Beata Marie Redublo Quinto

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 and intracellular ACE forms from mesangial cells in culture. Methods and results Medium from Wistar 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 (ACE1, mol. wt 130 000 and ACE2, 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 (ACEInt1, mol. wt 130 000 and ACEInt2, 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 Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 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 ACE1, ACE2, ACEInt1 and ACEInt2 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.

Uric acid as a marker for renal dysfunction in hypertensive women on diuretic and nondiuretic therapy.

Hyperuricemia is a common finding in hypertensive patients, especially among those who are on diuretic therapy. However, its clinical relevance regarding cardiovascular and chronic kidney disease (CKD) has not clearly been established. The authors assessed whether, in a population of 385 hypertensive women categorized according to diuretic therapy, the stratification in quartiles by uric acid levels would identify a gradient of changes in renal function and in risk factors for cardiovascular disease. The following were evaluated: serum uric acid, glycemia, total and fractional cholesterol, triglycerides, apolipoprotein (Apo) B, Apo A‐I, and C‐reactive protein. Renal function was assessed by serum creatinine, albuminuria, and estimated glomerular filtration rate (eGFR) by the Modification of Diet in Renal Disease equation, whereas cardiovascular risk was estimated through the Framingham score. A total of 246 women were on diuretic therapy; 139 were taking other antihypertensive medications. There was a reduction in eGFR parallel to the increase in uric acid levels, regardless of diuretic use and without a concomitant increase in albuminuria. In both groups, higher uric acid levels translated into an increase in metabolic syndrome components, in markers of insulin resistance, triglyceride/high‐density lipoprotein levels, and Apo B/Apo A‐I ratios, as well as in Framingham scores. Hyperuricemia was associated with an increase in inflammatory markers only in patients on diuretic therapy. In a binary logistic regression, hyperuricemia (uric acid >6.0 mg/dL) was independently associated with CKD (eGFR <60 mL/min/1.73 m²) (odds ratio, 2.63; 95% confidence interval, 1.61–4.3; P<.001). In hypertensive women, the presence of hyperuricemia indicated a substantial degree of kidney dysfunction as well as a greater cardiovascular risk profile.

Expression of angiotensin I-converting enzymes and bradykinin B2 receptors in mouse inner medullary-collecting duct cells

We described in mouse inner medullary-collecting duct cells (mIMCD-3) the somatic and the N-domain ACE synthesis and its interaction with the kallikrein-kinin system co-localized in the same cells. We purified two ACE forms from culture medium, M1 (130 kDa) and M2 (N-domain, 60 kDa), and cellular lysate, C1 (130 kDa) and C2 (N-domain, 60 kDa). Captopril and enalaprilat inhibited the purified enzymes. The immunofluorescence studies indicated that ACE is present in the membrane, cytoplasm and in the cell nucleus. Kinin B1 and B2 receptors were detected by immunofluorescence and showed to be activated by BK and DesR9 BK, increasing the acidification rate which was enhanced in the presence of enalaprilat. The presence of secreted and intracellular ACE in mIMCD-3 confirmed the hypothesis previously proposed by our group for a new site of ACE secretion in the collecting duct.

N-Domain Angiotensin I-Converting Enzyme With 80 kDa as a Possible Genetic Marker of Hypertension

Abstract—We have previously described angiotensin I-converting enzyme (ACE) forms in urine of normotensive (190 and 65 kDa) and hypertensive patients (90 and 65 kDa, N-domain ACEs). Based on the results described above, experimental and genetic models of hypertension were investigated to distinguish hemodynamic and genetic influence on the generation of ACE profile in urine: Wistar-Kyoto and Brown Norway rats (WKY and BN), spontaneously and stroke-prone spontaneously hypertensive rats (SHR and SHR-SP), one kidney/one clip rats (1K1C), deoxycorticosterone acetate (DOCA) salt-treated and untreated rats, and enalapril-treated SHR (SHRen). Two peaks with ACE activity were separated from the urine of WKY and BN rats submitted to an AcA-44 column, WK-1/BN-1 (190 kDa), and WK-2/BN-2 (65 kDa), as described for urine of normotensive subjects. The same results were obtained for urine of 1K1C and DOCA salt-treated and untreated rats, analyzed to evaluate the influence of hemodynamic factors in the ACE profile in urine. The urine from SHR, SHR-SP, and SHRen presented 80 (S-1, SP-1, Sen-1) and 65 (S-2, SP-2, Sen-2) kDa ACE forms, differing from the urine profile of normotensive rats, but similar to that described for hypertensive patients. The presence of 80 kDa ACE in urine of SHR, SHR-SP, and SHRen and its absence in urine of experimental hypertensive rats (1K1C and DOCA salt) support the hypothesis that this enzyme could be a possible genetic marker of hypertension. Taken together, our results provide evidence that ACE forms with 90/80 kDa isolated from the urine of hypertensive subjects and genetic hypertensive animals behaves as a possible genetic marker of hypertension and not as a marker of high blood pressure.

