ON HUMAN TISSUE ACTIVITY IN URINE OF BRAZILIAN WHITE AND BLACK PRIMARY HYPERTENSIVE PATIENTS

Objective: To evaluate the possible relation- Andreza Alves Belo, MSc; Marinez de Oliveira Sousa, PhD; ship between the human tissue kallikrein Eduardo Luis Guimara˜es Machado, MD; amidase activity with sex and ethnicity in Brazilian primary hypertensive patients. Amintas Fabiano de Souza Figueiredo, PhD

Design: Population-based study. INTRODUCTION Participants: One hundred men and women, Abnormality of the tissue Black and White primary hypertensive patients Primary (essential) hypertension is a aged 20 years and older were selected. Eighty kallikrein-kinin system has nine healthy individuals, paired according to multifactorial disease influenced by a age, sex, and ethnics were used as controls. combination of genetic and environ- long been documented in the mental factors.1 Once hypertension 4 Methods: Early-morning midstream urine was develops, it tends to become self- pathogenesis of hypertension. used. Human tissue kallikrein amidase activity perpetuating by amplifying mechanisms was estimated with D-Val-Leu-Arg 4-nitroani- lide substrate. Creatinine was determined by a mediated by secondary structural chang- method based on Jaffe’s reaction. hK1 amidase es in blood vessels, heart, and kidney. activity is expressed in mM/(min . mg creati- The long-term sequelae from hyperten- 3.4.21.34) and tissue (EC 3.4.21.35) 4 nine) to correct for differences in urine flow sion are atherosclerotic vascular disease . The KLK1 gene, located on rate. Data are expressed as medians. complications, cardiac hypertrophy and chromosome 19q13.4, expresses human failure, stroke, and renal failure.2 The tissue kallikrein (hK1), whose principal Results: Human tissue kallikrein amidase known biochemical function is to activity was significantly lower in the urine of primary cause of human essential hy- hypertensive patients (0.210 mM/(min . mg pertension is not yet known, despite release the vasoactive and spasmogenic creatinine) than in the urine of control subjects intensive research on the various mech- decapeptide kallidin (lysyl-bradykinin) (0.260 mM/(min . mg creatinine) (P5.010). anisms that may be involved in its (Lys-BK) from the plasma protein This result supports data from the literature. development.3 low molecular weight kininogen Contrasting to what was already reported, (LMWK).5,6 Lys-BK is a potent vaso- namely that human tissue kallikrein excretion Abnormality of the tissue kallikrein- is higher in females than in males, and kinin system has long been documented dilator and also promotes diuresis and 7 especially higher in Caucasians than in African in the pathogenesis of hypertension.4 natriuresis. Urinary excretion of renal Americans, our results show that, in the urine Epidemiological studies have identified (tissue) kallikrein has emerged as an of Brazilian hypertensive patients and control an inverse relationship between urinary intermediate phenotype for hyperten- subjects, no significant effect of sex and sion, because of the association of ethnicity on human tissue kallikrein amidase or renal kallikrein levels and blood activity was observed. pressure in patients with primary (es- diminished kallikrein excretion with sential) hypertension.4 Reduced urinary the disease state, bimodal frequency Conclusions: The lack of ethnicity effect kallikrein excretion has also been de- distribution in the population, familial supports what was already asserted, namely, scribed in a number of genetically aggregation, and early diminution even that, in , at an individual level, color, as 4 determined by physical evaluation, is a poor hypertensive rat models. in the still-normotensive offspring of 8 predictor of genomic African ancestry, estimat- Kallikreins (EC 3.4.21.8) are a patients with hypertension. ed by molecular markers. (Ethn Dis. 2009;19: subgroup of the family Kallikrein excretion is diminished in 265–270) known to have