Hypertension in Pregnancy, 2014; 33(3): 349–359 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.898305
ORIGINAL ARTICLE
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
Urinary lysosomal enzyme excretion in pregnant women with hypertensive disorders Andrzej Torbe ´ ,1 Ewelina Chłapowska,1 Jolanta Szyman´ska-Pasternak,2 Aneta Sulecka,2 Joanna Bober,2 Ewa Kwiatkowska,3 Sebastian Kwiatkowski,1 Rafał Rzepka,1 Wioletta Konstanty-Kurkiewicz,4 and Bogdan Torbe ´5 1
Department of Obstetrics and Gynecology, Department of Medical Chemistry, 3 Department of Nephrology, Transplantology and Internal Medicine, 4 Department of Gynecological Surgery and Gynecological Oncology of Adults and Adolescents, and 5 Department of Radiotherapy, Pomeranian Medical University, Szczecin, Poland 2
Background: The authors assessed proximal renal tubular dysfunction and/or damage in pregnant women with various types of hypertension by measuring the three urinary lysosomal enzyme levels: N-acetyl-b-D-glucosaminidase (NAG), arylsulfatase A and b-glucuronidase. Methods: The study consisted of 120 pregnant women divided into four groups: 41 women in 20th week of gestation or more, with pregnancy-induced hypertension (PIH group), 28 pregnant women after 20 weeks of pregnancy with preeclampsia (PE group), 21 pregnant women with chronic hypertension, identified before 20th week of pregnancy (CH group) and 30 healthy, pregnant women (healthy controls (HC) group). Results: Statistical analysis showed significantly higher levels of all the three of lysosomal enzymes in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and the ones with chronic hypertension. Additionally, significantly higher values of NAG were found in the group of pregnant women with PIH compared with healthy pregnancies. No correlation was found between the concentration of enzymes in urine and values of blood pressure in any of the analyzed groups of pregnant women. Conclusions: The authors conclude that higher values of all the studied enzymes in PE group, in the comparison with the other groups, indicate proximal tubular damage at the cellular level. The lack of correlation between the concentration of lysosomal enzymes and blood pressure suggests that the damage to these parts of kidney is complex. In addition, mechanisms other than hypertension realizing intracellular enzymes may be involved in this process. Keywords Hypertension, Lysosomal enzymes, Pre-eclampsia, Pregnancy.
Correspondence: Andrzej Torbe´, Department of Obstetrics and Gynecology, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland. Tel: +48 91 4661350. Fax: +48 91 4661350. E-mail: [email protected]
350
A. Torbe ´ et al.
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
INTRODUCTION Hypertension is the most common issue during pregnancy. It complicates up to 10% of pregnancies and is associated with a significantly increased risk of perinatal complications for both mother and child (1–3). Hypertensive disorders, with (4) or without (5) liver abnormalities, are one of the major causes of pregnancy-related maternal and babies deaths (6). Hypertension leads to the development of pre-eclampsia (PE) in 15–20% cases and the reasons for this phenomenon remain unexplained (7–9). PE is a multifactorial and multisystem disease of unknown etiology (10,11). It leads to serious consequences for both pregnant women and fetuses. PE is commonly associated with intrauterine growth restriction and prematurity with its consequences, including hypoxia and perinatal death. Activation of the coagulation system and vasoconstriction in pregnancy lead to a severe and even fatal complications such as pulmonary and cerebral edema, heart and renal failure, intracranial bleeding, or damage of the liver (12,13). Kidney function dramatically increases during pregnancy and renal lesion is characteristic for PE. This process induces changes in the structure and function of both glomeruli and tubules (14,15), which is reflected in change of the composition of urine. Researchers identified various enzymes in the lysosomes of proximal tubular cells. These include N-acetyl-b-D-glucosaminidase (NAG), arylsulfatase A (ASA) and b-glucuronidase (BGR). The change of their concentrations in the urine indicates damage to the tubular cells (15–17). The literature provides few reports regarding the increased activity of lysosomal enzymes in urine in the course of PE (18–23). NAG is an enzyme that catalyzes the breakdown of glycoproteins and glycosaminoglycans. It can be found in the lysosomes of cells of many tissues. Its high activity can be noticed particularly in cells of renal proximal tubules from where it is released into the urine. Low concentration of NAG in the urine of healthy people is the result of exocytosis and pinocytosis processes. The high activity of this enzyme in the urine is the evidence of renal tubular damage at the cellular level (14,16,17). BGR is a lysosomal enzyme hydrolyzing b-D-glucuronides into glucuronic acid and the aglycone. According to Refaie et al. (24), the increased activity of this enzyme in the urine may indicate the damage of the renal proximal tubular cells as well. BGR is also involved in inflammatory processes. It is one of the components of granules which are present inside the neutrophils. It is reported by Florian´czyk et al. (25) that the above-mentioned enzyme participates in the phagocytosis and degradation of microorganisms. Another enzyme, called ASA, is a lysosomal enzyme that is present in various tissues like liver, kidney, pancreas and body fluids such as saliva, urine, blood serum or amniotic fluid. It breaks down aryl sulfate esters (26). All the three listed enzymes have a high molecular weight. This excludes their filtration through a primary membrane of the renal glomeruli to the filtrate. The aim of this study was to assess proximal renal tubular dysfunction and/ or damage in pregnant women with various types of hypertension by measuring the urinary lysosomal enzyme levels.