TNF-α modulates statin effects on secretion and expression of MCP-1, PAI-1 and adiponectin in 3T3-L1 differentiated adipocytes

PURPOSE Systemic inflammatory conditions, as seen in obesity and in the metabolic syndrome, are associated with high plasmatic levels of proatherogenic and prothromboticadipokines and low levels of adiponectin. Inhibitors of HMG-CoA reductase have beneficial effects in reducing cardiovascular events attributed predominantly to its lipid-lowering effects and recent studies suggest that these effects might be due to its anti-inflammatory properties. Based on the pleiotropic properties of simvastatin we studied the effects of this drug on the secretion and expression of adiponectin, PAI-1 and MCP-1 in mature adipocytes under baseline conditions and after an inflammatory stimulation. MATERIALS AND METHODS The differentiated adipocytes were incubated with 10 μM simvastatin or vehicle and TNF-α 10 ng/mL or vehicle were added to treatment media. After 24h of incubation, the media was harvested and the proteins of interest were analyzed by Multiplex method. Gene expression was analyzed by real time-PCR. RESULTS The addition of TNF-α increased the expression and secretion of MCP-1 and PAI-1. However, stimulation did not interfere with the secretion of adiponectin, despite having significantly reduced its expression. Our data also demonstrated that simvastatin reduced the expression and secretion of MCP-1, under baseline (770.4 ± 199.9 vs 312.7 ± 113.7 and 1.00 ± 0.14 vs 0.63 ± 0.13, p<0.05, respectively) and inflammatory conditions (14945 ± 228.7 vs 7837.6 ± 847.4 and 24.16 ± 5.49 vs 14.97 ± 2.67, p<0.05, p<0.05, respectively). Simvastatin also attenuated the increase in expression and secretion of PAI-1 induced by TNF-α (16898.6 ± 1663.3 vs 12922.1 ± 843.9 and 5.19 ± 3.12 vs 0.59 ± 0.16, respectively p<0.05), but under baseline conditions had no effect on the expression or secretion of PAI-1. The statin increased the expression of adiponectin under baseline conditions and inflammatory stimulation (1.03 ± 0.08 vs 4.0 ± 0.96 and 0.77 ± 0.19 vs 2.16 ± 0.23, respectively, p<0.05) and also increased the secretion of this adipokine but only with the inflammatory stimulus (5347.7 ± 1789.3 vs 7327.3 ± 753.6, p<0.05). CONCLUSIONS Our findings suggested that simvastatin counteracted the stimulatory effect of TNF-α on secretion and expression of MCP-1, PAI-1 and adiponectin, implying a potential anti-atherogenic effect during the inflammatory process; these pleitropic effects were more pronounced with HMG-CoA reductase inhibitor.

Tumour necrosis factor-α plus interleukin-10 low producer phenotype predicts acute kidney injury and death in intensive care unit patients