several physiological African Americans, especially those with functions.5 The kallikreins are divided hypertension, suggesting a potential Key Words: Human Kallikrein, Human Tis- sue Kallikrein, Hypertension, Ethnic Groups, into two main groups: plasma (EC pathophysiologic mechanism for the Brazilian frequent development of hypertension in this population.8 Several studies have Address correspondence and reprint demonstrated ethnic differences in uri- requests to Amintas Fabiano de Souza nary kallikrein excretion, with African From the Department of Ana´lises Clı´n- Figueiredo, PhD; Departamento de Ana´- Americans consistently having lower icas e Toxicolo´gicas, Faculdade de Farm- lises Clı´nicas e Toxicolo´gicas, Faculdade de urinary kallikrein excretion when com- ´acia, Universidade Federal de , Farma´cia, Universidade Federal de Minas pared to Caucasians, across both normal 31270-901 , MG, Brazil Gerais, Av. Presidente Antoˆnio Carlos, 1 (AAB, MOS, AFSF); Faculdade de Cieˆncias 6627; 31270-901 Belo Horizonte, MG, and high blood pressure strata. Anoth- Me´dicas de Minas Gerais, Belo Horizonte, Brazil; 55-31-3409-6897; 55-31-3409-6985 er study showed that the urinary tissue MG, Brazil (ELGM). (fax); [email protected] kallikrein levels were, in general, lower

Ethnicity & Disease, Volume 19, Summer 2009 265 TISSUE KALLIKREIN AND HYPERTENSION - Belo et al in Indian hypertensives when compared sive patients, considering healthy sub- was divided into two subgroups and to the Black hypertensive patients.9 On jects as controls. were compared according to the follow- the other hand, another study reported ing variables: systolic arterial pressure, no difference in urinary kallikrein excre- diastolic arterial pressure and hK1 tion between Caucasian and .1 METHODS amidase activity. The hK1 amidase According to the Brazilian Institute activity was expressed in mM/(min . mg of Geography and Statistics (IBGE, in This study was approved by the creatinine). Portuguese),10 the Brazilian population ethical committee of the Federal Uni- in 2000, based on skin color, com- versity of Minas Gerais (UFMG). All Chemicals prised: White - 53.7%; patients and control subjects gave D-valyl-L-leucyl-L-arginine 4-ni- ‘Pardo’ Brazilians (ie, non-White and written informed consent. troanilide (D-Val-Leu-Arg-Nan) was non-Black) - 38.4%; African Brazilians - Patients of both sexes were eligible purchased from Chromogenix AB 7.2%; Asian Brazilians - 0.4% and for inclusion in the study as long as they (Italy). TrasylolH was provided by Aboriginal Brazilians - 0.4%. were older than 20 years of age and had courtesy of Bayer (Brazil). Bovine serum On the other hand, studies have primary hypertension. All subjects were albumin and soybean shown that Brazilians are one of the studied as outpatients. Patients and (SBTI) were purchased from Sigma most heterogeneous populations in the control subjects were consecutively Chemical Company (St. Louis, MO, world, resulting from five centuries of screened by the same physician USA). Dipstick tests (Urofita 10U interethnic crosses among peoples from (ELGM) for a two-year period, and bioBRA´ SDiagno´sticos, Biobra´sS.A., three continents: the European coloniz- underwent a thorough clinical interview Belo Horizonte, MG, Brazil) were pur- ers, mainly represented by the Portu- as well as physical examination. All of chased from Biobra´s. The diagnostic kit guese, the African slaves, and the their symptoms and signs indicative of for creatinine determination was freely autochthonous Amerindians. Therefore, either primary hypertension or any provided courtesy of KATAL Biotecno- in Brazil, at an individual level, color, as other cardiovascular disease were ana- lo´gica Indu´stria e Come´rcio Ltda (Belo determined by physical evaluation, is a lyzed, as well as the patients’ personal Horizonte, MG, Brazil). All other re- poor predictor of genomic African antecedents and the types of (cardiovas- agents were from Sigma