Lysosomal enzymes in preeclamsia
MATERIALS AND METHODS
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
The control group consisted of a total of 120 women with singleton pregnancies, of whom 90 women were hospitalized between December 2008 and January 2012 in the Department of Obstetrics and Gynecology of Pomeranian Medical University, due to high blood pressure. The remaining 30 healthy pregnant women, in the corresponding gestational age, were in the care of ambulatory services over the same period. The study population was divided into four groups: (1) PIH (pregnancy-induced hypertension): 41 women in 20th week of gestation or more, 1. with pregnancy-induced hypertension. (2) PE (pre-eclampsia): 28 pregnant women after 20 weeks of pregnancy with pre-eclampsia defined as hypertension and proteinuria (4300 mg/day) without (25–89.3%) or with mild (3–10.7%) liver dysfunction. (3) CH (chronic hypertension): 21 pregnant women with chronic hypertension, identified 3. before 20th week of pregnancy. (4) HC (healthy controls): a control group, consisting of 30 healthy, pregnant women. A person was qualified to a group with PIH or a group with PE on the basis of a measurement of the blood pressure values 140/90 mm Hg, measured twice at intervals of 4–6 h, on a day on admission to the hospital. Moreover, such symptoms had to be observed for the first time after 20th week of gestation. In the PE group, the proteinuria was diagnosed subsequently. An inclusive criterion into CH group was high blood pressure requiring hospitalization before 20th week of pregnancy. The study was approved by the University Human Subject Research Review Committee (KB-0080/165/09). First morning mid-stream urine samples were collected in sterile bottles, immediately centrifuged in 15 mL glass test tubes at 5000 rpm for 10 min and then kept frozen in 1.5 mL polypropylene tubes at 70 C until assayed. The enzymatic activity was detected with colorimetric method using kit assays based on the hydrolysis of NAG, BGR and ASA substrates (Sigma–Aldrich Corporation, Poznan´, Poland) by the enzymes. The reaction product was detected colorimetrically at 405 nm for NAG and BGR and at 515 nm for ASA and quantitatively measured using Marcel Media Bio spectrophotometer (Merazet, Poland). Urine examination was also performed in every pregnant woman using LabUReader Plus analyzer (Allmed, Warsaw, Poland). In case of proteinuria 425 mg/dL in a single urine sample, daily urine collection was performed with the loss of protein checked turbimetrically, using Architect c4000 clinical chemistry analyzer (Abbot Diagnostics, Wiesbaden, Germany). Maternal serum uric acid was measured using Architect c4000 clinical chemistry analyzer (Abbot Diagnostics). The urinary creatinine were estimated with tests (Pointe Scientific, Warszawa, Poland) based on Jaffe’s kinetic method. The urinary enzymes’ values were expressed as a ratio to urinary creatinine concentration. This relationship shows less variability than the urinary enzyme excretions related to volume or time.
351
352
A. Torbe ´ et al.
The results were statistically analyzed by using STATA 11 software (License No. 30110532736) and presented as box and whiskers plots. The Shapiro–Wilk test showed non-normal distribution of parameters. To identify a statistical significance for multiple comparisons, the ANOVA test was used. Analyses of differences between individual groups were assessed by Mann–Whitney U test. The strength of correlation between the parameters was measured with the use of the Spearman’s rank correlation’s coefficient. The p values were considered significant at a level 50.05.