Genetic polymorphism studies of cytokines may provide an insight into the understanding of acute kidney injury (AKI) and death in intensive care unit (ICU) patients. The aim of this study was to investigate whether the genetic polymorphisms of −308 G < A tumour necrosis factor (TNF)‐α, −174 G > C interleukin (IL)‐6 and −1082 G > A IL‐10 may predispose ICU patients to the development of AKI and/or death. In a prospective nested case–control study, 303 ICU patients and 244 healthy individuals were evaluated. The study group included ICU patients who developed AKI (n = 139) and 164 ICU patients without AKI. The GG genotype of TNF‐α (low producer phenotype) was significantly lower in the with AKI than without AKI groups and healthy individuals (55 versus 62 versus 73%, respectively; P = 0·01). When genotypes were stratified into four categories of TNF‐α/IL‐10 combinations, it was observed that low TNF‐α plus low IL‐10 producer phenotypes were more prevalent in patients with AKI, renal replacement therapy and death (P < 0·05). In logistic regression analysis, low TNF‐α producer plus low IL‐10 producer phenotypes remained as independent risk factors for AKI and/or death [odds ratio (OR) = 2·37, 95% confidence interval (CI): 1·16–4·84; P = 0·02] and for renal replacement therapy (RRT) and/or death (OR = 3·82, 95% CI: 1·19–12·23; P = 0·02). In this study, the combination of low TNF‐α plus low IL‐10 producer phenotypes was an independent risk factor to AKI and/or death and RRT and/or death in critically ill patients. Our results should be validated in a larger prospective study with long‐term follow‐up to emphasize the combination of these genotypes as potential risk factors to AKI in critically ill patients.

Is Cystatin C a Useful Marker in the Detection of Diabetic Kidney Disease

Background/Aims: To evaluate cystatin C as a marker of diabetic kidney disease in normoalbuminuric diabetic patients without chronic kidney disease (CKD). Methods: A cross- sectional study was carried out comprising 243 hypertensive patients, 61 of them with type 2 diabetes, presenting normoalbuminuria and an estimated glomerular filtration rate (eGFR) ≧60 ml/min/1.73 m2. Renal function assessment included determinations of serum creatinine and cystatin C levels, microalbuminuria, as well as eGFR through Cockcroft-Gault and Modification of Diet in Renal Disease equations. Results: Diabetic patients presented higher cystatin C levels than nondiabetic patients (0.95 ± 0.19 vs. 0.89 ± 0.17 mg/l; p < 0.05). In the binary logistic regression, the presence of diabetes and metabolic syndrome was significantly associated with elevated cystatin C levels. Diabetic patients also presented a slightly greater albuminuria (6.72 ± 4.43 vs. 5.07 ± 3.59 µg/min; p < 0.05). Conclusions: Our results suggest that elevated cystatin C levels in diabetic patients may identify a certain degree of renal dysfunction even when albuminuria and eGFR do not mirror CKD. Longitudinal studies with direct GFR measures need to be done in order to confirm the value of cystatin C as an indicative of worse renal outcomes in the diabetic population.

Purification and characterization of a neutral endopeptidase-like enzyme from human urine.

Objective The aims of this study were to purify and characterize a neutral endopeptidase-like enzyme (NEP- like) in human urine and propose a rapid, sensitive and specific assay for this enzyme using the fluorogenic substrate Abz-FDQ-EDDnp, where Abz = O-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine. Methods Soluble urinary NEP was purified from human urine using a DEAE-cellulose Cellex D column and gel filtration on an AcA-44 column. NEP-like activity was assayed by its ability to hydrolyse bradykinin (BK) and the fluorogenic substrates Abz-BKQ-EDDnp and Abz- FDQ-EDDnp. The Km was determined using Abz-FDQEDDnp as a substrate. The hydrolysis products of BK and Abz-FDQ-EDDnp were analysed by high-performance liquid chromatography (HPLC). The mol. wt was estimated by polyacrylamide gel electrophoresis and the enzyme analysed by Western blot using the antibody obtained from purified recombinant NEP expressed in Pichia pastoris yeast. Results The NEP-like was purified from human urine until homogeneity and presented a mol. wt of 94 000. The substrate Abz-FDQ-EDDnp was selectively hydrolysed at the F-D bond by NEP-like and by recombinant NEP. For this substrate, the NEP-like activity was maximal at pH 7.0, although a small peak of activity was observed at pH 8.0, and the determined Km was 14 μM. The enzymatic activity was inhibited by thiorphan and phosphoramidon. In Western blot analysis, NEP-like reacted strongly with a polyclonal antibody for NEP. Conclusion A NEP-like enzyme was purified from human urine. Based on the mol. wt of the isolated NEP-like enzyme, it was concluded that this enzyme was produced in the kidney. In the kidney, this enzyme may cleave the kinins filtered through the glomerulus and also the kinins produced in the distal nephron. An internally quenched fluorogenic peptide, Abz-FDQ-EDDnp, was selectively hydrolysed by NEP-like and by recombinant NEP.

