RESUMEN
Treatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.
Asunto(s)
Aprotinina/metabolismo , Túbulos Renales/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Animales , Aprotinina/administración & dosificación , Aprotinina/efectos adversos , Relación Dosis-Respuesta a Droga , Femenino , Inyecciones Subcutáneas , Túbulos Renales/patología , Masculino , Ratones , Ratones Transgénicos , Estructura Molecular , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/efectos adversos , Relación Estructura-ActividadRESUMEN
Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Síndrome Nefrótico/etiología , Síndrome Nefrótico/metabolismo , Aldosterona/farmacología , Animales , Antibióticos Antineoplásicos/toxicidad , Aprotinina/farmacología , Doxorrubicina/toxicidad , Canales Epiteliales de Sodio/genética , Femenino , Humanos , Riñón/metabolismo , Masculino , Ratones , Subunidades de Proteína , Proteolisis , Triantereno/farmacologíaRESUMEN
Several studies have implicated a remarkable dysfunctional apoptotic state and/or response in ESRD patients. Previously published studies are controversial with respect to acute effects of haemodialysis (HD) treatment on up- or downregulation of apoptotic genes. Twenty-eight chronic HD patients were haemodialysed for 4 hours with a 4008 dialyser using high-flux membranes. For subgroup analysis, patients were separated into a low (up to 0.5 mg/dl) and a high (0.5 to 5.0 mg/dl) CRP group. Blood was drawn prior to HD and 240 min after initiation of HD. Acute changes of transcript levels encoding pro- or anti-apoptotic genes were analyzed in RNA immediately isolated from blood leukocytes using quantitative real-time PCR. In the present study, we detected a significant elevation of the death receptor CD95/Fas (induction factor (IF) 1.55 +/- 0.16), the death receptor 5 (DR5) (IF 1.17 +/- 0.08), and caspase 8 (IF 1.37 +/- 0.14) gene expression during HD. mRNA levels of the respective ligands (CD95L, TRAIL), of the caspase 5 and anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl2l2 were slightly, but not significantly, increased after HD treatment. An additional anti-apoptotic molecule, BAG3, was found to be slightly, but significantly, induced after HD (IF 1.16 +/- 0.07). In addition to being an activator of immune cells, CD40L has been shown to be strongly induced after HD treatment (IF 1.70 +/- 0.20). Subgroup analysis revealed no significant differences between low vs. high CRP patient groups or diabetic vs. non-diabetic patients. These results indicate a marked influence of routine haemodialysis treatment on the transcription of pro- and anti-apoptotic molecules and the involvement of the extrinsic pathway for apoptosis through the activation of death receptors and the initiator caspase 8. Furthermore, following dialysis, lymphocytes seem to be activated by CD40L, which represents an early T-cell activation marker.