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
RESULTS The demographic characteristics of the study groups are shown in Table 1. The ANOVA test showed that there was a statistically significant difference between the groups that concerned urinary levels of all the three of lysosomal enzymes (Figures 1–3). The comparison between individual groups confirmed that NAG, BGR (p50.001) and ASA (p50.05) levels were significantly higher in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and those with chronic hypertension. In addition, significantly higher NAG values (p50.05) were found in the group of pregnant women with PIH compared with healthy pregnant women. No correlation was found between the concentration of enzymes in urine and values of blood pressure of any of the analyzed groups of pregnant women. There was also significant difference in blood uric acid levels between the groups (Figure 4). The Mann–Whitney U test revealed that the value of the uric acid in the PE group was significantly higher than that in PIH (p50.01), CH (p50.05) and the control group (p50.001). There was no correlation between the values of enzymes derived from the urine and the concentration of uric acid from the blood, which is an indicator of the severity of the PE. Table 1. Clinical characteristics of the groups. Group
PIH (N = 41)
PE (N = 28)
CH (N = 21)
Maternal age (yrs) Primiparous Gestational age at sampling (wks) Mean systolic blood pressure (mmHg) Mean diastolic blood pressure (mmHg) Daily urinary protein loss (g/L) Birth weight (g) Gestational age at delivery (wks) Caesarean section delivery 5-Min Apgar score (points)
28.4 ± 6.1 23 (56.1) 35.66 ± 3.89
30.5 ± 6.7 22 (78.57) 34.11 ± 3.89
32.76 ± 4.3 13 (61.9) 32.48 ± 5.3
151.4 ± 7.9
153.3 ± 7.48
95.58 ± 5.4
98.18 ± 4.89
97.99 ± 7.4
74.68 ± 8.3
0 3086 ± 770 37.39 ± 3.05
3.88 ± 3.48 2188 ± 880 34.7 ± 3.83
1.23 ± 1.3 2478 ± 1040 35.38 ± 3.6
0 3343 ± 440 39.23 ± 1.65
21 (75.0) 8.92 ± 1.62
17 (80.9) 8.85 ± 1.42
11 (36.6) 9.83 ± 0.38
20 (48.78) 9.44 ± 1.34
HC (N = 30) 28.8 ± 5.55 20 (66.67) 33.9 ± 5.5
154.16 ± 12.27 121.67 ± 14.5
Values are given as mean ± SD and n (%). PIH, pregnancy-induced hypertension; PE, preeclampsia; CH, chronic hypertension; HC, healthy controls.
Lysosomal enzymes in preeclamsia Anova test: F(3;116) = 17,319; p
ORIGINAL ARTICLE
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
Urinary lysosomal enzyme excretion in pregnant women with hypertensive disorders Andrzej Torbe ´ ,1 Ewelina Chłapowska,1 Jolanta Szyman´ska-Pasternak,2 Aneta Sulecka,2 Joanna Bober,2 Ewa Kwiatkowska,3 Sebastian Kwiatkowski,1 Rafał Rzepka,1 Wioletta Konstanty-Kurkiewicz,4 and Bogdan Torbe ´5 1
Department of Obstetrics and Gynecology, Department of Medical Chemistry, 3 Department of Nephrology, Transplantology and Internal Medicine, 4 Department of Gynecological Surgery and Gynecological Oncology of Adults and Adolescents, and 5 Department of Radiotherapy, Pomeranian Medical University, Szczecin, Poland 2
Background: The authors assessed proximal renal tubular dysfunction and/or damage in pregnant women with various types of hypertension by measuring the three urinary lysosomal enzyme levels: N-acetyl-b-D-glucosaminidase (NAG), arylsulfatase A and b-glucuronidase. Methods: The study consisted of 120 pregnant women divided into four groups: 41 women in 20th week of gestation or more, with pregnancy-induced hypertension (PIH group), 28 pregnant women after 20 weeks of pregnancy with preeclampsia (PE group), 21 pregnant women with chronic hypertension, identified before 20th week of pregnancy (CH group) and 30 healthy, pregnant women (healthy controls (HC) group). Results: Statistical analysis showed significantly higher levels of all the three of lysosomal enzymes in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and the ones with chronic hypertension. Additionally, significantly higher values of NAG were found in the group of pregnant women with PIH compared with healthy pregnancies. No correlation was found between the concentration of enzymes in urine and values of blood pressure in any of the analyzed groups of pregnant women. Conclusions: The authors conclude that higher values of all the studied enzymes in PE group, in the comparison with the other groups, indicate proximal tubular damage at the cellular level. The lack of correlation between the concentration of lysosomal enzymes and blood pressure suggests that the damage to these parts of kidney is complex. In addition, mechanisms other than hypertension realizing intracellular enzymes may be involved in this process. Keywords Hypertension, Lysosomal enzymes, Pre-eclampsia, Pregnancy.
