Hypertension and Urine Protease Activity in Preeclampsia
Condition: Preeclampsia; Hypertension; Proteinuria; Pregnancy
MeSH terms: Pre-Eclampsia; Proteinuria; Hypertension
Study type: Interventional

Summary: 
      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
    

Detailed description: 
      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.
    

Intervention name(s): Sodium; Placebo
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): supplemental sodium tablets 150-200 mmol/day in 5 days; Placebo are given in 5 days
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Treatment
Keywords: Proteinuria; Pregnancy; Hypertension; Plasmin; Plasminogen; Aldosterone; Angiotensin; Renin; sodium chloride; epithelial sodium channel; Placenta