Effects of simvastatin on cytokines secretion from mononuclear cells from critically ill patients with acute kidney injury.

PURPOSE To assess the in vitro effects of simvastatin on IL-10 and TNF-α secretion from peripheral blood mononuclear cells (PBMC) of critically ill patients with and without acute kidney injury (AKI). METHODS PBMC were collected from 63 patients admitted to the intensive care unit (ICU) and from 20 healthy controls. Patients were divided in 3 subgroups: with AKI, with sepsis and without AKI and with AKI and sepsis. After isolation by ficoll-gradient centrifugation cells were incubated in vitro with LPS 1 ng/mL, simvastatin (10(-8)M) and with LPS plus simvastatin for 24h. TNF-α and IL-10 concentrations on cells surnatant were determined by ELISA. RESULTS Cells isolated from critically ill patients showed a decreased spontaneous production of TNF-α and IL-10 compared to healthy controls (6.7 (0.2-12) vs 103 (64-257) pg/mL and (20 (13-58) vs 315 (105-510) pg/mL, respectively, p<0.05). Under LPS-stimulus, IL-10 production remains lower in patients compared to healthy control (451 (176-850) vs 1150 (874-1521) pg/mL, p<0.05) but TNF-α production was higher (641 (609-841) vs 406 (201-841) pg/mL, p<0.05). The simultaneous incubation with LPS and simvastatin caused decreased IL-10 production in cells from patients compared to control (337 (135-626) vs 540 (345-871) pg/mL, p<0.05) and increased TNF-α release (711 (619-832) vs 324 (155-355) pg/mL, p<0.05). Comparison between subgroups showed that the results observed in TNF-α and IL-10 production by PBMC from critically ill patients was independent of AKI occurrence. CONCLUSIONS The PBMC treatment with simvastatin resulted in attenuation on pro-inflammatory cytokine spontaneous production that was no longer observed when these cells were submitted to a second inflammatory stimulus. Our study shows an imbalance between pro and anti-inflammatory cytokine production in PBMC from critically ill patients regardless the presence of AKI.

Characterization of a prolyl endopeptidase (kininase) from human urine using fluorogenic quenched substrates.

A prolyl endopeptidase (PE) was purified 83 times from human urine by DEAE-cellulose and Sepharose Mercurial chromatographies. In this work we studied the specificity of PE using different fluorogenics substrates. Further characterization of the enzyme was carried out using BK and its analogue, Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp, for measure of enzymatic activity of prolyl endopeptidase (Abz=ortho-aminobenzoic acid; EDDnp=N-[2, 4-dinitrophenyl]ethylenediamine). The substrate Abz-FPQ-EDDnp was considered as specific for PE. The endopeptidase PE, with a molecular weight of 45 kDa, was inhibited 100% by EDTA and pOHMB and resistant to PMSF, thyorphan, E64 and phosphoramidon, when we used the mentioned substrates. These results suggest that PE is a metallo endopeptidase that contains a thiol group important for its activity. It was also able to hydrolyze in Abz-RPPGFSPFRQ-EDDnp the F-R peptide bound, differing from those obtained upon BK molecule, where the enzyme prefer the peptide bound located after double proline. In the substrate Abz-FPQ-EDDnp PE hydrolyzes the P-Q peptide bound. Furthermore the urinary PE is particularly unable to hydrolyze peptides with single prolines such as substance P, neurotensin and LHRH. The determined K(m) for Abz-RPPGFSPFRQ-EDDnp and Abz-FPQ-EDDnp were 0.74 and 0.65 uM, respectively. The optimum pH for the PE activity, using the substrate Abz-RPPGFSPFRQ-EDDnp was approximately 9.0, but using the specific substrate Abz-FPQ-EDDnp was 6.5 and 8.0. Endopeptidases, which are situated at brush border surface from proximal tubules, have an important role in kidney handling of many peptides, which are filtered by the glomerulus. The prolyl endopeptidase located at distal tubule could have an important physiological function in control of kinin formed in this portion. Its known that all components from kallicrein-kinin system like low molecular weigh kininogen and kallikrein are presents in this portion.