Correspondence: Andrzej Torbe´, Department of Obstetrics and Gynecology, Pomeranian Medical University, Powstancow Wielkopolskich Av. 72, 70-111 Szczecin, Poland. Tel: +48 91 4661350. Fax: +48 91 4661350. E-mail: [email protected]
350
A. Torbe ´ et al.
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
INTRODUCTION Hypertension is the most common issue during pregnancy. It complicates up to 10% of pregnancies and is associated with a significantly increased risk of perinatal complications for both mother and child (1–3). Hypertensive disorders, with (4) or without (5) liver abnormalities, are one of the major causes of pregnancy-related maternal and babies deaths (6). Hypertension leads to the development of pre-eclampsia (PE) in 15–20% cases and the reasons for this phenomenon remain unexplained (7–9). PE is a multifactorial and multisystem disease of unknown etiology (10,11). It leads to serious consequences for both pregnant women and fetuses. PE is commonly associated with intrauterine growth restriction and prematurity with its consequences, including hypoxia and perinatal death. Activation of the coagulation system and vasoconstriction in pregnancy lead to a severe and even fatal complications such as pulmonary and cerebral edema, heart and renal failure, intracranial bleeding, or damage of the liver (12,13). Kidney function dramatically increases during pregnancy and renal lesion is characteristic for PE. This process induces changes in the structure and function of both glomeruli and tubules (14,15), which is reflected in change of the composition of urine. Researchers identified various enzymes in the lysosomes of proximal tubular cells. These include N-acetyl-b-D-glucosaminidase (NAG), arylsulfatase A (ASA) and b-glucuronidase (BGR). The change of their concentrations in the urine indicates damage to the tubular cells (15–17). The literature provides few reports regarding the increased activity of lysosomal enzymes in urine in the course of PE (18–23). NAG is an enzyme that catalyzes the breakdown of glycoproteins and glycosaminoglycans. It can be found in the lysosomes of cells of many tissues. Its high activity can be noticed particularly in cells of renal proximal tubules from where it is released into the urine. Low concentration of NAG in the urine of healthy people is the result of exocytosis and pinocytosis processes. The high activity of this enzyme in the urine is the evidence of renal tubular damage at the cellular level (14,16,17). BGR is a lysosomal enzyme hydrolyzing b-D-glucuronides into glucuronic acid and the aglycone. According to Refaie et al. (24), the increased activity of this enzyme in the urine may indicate the damage of the renal proximal tubular cells as well. BGR is also involved in inflammatory processes. It is one of the components of granules which are present inside the neutrophils. It is reported by Florian´czyk et al. (25) that the above-mentioned enzyme participates in the phagocytosis and degradation of microorganisms. Another enzyme, called ASA, is a lysosomal enzyme that is present in various tissues like liver, kidney, pancreas and body fluids such as saliva, urine, blood serum or amniotic fluid. It breaks down aryl sulfate esters (26). All the three listed enzymes have a high molecular weight. This excludes their filtration through a primary membrane of the renal glomeruli to the filtrate. The aim of this study was to assess proximal renal tubular dysfunction and/ or damage in pregnant women with various types of hypertension by measuring the urinary lysosomal enzyme levels.
Lysosomal enzymes in preeclamsia
MATERIALS AND METHODS
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
The control group consisted of a total of 120 women with singleton pregnancies, of whom 90 women were hospitalized between December 2008 and January 2012 in the Department of Obstetrics and Gynecology of Pomeranian Medical University, due to high blood pressure. The remaining 30 healthy pregnant women, in the corresponding gestational age, were in the care of ambulatory services over the same period. The study population was divided into four groups: (1) PIH (pregnancy-induced hypertension): 41 women in 20th week of gestation or more, 1. with pregnancy-induced hypertension. (2) PE (pre-eclampsia): 28 pregnant women after 20 weeks of pregnancy with pre-eclampsia defined as hypertension and proteinuria (4300 mg/day) without (25–89.3%) or with mild (3–10.7%) liver dysfunction. (3) CH (chronic hypertension): 21 pregnant women with chronic hypertension, identified 3. before 20th week of pregnancy. (4) HC (healthy controls): a control group, consisting of 30 healthy, pregnant women. A person was qualified to a group with PIH or a group with PE on the basis of a measurement of the blood pressure values 140/90 mm Hg, measured twice at intervals of 4–6 h, on a day on admission to the hospital. Moreover, such symptoms had to be observed for the first time after 20th week of gestation. In the PE group, the proteinuria was diagnosed subsequently. An inclusive criterion into CH group was high blood pressure requiring hospitalization before 20th week of pregnancy. The study was approved by the University Human Subject Research Review Committee (KB-0080/165/09). First morning mid-stream urine samples were collected in sterile bottles, immediately centrifuged in 15 mL glass test tubes at 5000 rpm for 10 min and then kept frozen in 1.5 mL polypropylene tubes at 70 C until assayed. The enzymatic activity was detected with colorimetric method using kit assays based on the hydrolysis of NAG, BGR and ASA substrates (Sigma–Aldrich Corporation, Poznan´, Poland) by the enzymes. The reaction product was detected colorimetrically at 405 nm for NAG and BGR and at 515 nm for ASA and quantitatively measured using Marcel Media Bio spectrophotometer (Merazet, Poland). Urine examination was also performed in every pregnant woman using LabUReader Plus analyzer (Allmed, Warsaw, Poland). In case of proteinuria 425 mg/dL in a single urine sample, daily urine collection was performed with the loss of protein checked turbimetrically, using Architect c4000 clinical chemistry analyzer (Abbot Diagnostics, Wiesbaden, Germany). Maternal serum uric acid was measured using Architect c4000 clinical chemistry analyzer (Abbot Diagnostics). The urinary creatinine were estimated with tests (Pointe Scientific, Warszawa, Poland) based on Jaffe’s kinetic method. The urinary enzymes’ values were expressed as a ratio to urinary creatinine concentration. This relationship shows less variability than the urinary enzyme excretions related to volume or time.
