate of absolute fluid reabsorption in the proximal tubule but enhanced sodium reabsorption in the thick ascending limb of Henle's loop (TAL). Expression analyses of sodium transporters at the same nephron segments further revealed a reduction in Na+-H+ exchanger isoform 3 (NHE3) in the renal cortex, while TAL-specific, furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and NHE3 were significantly upregulated in the inner stripe of outer medulla. CsA-treated patients had a larger excretion of urinary NKCC2 protein at basal condition, and higher diuretic response to furosemide, showing increased FeNa+, FeCl- and FeCa2+ compared with both healthy controls and FK506-treated transplanted patients. CONCLUSION: Altogether, these findings suggest that up-regulation of NKCC2 along the TAL facilitates sodium retention and contributes to the development of CsA-induced hypertension.
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Ciclosporina , Hipertensión , Adulto , Humanos , Masculino , Ratas , Animales , Ciclosporina/efectos adversos , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Regulación hacia Arriba , Furosemida , Ratas Sprague-Dawley , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
Electroneutral NaCl transport by Na+/H+ exchanger 3 (NHE3, SLC9A3) is the major Na+ absorptive mechanism in the intestine and decreased NHE3 activity contributes to diarrhea. Patients with diabetes often experience gastrointestinal adverse effects and medications are often a culprit for chronic diarrhea in type 2 diabetes (T2D). We have shown previously that metformin, the most widely prescribed drug for the treatment of T2D, induces diarrhea by inhibition of Na+/H+ exchanger 3 (NHE3) in rodent models of T2D. Metformin was shown to activate AMP-activated protein kinase (AMPK), but AMPK-independent glycemic effects of metformin are also known. The current study is undertaken to determine whether metformin inhibits NHE3 by activation of AMPK and the mechanism by which NHE3 is inhibited by AMPK. Inhibition of NHE3 by metformin was abolished by knockdown of AMPK-α1 or AMPK-α2. AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) phosphorylated NHE3 at S555. S555 is the primary site of phosphorylation by protein kinase A (PKA), but AMPK phosphorylated S555 independently of PKA. Using Mass spectrometry, we found S563 as a newly recognized phosphorylation site in NHE3. Altering either S555 or S563 to Ala was sufficient to block the inhibition of NHE3 activity by AMPK. NHE3 inhibition is dependent on ubiquitination by the E3 ubiquitin ligase Nedd4-2 and metformin was shown to induce NHE3 internalization via Nedd4-2-mediated ubiquitination. AICAR did not increase NHE3 ubiquitination when S555 or S563 was mutated. We conclude that AMPK activation inhibits NHE3 activity and NHE3 inhibition is associated with phosphorylation of NHE3 at S555 and S563.NEW & NOTEWORTHY We show that AMP-activated protein kinase (AMPK) phosphorylates NHE3 at S555 and S563 to inhibit NHE3 activity in intestinal epithelial cells. Phosphorylation of NHE3 by AMPK is necessary for ubiquitination of NHE3.
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Diabetes Mellitus Tipo 2 , Metformina , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Fosforilación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Metformina/farmacología , Intestinos , Diarrea , Aminoimidazol Carboxamida/farmacologíaRESUMEN
Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes high mortality in piglets, thus posing a serious threat to the world pig industry. Porcine epidemic diarrhea (PED) is related to the imbalance of sodium absorption by small intestinal epithelial cells; however, the etiology of sodium imbalanced diarrhea caused by PEDV remains unclear. Herein, we first proved that PEDV can cause a significant decrease in Na+/H+ exchanger 3 (NHE3) expression on the cell membrane, in a viral dose-dependent manner. Further study showed that the PEDV nucleocapsid (N) protein participates in the regulation of NHE3 activity through interacting with Ezrin. Flame atomic absorption spectroscopy results indicated a serious imbalance in Na+ concentration inside and outside cells following overexpression of PEDV N. Meanwhile, molecular docking technology identified that the small molecule drug Pemetrexed acts on the PEDV N-Ezrin interaction region. It was confirmed that Pemetrexed can alleviate the imbalanced Na+ concentration in IPEC-J2 cells and the diarrhea symptoms of Rongchang pigs caused by PEDV infection. Overall, our data suggest that the interaction between PEDV N and Ezrin reduces the level of phosphorylated Ezrin, resulting in a decrease in the amount of NHE3 protein on the cell membrane. This leads to an imbalance of intracellular and extracellular Na+, which causes diarrhea symptoms in piglets. Pemetrexed is effective in relieving diarrhea caused by PEDV. Our results provide a reference to screen for anti-PEDV targets and to develop drugs to prevent PED.IMPORTANCEPorcine epidemic diarrhea (PED) has caused significant economic losses to the pig industry since its initial outbreak, and the pathogenic mechanism of porcine epidemic diarrhea virus (PEDV) is still under investigation. Herein, we found that the PEDV nucleocapsid protein interacts with Ezrin to regulate Na+/H+ exchanger 3 activity. In addition, we screened out Pemetrexed, a small molecule drug, which can effectively alleviate pig diarrhea caused by PEDV. These results provide support for further exploration of the pathogenesis of PEDV and the development of drugs to prevent PED.
