RESUMEN
AIM: In nephrotic syndrome, aberrantly filtered plasminogen (plg) is converted to active plasmin by tubular urokinase-type plasminogen activator (uPA) and thought to lead to sodium retention by proteolytic activation of the epithelial sodium channel (ENaC). This concept predicts that uPA is an important factor for sodium retention and that inhibition of uPA might be protective in nephrotic syndrome. METHODS: Activation of amiloride-sensitive currents by uPA and plg were studied in Xenopus laevis oocytes expressing murine ENaC. In doxorubicin-induced nephrotic mice, uPA was inhibited pharmacologically by amiloride and genetically by the use of uPA-deficient mice (uPA-/- ). RESULTS: Experiments in Xenopus laevis oocytes expressing murine ENaC confirmed proteolytic ENaC activation by a combination of plg and uPA which stimulated amiloride-sensitive currents with concomitant cleavage of the ENaC γ-subunit at the cell surface. Treatment of nephrotic wild-type mice with amiloride inhibited urinary uPA activity, prevented urinary plasmin formation and sodium retention. In nephrotic mice lacking uPA (uPA-/- ), urinary plasmin formation from plg was suppressed and urinary uPA activity absent. However, in nephrotic uPA-/- mice, sodium retention was not reduced compared to nephrotic uPA+/+ mice. Amiloride prevented sodium retention in nephrotic uPA-/- mice which confirmed the critical role of ENaC in sodium retention. CONCLUSION: uPA is responsible for the conversion of aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo. However, uPA-dependent plasmin generation is not essential for ENaC-mediated sodium retention in experimental nephrotic syndrome.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Amilorida/administración & dosificación , Amilorida/farmacología , Animales , Relación Dosis-Respuesta a Droga , Bloqueadores del Canal de Sodio Epitelial/administración & dosificación , Bloqueadores del Canal de Sodio Epitelial/farmacología , Canales Epiteliales de Sodio/genética , Regulación de la Expresión Génica/efectos de los fármacos , Activación del Canal Iónico , Ratones , Ratones Noqueados , Síndrome Nefrótico , Oocitos , Activador de Plasminógeno de Tipo Uroquinasa/genética , Xenopus laevisRESUMEN
Doxorubicin-induced nephropathy in mice is a model for studying experimental nephrotic syndrome. It corresponds to puromycin aminonucleoside nephrosis in rats. In this model, susceptible 129 S1/SvImJ mice are administered a rapid intravenous injection that can be accomplished via either the lateral tail vein or the retrobulbar sinus. Because doxorubicin is a highly toxic substance, extravasation must be avoided during the administration of the intravenous injection to prevent the development of large necrotizing lesions and exacerbation of the animals' stress. In the present study, we compared the safety and stress of these two injection routes by using histopathological analyses of the animals' orbital cavities or tails, respectively. The injection of 14.5 µg/g body weight doxorubicin into the mice's lateral tail veins (n = 9) or retrobulbar sinuses (n = 19) caused no clinically detectable stress or impairment. Histopathologies of the specimens five days after doxorubicin injection revealed inflammatory lesions at the injection sites in both groups. In the orbital sinus specimens from the retrobulbar-injected group, fibrosis was evident 25 days after injection. Moreover, while all of the retrobulbar-injected mice (100%) developed nephrotic syndrome, tail vein-injected mice had a significantly lower response rate (66%, p = 0.047, Fisher's exact test) and exhibited only attenuated features of nephrotic syndrome. It was therefore concluded that doxorubicin administration via either lateral tail vein or retrobulbar sinus injections led to a similar induction of histopathological changes with no effects on the clinical well-being of the mice. However, retrobulbar sinus injections were more efficient for inducing experimental nephrotic syndrome.
Asunto(s)
Administración Intranasal , Doxorrubicina/efectos adversos , Inyecciones Intravenosas , Síndrome Nefrótico/inducido químicamente , Animales , Femenino , Masculino , Ratones , Proyectos Piloto , Cola (estructura animal)RESUMEN
Volume retention in nephrotic syndrome has been linked to activation of the epithelial sodium channel (ENaC) by proteolysis of its γ-subunit following urinary excretion of serine proteases such as plasmin. Here we tested whether pharmacological inhibition of urinary serine protease activity might protect from ENaC activation and volume retention in nephrotic syndrome. Urine from both nephrotic mice (induced by doxorubicin injection) and nephrotic patients exhibited high aprotinin-sensitive serine protease activity. Treatment of nephrotic mice with the serine protease inhibitor aprotinin by means of subcutaneous sustained-release pellets normalized urinary serine protease activity and prevented sodium retention, as did treatment with the ENaC inhibitor amiloride. In the kidney cortex from nephrotic mice, immunofluorescence revealed increased apical γ-ENaC staining, normalized by aprotinin treatment. In Xenopus laevis oocytes heterologously expressing murine ENaC, aprotinin had no direct inhibitory effect on channel activity but prevented proteolytic channel activation. Thus, our study shows that volume retention in experimental nephrotic syndrome is related to proteolytic ENaC activation by proteasuria and can be prevented by treatment with aprotinin. Hence, inhibition of urinary serine protease activity might become a therapeutic approach to treat patients with nephrotic-range proteinuria.