reagent grade. ancestry, when the latter is estimated cular or non-cardiovascular) medication by molecular markers.11 they were making use of. Individuals Assessments Sex was also reported to be a were stratified according to ethnicity by A random urine sample, the early- substantial influence on human tissue the physician (ELGM). morning midstream specimen, was used. kallikrein excretion, with women ex- In the laboratory, the urine sample was creting more kallikrein than men, Patient Criteria visually and chemically examined with 1 regardless of ethnicity. To be included in the study, patients dipstick test. All the urine samples were The following facts laid the ground- had primary hypertension, either dia- negative for blood and for all chemical work for our research: 1) renal kallikrein stolic blood pressure of $90 mm Hg or compounds evaluated. Hydrolysis of the excretion varies according to ethnicity, systolic blood pressure of $140 chromogenic substrate D-Val-Leu-Arg- with especially low values in African- mm Hg, on three separate determina- Nan was assayed spectrophotometrically American individuals;8 2) the special tions. The criteria for patient exclusion at 410 nm, to allow the release of 4- ethnic heterogeneity of Brazilian popu- included: non-agreement for participat- nitroaniline (4-NAn) (e410 5 8800/(M . lation; and 3) the influence of ethnicity ing in the study, secondary hyperten- cm) to be monitored as previously and sex on the hK1 amidase activity sion, heart failure, renal failure (serum described.12,13 The hK1 amidase activity both in normotensive subjects and in creatinine level $ 1.5 mg/dL or assay was carried out as already de- hypertensive patients has never been 133 mmol/L in man and $ 1.4 mg/dL scribed.14 The reaction rate (v) was described for the Brazilian population. or 124 mmol/L in woman), hepatic expressed in mM/(min . mL urine). Thus, our work had the following aims: alterations and diabetes mellitus. Pre- Creatinine concentration was deter- 1) to evaluate the behavior of urinary menopausal women were not studied mined spectrophotometrically using a kit hK1 amidase activity among Brazilian during their menstruation phase of their of reagents based on the Jaffe´’s reaction primary hypertensive patients, consider- menstrual cycle. Because hK1 amidase and was expressed in mg/mL urine. ing healthy subjects as controls; 2) to activity and creatinine determinations Kallikrein specific amidase activity evaluate whether urinary hK1 amidase are spectrophotometric assays, the pres- was calculated dividing the reaction rate activity is influenced by sex and ethnic- ence of blood in urine could interfere (v) [mM/(min . mL urine)] by creatinine ity among Brazilian primary hyperten- with the results. The population studied concentration (mg/mL urine). The re-

266 Ethnicity & Disease, Volume 19, Summer 2009 TISSUE KALLIKREIN AND HYPERTENSION - Belo et al sult was expressed as mM/(min . mg Table 1. Baseline characteristicsof the study population creatinine) to correct for differences in urine flow rate. Parameter Patients Controls P Number 100 89 – Statistical Analysis Demographic Descriptive statistics are reported as Age, y* 52 (39–61) 48 (35–57) – Sex (male/female) 50/50 48/41 – medians from the irregular distribution Race (White/Black) 26/74 31/58 – of the variables studied. Differences Physiologic between the groups were evaluated by SBP, mm Hg* 160 (150–170) 120 (120–130) ,.001 the non-parametric Kruskall-Wallis test, DBP, mm Hg* 90 (85–100) 80 (80–80) ,.001 since the population studied had a non- Biochemical Gaussian distribution with non-homo- hK1 am act*,** 0.210 (0.100–0.395) 0.260 (0.180–0.445) .010 geneous variance. A P value of ,.05 was Abbreviations: DBP, diastolic blood pressure; hK1 am act, hK1 amidase activity; SBP, systolic blood pressure; considered statistically significant. *median value; numbers in parentheses are the interquartile ranges 25% to75%; ** mM/(min . mg creatinine).