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Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Animales , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/veterinaria , Diarrea/tratamiento farmacológico , Diarrea/veterinaria , Simulación del Acoplamiento Molecular , Proteínas de la Nucleocápside/metabolismo , Pemetrexed/metabolismo , Virus de la Diarrea Epidémica Porcina/fisiología , Sodio/metabolismo , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Porcinos , Enfermedades de los Porcinos/tratamiento farmacológicoRESUMEN
Small organic molecules in the intestinal lumen, particularly short-chain fatty acids (SCFAs) and glucose, have long been postulated to enhance calcium absorption. Here, we used 45Ca radioactive tracer to determine calcium fluxes across the rat intestine after exposure to glucose and SCFAs. Confirming previous reports, glucose was found to increase the apical-to-basolateral calcium flux in the cecum. Under apical glucose-free conditions, SCFAs (e.g., butyrate) stimulated the cecal calcium fluxes by approximately twofold, while having no effect on proximal colon. Since SCFAs could be absorbed into the circulation, we further determined whether basolateral SCFA exposure rendered some positive actions. It was found that exposure of duodenum and cecum on the basolateral side to acetate or butyrate increased calcium fluxes. Under butyrate-rich conditions, cecal calcium transport was partially diminished by Na+/H+ exchanger 3 (NHE3) inhibitor (tenapanor) and nonselective transient receptor potential vanilloid subfamily 6 (TRPV6) inhibitor (miconazole). To confirm the contribution of TRPV6 to SCFA-stimulated calcium transport, we synthesized another TRPV6 inhibitor that was demonstrated by in silico molecular docking and molecular dynamics to occlude TRPV6 pore and diminish the glucose- and butyrate-induced calcium fluxes. Therefore, besides corroborating the importance of luminal molecules in calcium absorption, our findings provided foundation for development of more effective calcium-rich nutraceuticals in combination with various absorptive enhancers, e.g., glucose and SCFAs.NEW & NOTEWORTHY Organic molecules in the intestinal lumen, e.g., glucose and short-chain fatty acids (SCFAs), the latter of which are normally produced by microfloral fermentation, can stimulate calcium absorption dependent on transient receptor potential vanilloid subfamily 6 (TRPV6) and Na+/H+ exchanger 3 (NHE3). A selective TRPV6 inhibitor synthesized and demonstrated by in silico docking and molecular dynamics to specifically bind to the pore domain of TRPV6 was used to confirm a significant contribution of this channel. Our findings corroborate physiological significance of nutrients and SCFAs in enhancing calcium absorption.
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Calcio , Ácidos Grasos Volátiles , Ratas , Animales , Intercambiador 3 de Sodio-Hidrógeno/metabolismo , Calcio/metabolismo , Simulación del Acoplamiento Molecular , Ácidos Grasos Volátiles/farmacología , Ácidos Grasos Volátiles/metabolismo , Butiratos/farmacología , Proteínas Portadoras/metabolismo , Duodeno/metabolismo , Glucosa/metabolismo , Absorción IntestinalRESUMEN
Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.