351
352
A. Torbe ´ et al.
The results were statistically analyzed by using STATA 11 software (License No. 30110532736) and presented as box and whiskers plots. The Shapiro–Wilk test showed non-normal distribution of parameters. To identify a statistical significance for multiple comparisons, the ANOVA test was used. Analyses of differences between individual groups were assessed by Mann–Whitney U test. The strength of correlation between the parameters was measured with the use of the Spearman’s rank correlation’s coefficient. The p values were considered significant at a level 50.05.
Hypertens Pregnancy Downloaded from informahealthcare.com by University of Exeter on 03/18/15 For personal use only.
RESULTS The demographic characteristics of the study groups are shown in Table 1. The ANOVA test showed that there was a statistically significant difference between the groups that concerned urinary levels of all the three of lysosomal enzymes (Figures 1–3). The comparison between individual groups confirmed that NAG, BGR (p50.001) and ASA (p50.05) levels were significantly higher in the urine of patients with PE compared with the healthy pregnant women, pregnant women with PIH and those with chronic hypertension. In addition, significantly higher NAG values (p50.05) were found in the group of pregnant women with PIH compared with healthy pregnant women. No correlation was found between the concentration of enzymes in urine and values of blood pressure of any of the analyzed groups of pregnant women. There was also significant difference in blood uric acid levels between the groups (Figure 4). The Mann–Whitney U test revealed that the value of the uric acid in the PE group was significantly higher than that in PIH (p50.01), CH (p50.05) and the control group (p50.001). There was no correlation between the values of enzymes derived from the urine and the concentration of uric acid from the blood, which is an indicator of the severity of the PE. Table 1. Clinical characteristics of the groups. Group
PIH (N = 41)
PE (N = 28)
CH (N = 21)
Maternal age (yrs) Primiparous Gestational age at sampling (wks) Mean systolic blood pressure (mmHg) Mean diastolic blood pressure (mmHg) Daily urinary protein loss (g/L) Birth weight (g) Gestational age at delivery (wks) Caesarean section delivery 5-Min Apgar score (points)
28.4 ± 6.1 23 (56.1) 35.66 ± 3.89
30.5 ± 6.7 22 (78.57) 34.11 ± 3.89
32.76 ± 4.3 13 (61.9) 32.48 ± 5.3
151.4 ± 7.9
153.3 ± 7.48
95.58 ± 5.4
98.18 ± 4.89
97.99 ± 7.4
74.68 ± 8.3
0 3086 ± 770 37.39 ± 3.05
3.88 ± 3.48 2188 ± 880 34.7 ± 3.83
1.23 ± 1.3 2478 ± 1040 35.38 ± 3.6
0 3343 ± 440 39.23 ± 1.65
21 (75.0) 8.92 ± 1.62
17 (80.9) 8.85 ± 1.42
11 (36.6) 9.83 ± 0.38
20 (48.78) 9.44 ± 1.34
HC (N = 30) 28.8 ± 5.55 20 (66.67) 33.9 ± 5.5
154.16 ± 12.27 121.67 ± 14.5
Values are given as mean ± SD and n (%). PIH, pregnancy-induced hypertension; PE, preeclampsia; CH, chronic hypertension; HC, healthy controls.
Lysosomal enzymes in preeclamsia Anova test: F(3;116) = 17,319; p