RESULTS women constituted the patient group. activity between White and Black On the other hand, 48 normotensive patients, either with hypertension Patients and Control men and 41 normotensive women (0.220 mM/(min . mg creatinine and Subjects Characteristics constituted the control group. Twenty 0.180 mM/(min . mg creatinine, respec- six hypertensive Whites and 74 hyper- tively) (P5.931), or without hyperten- One hundred patients (26 White, 74 tensive Blacks constituted the patient sion (0.280 mM/(min . mg creatinine Black, stratified with basis on their group. On the other hand, 31 normo- and 0.260 mM/(min . mg creatinine, identification by physician) fulfilled the tensive Whites and 58 normotensive respectively) (P5.914). study criteria. Patients’ continued their Blacks constituted the control group. diets of customary intake of fluid, All 100 patients had primary hyperten- electrolytes, calories, or other macronu- sion. The systolic blood pressure (SBP) DISCUSSION trient/micronutrient composition. The and diastolic blood pressure (DBP) were serum sodium, chloride, potassium, significantly higher in hypertensive pa- To our knowledge, this is the first blood urea and serum creatinine values tients than in controls. The hK1 study concerning the evaluation of the were normal in all the patients. Twenty- amidase activity was significantly lower effects of sex and ethnicity on the nine (29%) patients were using a in primary hypertensive patients than in activity of human tissue kallikrein diuretic, 25% an ACE inhibitor, 10% a controls (Table 1, Figure 1). (hK1) in the urine of Brazilian normo- calcium channel blocker, 9% a b- tensive subjects and primary hyperten- blocker, 7% a selective a -agonist, 2 sive patients. We have shown that hK1 whereas 20 patients (20%) were using Effect of Gender on Human amidase activity is significantly lower in no medication. Patients in this study Tissue Kallikrein Amidase Activity the urine of primary hypertensive pa- were otherwise healthy, but for their tients than in the urine of control We did not observe a statistically hypertension. Eighty-nine healthy nor- subjects. This result is in agreement significant difference in hK1 amidase motensive (diastolic blood pressure con- with data previously published. Indeed sistently , 90 mm Hg or systolic blood activity between men and women, either pressure , 140 mmHg on three separate with hypertension (0.185 mM/(min . mg determinations) individuals (31 White creatinine and 0.210 mM/(min . mg We have shown that hK1 and 58 Black, stratified with basis on creatinine, respectively) (P5.992), or their identification by physician), paired without hypertension (0.206 mM/(min amidase activity is according to ethnicity, sex and age (6 5), . mg creatinine and 0.260 mM/(min . mg were used as normal controls, thereby creatinine, respectively) (P5.795). significantly lower in the constituting the control subgroup. urine of primary hypertensive The baseline characteristics of the Effect of Ethnicity on Human two groups are shown in Table 1. The Tissue Kallikrein patients than in the urine of age medians of the patients and controls Amidase Activity control subjects. were 52 and 48 years, respectively. Fifty We did not observe a statistically hypertensive men and 50 hypertensive significant difference in hK1 amidase

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Sex Effect The influence of sex on hK1 amidase activity is not clear. Ash et al22 reported that the human urinary kallikrein (hK1) output values among young men and women were similar. On the other hand, Hughes Jr. et al23 found sex differences in tissue kallikrein levels in urine in groups of young, White subjects without hypertension and patients with normal renin essential hypertension. According to these au- thors, urinary kallikrein excretion was higher in women than in men regardless of sodium intake or level of blood pressure. Lieberthal et al24 found larger urinary kallikrein excretion in White men with hypertension than in White women with hypertension. On the other hand, Song et al1 reported