Hypertension and Urine Protease Activity in Preeclampsia
Hypertension and Urine Protease Activity in Preeclampsia
Preeclampsia (PE) is a common disorder of pregnancy that complicates 4-7% of all pregnancies.
 It is a serious condition with acute proteinuria and hypertension and varying degrees of
 edema after 20 weeks of gestation. PE leads to a severe risk of low birth weight because of
 prematurity with inherent complications. The pathogenesis is unknown but is assumed to
 involve placental ischemia.The primary placental disorder results in renal glomerular injury.
 Established PE is associated with paradoxical suppression of the
 renin-angiotensin-aldosterone system, RAAS.

 Despite suppressed RAAS, patients with PE retain NaCl(sodium chloride) after an intravenous
 isotonic NaCl overload compared to healthy pregnant women on a low NaCl diet. The
 investigators believe to have data that provide a possible explanation for the overall
 relationship between proteinuria, NaCl retension, suppression of RAAS, hypertension and
 underdevelopment of placenta. Earlier data, which the investigators have confirmed, shows
 abnormal glomerular loss of the enzyme plasmin/plasminogen from plasma to the urine in PE.
 Active plasmin in urine from patients with nephrotic syndrome and PE activates the epithelial
 sodium channel ( ENaC ) in renal collecting duct cells. The investigators hypothesize that
 loss of plasmin/plasminogen are shared for the diseases with proteinuria, including PE, and
 that plasmin- driven ENaC (epithelial sodium channel) activation is a causal factor in the
 pathophysiology of established PE. Hyperactive ENaC causes primary renal sodium retention
 with secondary suppression of the renin-angiotensin-aldosterone system. Aldosterone is
 recently established as a placental growth factor.

 Plasma-aldosterone levels are significant higher in normal pregnant women. PE is
 characterized by low aldosterone levels (a discovery the investigators have also confirmed)
 and by placental underdevelopment.

 Study Aim: To test specific hypothesis regarding established PE´s pathophysiological
 mechanisms.

 Study Hypothesis:

 1. Excretion of urine proteases (plasmin/plasminogen) in PE leads to an activation of ENaC
 and hence RAAS is less NaCl sensitive while the blood pressure is more NaCl sensitive
 compared to healthy pregnant women.

 2. The degree of aldosterone suppression in PE determines placental development
Selection of patients:

 The selection of patients is based on outpatients with preeclampsia and patients with normal
 pregnancies recruited from gynecological-obstetric department, Aarhus University Hospital -
 Skejby, Denmark. Non- pregnant woman are recruited by posting notices at the workplace.
 Specifically by office facilities, canteen and in gynecological department at Skejby
 hospital, Aarhus.

 Background information:

 Registration of date of birth, sex, weight, height, abdominal circumference, and smoking
 status will be noted.

 Furthermore, we will register current antihypertensive-, diuretic-, antidiabetic- and
 antiepileptic medicine and other current use of medicine. Also post-partum registration of
 gestation length, placentas weight and the infant weight will be noted.

 Effect variable:

 Clinical:

 Weight, height, BMI, abdominal circumference. Blood pressure (systolic, diastolic, mean
 arterial pressure). Weight of placenta, gestation length, Infant weight

 Measurements in blood tests:

 Se-creatinine, p-Na+, p-K+. P-plasminogen. P-albumin. P-renin concentration (not activity!),
 angiotensin II, aldosterone and arterial natriuretic peptide (ANP).

 Measurements in 50 ml newly "spot urine":

 Plasmin, plasminogen, ENaC peptide fragment (analyses in location of development),
 Proteolytic activity, Prostatin, Creatinine, Na+, K+.

 If findings dictate it we may have to examine the following on chosen urine tests:

 -  The ability to activate ENaC- current in M-1-collecting duct cells by patch clamp
 electrophysiology on complete cells.

 -  Detection of different proteolytic forms of the ENaC gamma-subunit through antibody.

 Measurements in 24- hour urine samples:

 Na+/K+ - concentration. Creatinine clearance. Albumin.

 Study process:

 The three groups, "cases" (patients with preeclampsia) and "pregnant controls" (patients with
 normal pregnancies) and non pregnant controls ( non pregnant woman), are provided with 10
 days of a healthy fully diet with a fixed low- normal NaCl content of 50 -60 mmol Na+/ day.
 The diet has the same content of calories per kg body weight and has the same content of
 potassium as far as possible. In this period of time the patients must only consume this diet
 and regularly drink water. Besides the fixed diet the patients are given a supplement with
 either sodium or placebo. The sequence is double randomized. The participants ingests either
 sodium tablets (150-200 mmol /day) or a corresponding number of placebo tablets. On the fifth
 day, the participants switch their daily supplement, so they now take placebo instead of
 sodium or the other way around.

