Identification of prolyl carboxypeptidase as an alternative enzyme for processing of renal angiotensin II using mass spectrometry.

Angiotensin-converting enzyme 2 (ACE2) catalyzes conversion of ANG II to ANG-(1-7). The present study uses newly established proteomic approaches and genetic mouse models to examine the contribution of alternative renal peptidases to ACE2-independent formation of ANG-(1-7). In situ and in vitro mass spectrometric characterization showed that substrate concentration and pH control renal ANG II processing. At pH ≥6, ANG-(1-7) formation was significantly reduced in ACE2 knockout (KO) mice. However, at pH <6, formation of ANG-(1-7) in ACE2 KO mice was similar to that in wild-type (WT) mice, suggesting alternative peptidases for renal ANG II processing. Furthermore, the dual prolyl carboxypeptidase (PCP)-prolyl endopeptidase (PEP) inhibitor Z-prolyl-prolinal reduced ANG-(1-7) formation in ACE2 KO mice, while the ACE2 inhibitor MLN-4760 had no effect. Unlike the ACE2 KO mice, ANG-(1-7) formation from ANG II in PEP KO mice was not different from that in WT mice at any tested pH. However, at pH 5, this reaction was significantly reduced in kidneys and urine of PCP-depleted mice. In conclusion, results suggest that ACE2 metabolizes ANG II in the kidney at neutral and basic pH, while PCP catalyzes the same reaction at acidic pH. This is the first report demonstrating that renal ANG-(1-7) formation from ANG II is independent of ACE2. Elucidation of ACE2-independent ANG-(1-7) production pathways may have clinically important implications in patients with metabolic and renal disease.

Administración de carboxipeptidasa tras altas dosis de metotrexato. Tratamiento e interacciones medicamentosas / Carboxypeptidase-G2 administration after high-dose methotrexate. Treatment and drug interactions

El metotrexato es un antineoplásico muy utilizado y eficaz en neoplasias como las leucemias, los linfomas y los osteosarcomas. La toxicidad renal es un efecto secundario indeseable que se pretende evitar con una alcalinización urinaria e hiperhidratación eficaz. En caso de intoxicación aguda se establece el uso de carboxipeptidasa G2, una enzima que provoca la hidrólisis del metotrexato en sus metabolitos inactivos. Por su parte, el uso de glutamina durante el tratamiento oncológico previene parte de los efectos indeseables secundarios a éste. Se presenta el caso clínico de un adolescente afectado de linfoma no hodgkiniano en tratamiento con glutamina, que tras la administración de un tercer ciclo de metotrexato (5g/m2) presentó un cuadro de insuficiencia renal que precisó la administración de carboxipeptidasa, con descenso no satisfactorio de las concentraciones de metotrexato en sangre, si bien no se pudo diferenciar la fracción activa del metabolito inactivo por carecer de esta técnica en España. Se revisó la bibliografía sobre la interacción de glutamina y metotrexato y se discutió su fisiopatología sobre un posible papel de la glutamina como favorecedora de la toxicidad por metotrexato (AU)

Methotrexate (MTX) is widely used as anticancer agent in various malignancies, including acute lymphoblastic leukaemia, lymphoma and osteosarcoma. High doses of MTX may cause acute renal dysfunction. Nephrotoxicity is prevented by the use of alkalinization and hydration. More recently Carboxypeptidase-G2, a recombinant bacterial enzyme that rapidly hydrolyzes MTX to inactive metabolites, has become available for the treatment of acute nephrotoxicity. On the other hand, glutamine is usually administered in oncology treatments to avoid other side effects. We report a case of an adolescent who was diagnosed with T lymphoblastic lymphoma. He was receiving treatment with glutamine when the third course of methotrexate was administered (5g/m2) and he suffered a deterioration in his renal function. Carboxypeptidase was used but the methotrexate serum concentration reduction was not satisfactory. The technique to assess the amount of enzyme-inactivated methotrexate by quantification of MTX metabolites is not available in our country, therefore, the concentrations of MTX may be overestimated. The literature was reviewed to study the influence of glutamine on delayed methotrexate elimination which may lead to acute toxicity (AU)

Functional amelioration of murine galactosialidosis by genetically modified bone marrow hematopoietic progenitor cells.

Protective protein/cathepsin A (PPCA), a lysosomal carboxypeptidase, is deficient in the neurodegenerative lysosomal disorder galactosialidosis (GS). PPCA(-/-) mice display a disease course similar to that of severe human GS, resulting in nephropathy, ataxia, and premature death. Bone marrow transplantation (BMT) in mutant animals using transgenic BM overexpressing the corrective enzyme in either erythroid cells or monocytes/macrophages has proven effective for the improvement of the phenotype, and encouraged the use of genetically modified BM cells for ex vivo gene therapy of GS. Here, we established stable donor hematopoiesis in PPCA(-/-) mice that received hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based, bicistronic retroviral vector overexpressing PPCA and the green fluorescent protein (GFP) marker. We observed complete correction of the disease phenotype in the systemic organs up to 10 months after transplantation. PPCA(+) BM-derived cells were detected in all tissues, with the highest expression in liver, spleen, BM, thymus, and lung. In addition, a lysosomal immunostaining was seen in nonhematopoietic cells, indicating efficient uptake of the corrective protein by these cells and cross-correction. Expression in the brain occurred throughout the parenchyma but was mainly localized on perivascular areas. However, PPCA expression in the central nervous system was apparently sufficient to delay the onset of Purkinje cell degeneration and to correct the ataxia. The long-term expression and internalization of the PPCA by cells of systemic organs and the clear improvement of the neurologic phenotype support the use of this approach for the treatment of GS in humans. (Blood. 2002;99:3169-3178)