that sex has a Fig 1. Comparison of hK1 amidase activity values between in the urine of hypertensive patients and in the urine of normotensive subjects. hK1 am act substantial influence on human kalli- indicates hK1 amidase activity, expressed as mM/(min . mg creatinine); the symbol krein excretion, with women excreting (*) indicates statistically significant difference between hypertensive and consistently higher activities than men, normotensive groups regardless of ethnicity. According to these authors, the sex difference in Elliot and Nuzum15 had already noticed blood pressure, with Black hypertensive kallikrein excretion has been attributed that hypertensive patients, with no subjects generally showing the lowest to an effect of the female’s menstrual clinically apparent renal disease, have measured levels.18 Wong et al8 reported cycle, wherein kallikrein rises ,50% significantly lower levels of urinary that renal kallikrein excretion is dimin- from the mid-follicular to the mid- kallikrein than normotensive subjects. ished in patients with hypertension, and luteal phase of the cycle. Black women Margolius et al16 reported lower levels perhaps even in the early, pre-hyperten- notably lack the cycle effect on kallikre- of urinary kallikrein in patients with sive phases of the syndrome. in. Although their female subjects were essential hypertension than in a control In our study, patients were submit- not studied at a particular phase during population. O’Connor17 reported that ted to a normal sodium diet (140 the menstrual cycle, they still observed kallikrein excretion is diminished in mmol/L). It was already reported that ,50% higher kallikrein excretion in hypertension (especially hypertension hK1 amidase activity is inhibited, in women than men, and sex effect was with reduced renal function), suggesting vitro, by sodium and other uni and independent of ethnicity. the involvement in the patho- divalent cations and, also, that bovine In our study, we did not observe a genesis of the disease. However, in serum albumin (0.2%) protected the statistically significant difference in hK1 1995, Margolius18 reported that, al- inhibition of hK1 amidase activity by amidase activity between male and though it is clear that patients with sodium and potassium.19,20 On the female, either with hypertension or essential hypertension excrete less kalli- other hand, it is known that only a without hypertension. Thus, our results krein than normotensive subjects, there small amount of protein is excreted agree with those reported by Ash et al22 is much overlap in the population normally in the human urine, most of and disagree with those reported by studies carried out over the past 25 which being albumin.21 Thus, it is Song et al1, Huges Jr et al23 and years. According to the author, many possible that urinary albumin can Lieberthal et al.24 hypertensive subjects show normal kal- protect hK1 from sodium and potassi- likrein excretion. In our study, three um inhibition. If this is correct, the hypertensive patients (6%) had normal decrease in hK1 amidase activity ob- ETHNICITY EFFECT hK1 amidase activity. Black people, served in our study is unlikely to be due adults and children excrete markedly to its inhibition by urinary sodium or Katori and Majima25 reported that less kallikrein than Whites, regardless of potassium. kallikrein excretion in White hyperten-

268 Ethnicity & Disease, Volume 19, Summer 2009 TISSUE KALLIKREIN AND HYPERTENSION - Belo et al sive men was lower than that in White potential pathophysiologic mechanism script. This research was presented in part at normotensives during normal sodium for the frequent development of hyper- the 63th Brazilian Meeting of Cardiology of intake, but was not different from that tension in this population. the Brazilian Society of Cardiology, Cur- itiba, PR, Brazil, September 06–10, 2008. in Black hypertensives and Black con- In our study, we did not observe a trol subjects under the same conditions. statistically significant difference in hK1 REFERENCES The kallikrein levels in the urine of amidase activity between White and 1. Song CK, Martinez JA, Kailasam MT, Dao normotensive Black subjects were sig- Black, either with hypertension or TT, Wong CM, Parmer RJ. Renal kallikrein nificantly lower than those in normo- without hypertension. Thus, our study excretion: role of ethnicity, gender, environ- tensive White subjects. does not support those previously ment, and genetic risk of hypertension. J Hum Seedat et al,9 in South Africa, reported.1,8,25 A similar result was Hypertens. 