 Day number 1 is a Monday and day number 5 is a Friday where the measurements are to be done.
 24-h urine samples are collected from Thursday 7 a.m. till Friday morning. On the 5th day the
 participants meet from 9 a.m. to 10 a.m. They are placed in a chair with elevated legs and
 relaxed for an hour in calm surroundings.

 From 7 a.m. they ingest 200 ml water per hour. Spot urine tests are collected. Blood pressure
 is measured with an automatic oscillometric device. Intravenous access is made and blood
 samples are taken to decide the plasma hormone concentration (renin, angiotensin,
 aldosterone, ANP) and albumin, plasminogen and electrolytes. Cardiac output is measured
 non-invasive by impedance cardiography. Placenta flow is measured by ultrasound. The
 procedure lasts approximately 2 hours.

 Hereafter a fixed, fully diet is given either with a sodium supply, a total of 250 mmol/day
 (corresponding to a high average sodium intake in Denmark) or placebo tablets. This diet is
 given in 4 days and measurements are made on the 5th day as described above.

 A fixed sodium intake is given to:

 1. Get valid steady-state values for the RAAS- components;

 2. To study reactivity in RAAS components by changes in the NaCl intake

 3. Decide changes in blood pressure during 2 different normal NaCl intakes - low and high
 normal

 4. Correlate RAAS reactivity to urine excretion of plasmin/plasminogen under the hypothesis
 at the correlation will be conversely and

 5. Achieve valid values for aldosterone in correlation to the placenta flow. Length of
 gestation and placenta´s weight is registered at birth.

 Data- analysis methods:

 Introduction:

 This is an interventional double blinded case-control, cross-over study which includes 10
 patients with preeclampsia, 10 patients with normal pregnancies and 10 non pregnant woman.

 Population size evaluation:

 The settings for a Ph.D. study makes it impossible to accomplish a fixed sodium diet
 intervention in 10 days with the amount of patients required to get a relevant change in the
 blood pressure detection.

 With a paired design it is estimated that to achieve an expected change in blood pressure at
 approximately 15 mmHg in the individual and a clinical relevant different in blood pressure
 at 8 mmHg the sample size must include 40 patients (5% percentage point - 90% strength).
 Therefore we have chosen that blood pressure is not the primary variable of the outcome,
 instead we choose the RAAS components.

 In previously similar designs we have measured hypertensive vs. normotensive patients and had
 enough statistic strength to detect differences in plasma concentrations of
 renin-angiotensin-aldosterone components and ANP by using 10 patients in each group.
Cases:

 Inclusion Criteria:

 1. Pregnancy week 28-36 (exclusion of patients with previously severe preeclampsia).

 2. Singleton pregnancy

 3. Preeclampsia- hypertension: repetitive high blood pressures (> 140/80 mm Hg) measured
 in the consultation and proteinuria (dip test, albumin).

 4. Pregnant with microalbuminuria and proteinuria, but without hypertension (and
 therefore do not meet the diagnostic criteria for preeclampsia) can also be included.
 Proteinuria is the most important factor.

 It is still possible to test our hypothesis with possible comorbidity such as diabetes,
 SLE(systemic lupus erythematosus), rheumatoid arthritis and therefore not a reason for
 exclusion.

 Exclusion Criteria:

 1. Hypertension in pregnancy without proteinuria.

 2. Pregestational nephropathy by other unknown reasons.

 3. Early severe preeclampsia.

 4. Organic or systemic disease of clinical relevance, such as malignancy.

 Pregnant controls-

 Inclusion Criteria:

 1. pregnancy week 28-36

 2. Singleton pregnancy

 3. Uncomplicated pregnancy

 Exclusion Criteria:

 1. Hypertension

 2. Any kind of nephropathy

 3. Organic or systemic disease of clinical relevance, such as malignancy.

 Non-pregnant controls:

 Inclusion Criteria:

 1. woman, not pregnant

 2. Matched by age and BMI

 Exclusion Criteria:

 1. Hypertension

 2. Any kind of nephropathy

 3. Organic or systemic disease of clinical relevance, such as malignancy.