Upregulation of intrarenal angiotensinogen in diabetes

Universidade Federal de São Paulo, Dept Med, Div Nephrol, BR-04023040 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, BR-04023040 São Paulo, BrazilWeb of Scienc

Characterization of a kinin inactivating serine endopeptidase H2 (kininase) from human urine using fluorogenic substrates

We have previously described a kinin-inactivating endopeptidase (H2), which was purified 19-fold from human urine by DEAE-cellulose chromatography and gel filtration. the enzyme was inhibited 100% by PMSF, TPCK and pOHMB. in the present communication, we further characterized this enzyme using the fluorogenic substrates Abz-RPPGFSPFRQ-EDDnp (Abz-BKQ-EDDnp) and Abz-FRQ-EDDnp (Abz = ortho-aminobenzoic acid; EDDnp = N-[2,4-dinitrophenyl] ethylenediamine). Also a rapid, sensitive and specific assay for the H2 was developed. the enzyme hydrolyzed bradykinin (BK = RPPGFSPFR) at the F-S peptide bond, differing from the cleavage site F-R, in the fluorogenic substrates Abz-BKQ-EDDnp and Abz-FRQ-EDDnp. Other enzymes present. in urine als the serine endopeptidase III, prolyl endopeptidase and neutral endopeptidase-like were not able to hydrolyze the related substrate Abz-FRQ-EDDnp. the determined K-m for Abz-BKQ-EDDnp and Abz-FRQ-EDDnp were 0.79 mu M and 3.02 mu M, respectively. Using the fluorogenic substrates, we observed that PMSF and p-hydroxymercuribenzoate irreversibly inhibited the enzyme H2. E-64 was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. the inhibition observed in the presence of pOHMB was partially reversed by 2 mM cysteine. These results suggest that the H2 enzyme belongs to the subfamily of SH-containing serine proteases. Based on the molecular weight of isolated H2 (60 kDa), we believe that this enzyme originated from the kidney and may cleave the kinins filtered through the glomerulus and also that produced in the kidney. (C) 1999 Published by Elsevier Science B.V. All rights reserved.Universidade Federal de São Paulo, Escola Paulista Med, Disciplina Nefrol, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Disciplina Nefrol, Dept Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04023900 São Paulo, BrazilWeb of Scienc

Neutral endopeptidase like (NEP-like) activity in human urine

Escola Paulista Med, Dept Med, UNIFESP, Div Nephrol, BR-04023900 Sao Paulo, BrazilEscola Paulista Med, UNIFESP, Dept Biophys, BR-04023900 Sao Paulo, BrazilEscola Paulista Med, Dept Med, UNIFESP, Div Nephrol, BR-04023900 Sao Paulo, BrazilEscola Paulista Med, UNIFESP, Dept Biophys, BR-04023900 Sao Paulo, BrazilWeb of Scienc

Purification and characterization of angiotensin I-converting enzymes from mesangial cells in culture

Objective Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. the aim of this study was to purify and characterize the secreted end intracellular ACE forms from mesangial cells in culture.Methods and results Medium from Wister rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. the concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE(1), mol. wt 130 000 and ACE(2), 60 000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. the mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. the supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACE(Int1), mol. wt 130 000 and ACE(Int2), 68 000) were separated. the purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. the K-m values were 2 mM for ACE(1) and ACE(2) and 3 mM for ACE(Int1) end ACE(Int2). the enzymes ACE(1) and ACE(2) presented an optimum pH of 8.0 and ACE(Int1) and ACE(Int2) an optimum pH of 7.5.Conclusion the activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. the ratios found for ACE(1), ACE(2), ACE(Int1) and ACE(Int2) in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation. I Hypertens 1998, 16:2063-2074 (C) 1998 Lippincott Williams & Wilkins.Universidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol,Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol,Div Nephrol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023900 São Paulo, BrazilWeb of Scienc