2000;14:461–468. 14 2. Krieger JE, Dzau VJ. Molecular biology of evaluated the excretion values for uri- observed by Figueiredo et al, who hypertension. Hypertension. 1991;18(Suppl nary tissue kallikrein in four groups: evaluated the hK1 amidase activity in I):I-3–I-17. Black normotensive, Black hypertensive, the urine of patients with systolic heart 3. Sun Z, Zhang Z. Historic perspectives and Indian normotensive and Indian hyper- failure (SHF), and in the urine of recent advances in major animal models of tensive. This research confirmed that healthy individuals, paired according hypertension. Acta Pharmacol Sin. 2005;26: 295–301. the renal excretion of tissue kallikrein to sex, ethnicity and age, as controls. 4. Bhoola KD, Figueroa CD, Worthy K. Bior- was reduced in Black hypertensives. The authors observed that the hK1 egulation of kinins: kallikreins, kininogens and According to the authors, the specific amidase activity was significantly lower kininases. Pharmacol Rev. 1992;44:1–80. activity of tissue kallikrein (ng/mg pro- in the urine of SHF patients. In that 5. Yousef GM, Diamandis EP. Tissue kallikreins: tein) was in general higher in the Black study, the majority of patients and new players in normal and abnormal cell growth? Thromb Haemost. 2003;90:7–16. controls were White and there was no hypertensives than in the Indian hyper- 6. MacDonald RJ, Margolius HS, Erdo¨s EG. tensives, whereas the normotensives in significant effect of ethnicity on hK1 Molecular biology of tissue kallikrein. the two groups showed variable trends amidase activity. The lack of ethnicity Biochem J. 1988;253:313–321. that were not significantly different. In effect on hK1 amidase activity reported 7. Campbell DJ. The kallikrein-kinin system in 14 conclusion, the authors reported that by Figueiredo et al, which is also humans. Clin Exp Pharmacol Physiol. 2001;28:1060–1065. their study showed that urinary tissue observed in this study, supports the data 11 8. Wong CM, O’Connor DT, Martinez JA, kallikrein levels were, in general, lower reported by Parra et al. The authors Kailasam MT, Parmer RJ. Diminished renal in Indian hypertensives compared to concluded that their data suggest that in kallikrein responses to mineralocorticoid stim- Black hypertensive patients. Brazil, at an individual level, color, as ulation in African Americans: determinats of an intermediate phenotype for hypertension. Song et al1 reported that several determined by physical evaluation, is a Am J Hypertens. 2003;16:281–289. poor predictor of genomic African ances- studies have demonstrated substantial 9. Seedat YK, Bhoola KD, Muller R, Randeree ethnic differences in urinary kallikrein try, estimated by molecular markers. IGH, Pillay D. Urinary levels of tissue excretion, African Americans consistent- In conclusion, the evidence present- kallikrein in the South African Black and ly have lower urinary kallikrein excretion ed here shows: 1) in Brazilian patients Indian hypertensives. J Hum Hypertens. 1999;13:771–775. when compared to Caucasians, across with primary hypertension, the hK1 10. Brazilian Institute of Geography and Statistics amidase activity was significantly lower both normal and high blood pressure (IBGE). Available at http://www.ibge.gov.br. strata. Song et al found a substantial than in healthy controls, supporting Last accessed 5/26/09. diminution in urinary kallikrein excre- data previously published involving 11. Parra FC, Amado RC, Lambertucci JR, Rocha tion in African Americans, but no studies with other populations; 2) in J, Antunes CM, Pena SDJ. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci. difference between Caucasian and Asian Brazilian subjects, the hK1 amidase 2003;100:177–182. activity was not influenced by sex; 3) values. 12. Erlanger BF, Kokowsky N, Cohen W. The 8 Wong et al evaluated the excretion in Brazilian subjects, the hK1 amidase preparation and properties of two new chro- of hK1, K+ and aldosterone in the urine activity was not influenced by skin mogenic substrates of trypsin. Arch Biochem of healthy young normotensive African color. Biophys. 1961;95:271–278. 13. Geiger R, Fritz H. Human urinary kallikrein. American (sub-Saharan African ances- Methods Enzymol. 1981;80:466–492. try) men and concluded that normo- 14. Figueiredo EL, Lea˜oFVG,OliveiraLV, tensive African Americans have dimin- ACKNOWLEDGMENTS Moreira MCV, Figueiredo AFS. The amidase ished urinary excretion of kallikrein, K+ Research supported by FAPEMIG (Proc. activity of human tissue kallikrein is signifi- CDS 923/98). A. A. Belo was a recipient of a and aldosterone. According to the cantly lower in the urine of patients with Graduate Student Fellowship from CAPES. systolic heart failure. J Card Fail. 2006;12: authors, kallikrein excretion was dimin- The authors are in debt to Aleida Nazareth 653–658. ished in African Americans, especially in Soares, for statistical support, and Erick 15. Elliot R, Nuzum F. Urinary excretion of a those with hypertension, suggesting a Ramalho, MLitt, for revising the manu- depressor substance (kallikrein of Frey and

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Kraut) in arterial hypertension. Endocrinology. magnesium. 2 - Linear mixed inhibition by 25. Katori M, Majima M. Pivotal role of renal 1934;18:462–474. aluminium. J Enz Inh Med Chem. 2004;19: kallikrein-kinin system in the development of 16. Margolius HS, Geller R, Pisano JJ, Sjoerdsma A. 317–325. hypertension and approaches to new drugs Altered urinary kallikrein excretion in human 21. Johnson AM. Amino acids, peptides, and based on this relationship. Jpn J Pharmacol. hypertension. Lancet. 1971;2:1063–1065. proteins. In: Burtis CA, Ashwood ER, Bruns 1996;70:95–128. 17. O’Connor DT. Response of renal kallikrein- DE, eds. Tietz Textbook of Clinical Chemistry kinin system, intravascular volume, and renal and Molecular Diagnostics. St. Louis: Elsevier AUTHOR CONTRIBUTIONS hemodynamics to sodium restriction and Saunders, 2006;533–595. Design concept of study: Belo, Sousa, Ma- diuretic treatment in essential hypertension. 22. Ash KO, Smith JB, Lynch M, Dadone M, chado, Figueiredo Hypertension. 1982;4(Suppl):III-72–III-78. Tolman KG, Williams RR. Urinary kallikrein: Acquisition of data: Belo, Sousa, Machado, 18. Margolius HS. Kallikreins and kinins. Some assay validation and physiological variability. Figueiredo unanswered questions about system character- Clin Chim Acta. 1985;151:133–140. Data analysis and interpretation: Belo, Sousa, istics and roles in human disease. Hypertension. 23. Huges Jr. GS, Margolius HS, Peters R, Machado, Figueiredo 1995;26:221–229. Oexmann MJ, Chao J, Lindenmayer G. Manuscript draft: Belo, Sousa, Machado, 19. Chao J, Shigemichi T, Margolius HS. Inhib- Gender differences of tissue kallikrein and an Figueiredo itory effects of sodium and other monovalent erythrocyte kallikrein-like enzyme in essential Statistical expertise: Belo, Sousa, Machado, cations on purified versus membrane- bound hypertension. J Lab Clin Med. 1988;112:612– Figueiredo kallikrein. J Biol Chem. 1983;258:6461–6465. 618. Acquisition of funding: Belo, Sousa, Ma- 20. Sousa MO, Santoro MM, Figueiredo AFS. 24. Lieberthal W, Arbeit L, Oza NB, Bernard DB, chado, Figueiredo The effect of cations on the amidase activity of Levinsky NG. Reduced ratio of active-to-total Administrative, technical, or material assis- human tissue kallikrein: 1 - linear competitive urinary kallikrein in essential hypertension. tance: Belo, Sousa, Machado, Figueiredo inhibition by sodium, potassium, calcium and Hypertension. 1983;5:603–609. Supervision: Belo, Sousa, Machado, Figueiredo

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