RESUMEN
Uric acid (urate) is the end product of purine metabolism in humans. Human kidneys reabsorb a large proportion of filtered urate. This extensive renal reabsorption, together with the fact that humans do not possess uricase that catalyzes the biotransformation of urate into allantoin, results in a higher plasma urate concentration in humans compared to other mammals. A major determinant of plasma urate concentration is renal excretion as a function of the balance between reabsorption and secretion. We previously identified that renal urate absorption in proximal tubular epithelial cells occurs mainly via apical urate/anion exchanger, URAT1/SLC22A12, and by facilitated diffusion along the trans-membrane potential gradient by the basolateral voltage-driven urate efflux transporter, URATv1/SLC2A9/GLUT9. In contrast, the molecular mechanism by which renal urate secretion occurs remains elusive. Recently, we reported a newly characterized human voltage-driven drug efflux transporter, hNPT4/SLC17A3, which functions as a urate exit pathway located at the apical side of renal proximal tubules. This transporter protein has been hypothesized to play an important role with regard to net urate efflux. An in vivo role of hNPT4 is supported by the fact that missense mutations in SLC17A3 present in hyperuricemia patients with urate underexcretion abolished urate efflux capacity in vitro. Herein, we report data demonstrating that loop diuretics and thiazide diuretics substantially interact with hNPT4. These data provide molecular evidence for loop and thiazide-diuretics-induced hyperuricemia. Thus, we propose that hNPT4 is an important transepithelial proximal tubular transporter that transports diuretic drugs and operates functionally with basolateral organic anion transporters 1/3 (OAT1/OAT3).
Asunto(s)
Polaridad Celular , Electricidad , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Transportadores de Anión Orgánico/genética , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/químicaRESUMEN
The current study describes the chemical synthesis of a series of (2-ethylbenzofuran-3-yl)(substituted-phenyl)methanone compounds and their subsequent in vitro testing via oocytes expressing hURAT1. The experimental data support the notion that a potent hURAT1 inhibitor requires an anion (i.e., a formal negative charge) to interact with the positively charged hURAT1 binding pocket. An anion appears to be a primary requirement in order to be a hURAT1 substrate (i.e., urate) or inhibitor. We discuss the inhibitor structure-activity relationship and how electronically donating or withdrawing groups attached to the B-ring can decrease or increase inhibitory potency, respectively.
Asunto(s)
Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/química , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/química , Aniones , Chalconas/química , Chalconas/farmacología , Humanos , Transportadores de Anión Orgánico/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Relación Estructura-Actividad , Ácido Úrico/química , Ácido Úrico/metabolismoRESUMEN
An inhibitory effect of steviol, metabolite of the natural sweetener stevioside, on transepithelial transport of p-aminohippurate (J(PAH)) was observed in isolated S(2) segments of rabbit renal proximal tubules using in vitro microperfusion. Addition of steviol (0.01--0.25 mM) to the bathing medium significantly depressed J(PAH) (approximately 50--90%). This inhibitory effect was dose-dependent and was maximum at a concentration of 0.05 mM. To further examine this effect, a steviol concentration (0.01 mM) that produced approximately 50% inhibition of J(PAH), was chosen. Addition of 0.01 mM steviol to the bathing medium significantly depressed J(PAH) by about 50 to 60%. Steviol at the same concentration (0.01 mM), when present in the tubule lumen, had no significant effect on J(PAH). Addition of 0.01 mM steviol to lumen and bath simultaneously, produced a slightly greater inhibitory effect compared with addition to bath alone (60 versus 70%). A higher concentration of steviol, 0.05 mM (which maximally inhibited J(PAH) when on the basolateral side), was required on the luminal side than on the basolateral side before an inhibitory effect was observed. To further examine the mechanism by which steviol inhibited J(PAH), its effect on Na(+)-K(+) ATPase activity and ATP content was determined. Steviol at concentrations of 0.01 and 0.05 mM had no effect on Na(+)-K(+) ATPase activity or cell ATP content. Kinetic analyses indicated that steviol can competitively inhibit PAH transport at the basolateral membrane. The present study clearly showed that steviol can have a direct inhibitory effect on renal tubular transport by competitive binding with organic anion transporter.
Asunto(s)
Diterpenos de Tipo Kaurano , Diterpenos/farmacología , Túbulos Renales Proximales/metabolismo , Ácido p-Aminohipúrico/metabolismo , Adenosina Trifosfato/metabolismo , Algoritmos , Animales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Técnicas In Vitro , Túbulos Renales Proximales/efectos de los fármacos , Cinética , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Perfusión , Conejos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Stevioside, a non-caloric sweetening agent, is used as a sugar substitute. An influence of stevioside on renal function has been suggested, but little is known about its effect on tubular function. Therefore, the present study was designed to explore the direct effect of stevioside on transepithelial transport of p-aminohippurate (PAH) in isolated S2 segments of rabbit proximal renal tubules using in vitro microperfusion. Addition of stevioside at a concentration of 0.45 mM to either the tubular lumen, bathing medium, or both at the same time had no effect on transepithelial transport of PAH. Similarly, a concentration of 0.70 mM (maximum solubility in the buffer) when present in the lumen, had no effect on PAH transport. However, this concentration in the bathing medium inhibited PAH transport significantly by about 25-35%. The inhibitory effect of stevioside was gradually abolished after it was removed from the bath. Addition of 0.70 mM stevioside to both lumen and bathing medium at the same time produced no added inhibitory effect. Stevioside at this concentration has no effect on Na+/K+-ATPase activity as well as cell ATP content. These findings suggest that stevioside, at a pharmacological concentration of 0.70 mM, inhibits transepithelial transport of PAH by interfering with the basolateral entry step, the rate-limiting step for transepithelial transport. The lack of effect of stevioside on transepithelial transport of PAH on the luminal side and its reversible inhibitory effect on the basolateral side indicate that stevioside does not permanently change PAH transport and should not harm renal tubular function at normal human intake levels.
Asunto(s)
Diterpenos de Tipo Kaurano , Diterpenos , Glucósidos/farmacología , Túbulos Renales Proximales/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Edulcorantes/farmacología , Terpenos/farmacología , Ácido p-Aminohipúrico/farmacocinética , Animales , Transporte Biológico , Túbulos Renales Proximales/metabolismo , Masculino , Conejos , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes.