Inactive Urinary Kallikrein Research Articles

Harnkallikrein zur Prädiktion hypertensiver Schwangerschaftserkrankungen

Objective: We studied whether urinary kallikrein levels can be used to predict the development of hypertensive disorders in pregnancy. Methods: We prospectively measured concentrations of total, active and inactive kallikrein in spontaneous urine samples from 410 unselected pregnant women at 16 to 20 weeks' gestation and calculated ensitivity, specificity and positive and negative predictive values. Results: The median concentrations of total, active and inactive urinary kallikrein were significantly higher in women who remained normotensive than in those who developed preeclampsia (3.9 vs. 2.7, 1.3 vs. 0.8, and 2.7 vs. 1.8 U/g creatinine, respectively; all P < 0.01). A total kallikrein cut-off level of 2.0 u/g creatinine was calculated. At this cut-off level, the sensitivity of total kallikrein for predicting preeclampsia was 44%, the specificity was 89%, the positive predictive value was 16%, and the negative predictive value was 97%. In patients without preexisiting hypertension, the sensitivity was 33%, the specificity was 89%, the positive predictive value was 9%, and the negative predictive value was 98%. Women with chronic hypertension had the lowest median kallikrein levels (1.95, 0.5 and 1.2 U/g creatinine, respectively). Conclusion: The low positive predictive value of the kallikrein/ creatinine ratio limits its usefulness as a screening test for detecting women at risk of developing preeclampsia.

Urinary kallikrein excretion in normotensive and hypertensive pregnancies: 8 years later

In a previous longitudinal study of urinary kallikrein (UK) excretion in pregnancy we reported that women with low inactive UK (IUK) to creatinine ratio (IUK:Cr), measured at 16–20 weeks gestation, were at increased risk of developing pregnancy induced hypertension. In this study, 8 years later, we have recalled 14 women who had an IUK:Cr ≤170 (Gp1) and became hypertensive in late pregnancy and 14 women who had IUK:Cr>170 and remained normotensive (Gp2). Resting blood pressure (BP) and BP response following application of cutaneous cold to assess vasoconstrictor reactivity were measured. A timed urine sample was also collected for measurements of components of the kallikrein kinin system (KKS): IUK, active UK (AUK), kininogen (UKg), urinary kinins (UKi). Urinary creatinine (UCr) was also measured. The correlation between IUK:Cr during the 1989–1990 study and current measurement was r2=0.52, p<0.0001. Although resting BP was higher in Gp1 than Gp2 this was not significantly different. However, there was a greater change in diastolic BP in Gp1 than in Gp2 in response to the cold pressor test (p<0.04). Excretion rates of IUK, UKg and UKi were significantly lower in Gp1, p<0.006, p<0.003, p<0.03, respectively. AUK was also reduced in Gp1 but did not reach statistical significance. Women with reduced activity of the renal KKS combined with increased sympathetic drive may be at increased risk of developing hypertension.

Prediction and prevention of pregnancy-induced hypertensive disorders

Because hypertensive disorders complicating pregnancy are common and constitute a leading cause of maternal, fetal and neonatal morbidity and mortality, prevention attempts appear to be justified. Primary prevention is only possible by avoiding pregnancy. Secondary prevention requires identification of patients at risk. A large number of predictive methods have been published and the majority appear to be of no or limited value. At present only the determination of inactive urinary kallikrein and uteroplacental colour–pulsed Doppler velocimetry show promise and require further assessment. Analysis of the many interventions advocated for prevention of pre-eclampsia reveals that only dietary calcium supplementation and prophylactic low-dose aspirin have shown promise of efficacy in small controlled clinical trials, but the results of large, multicentre trials are disappointing. The disappointing results obtained in large, multicentre trials may be explained, at least in part, by the lack of strict eligibility criteria and end-points and by low patient compliance. Prophylactic low-dose aspirin is recommended in women at high risk because it is associated with a moderate reduction in risk, may reduce the severity of pre-eclampsia if it develops and appears to be safe for mother and infant. The present data do not support any prophylactic intervention in pregnant women at low or medium risk.

Active and Inactive Urinary Kallikrein in Man: Effects of Diuresis and Antidiuresis

1. The urinary excretion of active and inactive kallikrein was studied in volunteers during diuresis induced by water loading or oral frusemide and during antidiuresis induced by desamino-D-arginine-vasopressin. 2. During acute oral water loading, excretion of active kallikrein was unchanged, despite high urine flow rates and low urine osmolalities being achieved. Excretion of inactive kallikrein correlated with the urine flow rate. 3. After desamino-D-arginine-vasopressin in eight water-loaded and six normally hydrated subjects, excretion of inactive kallikrein also correlated with the urine flow rate. There were no significant changes in the excretion of active kallikrein. 4. After frusemide there was a small transient increase in excretion of active kallikrein 1-2 h after dosing which coincided with the maximum diuresis and natriuresis. Excretion of inactive kallikrein again correlated with urine flow rate but the regression relationship between the two variables was different for water-load-induced and frusemide-induced diuresis. 5. These studies do not support a role for urinary kallikrein in the modulation of the antidiuretic action of vasopressin, but suggest that it may contribute to the natriuretic action of frusemide.

Role of renal kallikrein in the increased fractional sodium excretion in the rat remnant kidney model of chronic renal failure.

To assess the potential role of renal kallikrein-kinin system in enhancing sodium excretion per nephron in chronic renal failure, we studied urinary excretion of active and inactive kallikrein for 3 weeks in Wistar-Kyoto rats subjected to 5/6 nephrectomy (5/6), 1/2 nephrectomy (1/2) or sham operation (Sham). We determined urinary active and inactive kallikrein by measuring kallikrein activity using a kininogenase assay before and after treatment with trypsin (200 micrograms/ml). Fractional sodium excretion was significantly increased in 5/6-rats as compared with 1/2- or sham-rats. On the contrary, urinary active kallikrein excretion per nephron was not different in the three models whereas a significant rise in urinary inactive kallikrein excretion per nephron was found in 5/6-rats as compared with 1/2- or sham-rats. Urinary total kallikrein excretion per nephron was significantly increased in 5/6-rats as compared with sham-rats. In addition, no correlation was found between fractional sodium excretion and urinary active kallikrein excretion corrected for creatinine clearance (Ccr) in 5/6-rats. These results indicate that decreased excretion of renal active kallikrein may not play a significant role in the increased sodium excretion per nephron in the rat remnant kidney model of chronic renal failure. Furthermore, it is suggested that in this model of rat there might be impaired production of renal active kallikrein although its exact mechanism remains to be determined.

Activation of inactive kallikrein in the rat kidney during low sodium intake.

A method was developed to measure the protease responsible for activation of inactive kallikrein in the rat kidney. The renal protease was evaluated by incubating the purified rat urinary inactive kallikrein with the renal cortical extract in the presence of cysteine at pH 5.0. The renal cortical extract produced a dose- and time-dependent activation of the inactive kallikrein. Levels of the renal protease responsible for activation of inactive kallikrein remained unchanged with dietary sodium restriction. On the other hand, urinary excretion of total, active, and inactive kallikrein increased significantly during low sodium intake, with no change in the ratio of active to total kallikrein. These results suggest that low sodium intake stimulates the biosynthesis of inactive kallikrein but not the activation of this kallikrein precursor in the kidney.

Purification of inactive kallikrein from rat urine.

An inactive kallikrein was purified from rat urine, and some of the properties of this enzyme were examined, in comparison with those of rat urinary kallikrein (RUK). The purified inactive kallikrein reacted with the antiserum against RUK and migrated slightly more slowly than RUK, on the immunoelectrophoresis. The molecular weights of the inactive kallikrein and RUK were estimated to be 44,000 and 38,000 by gel filtration, respectively. These results indicate that the rat urinary inactive kallikrein is immunologically identical with RUK, but this inactive enzyme has biochemical properties different from those of RUK, with respect to molecular weight and electrophoretical mobility.

Inactive Urinary Kallikrein Excretion and Urine Flow Rate

Inactive Urinary Kallikrein Excretion and Urine Flow Rate

Activation of urinary inactive kallikrein by an extract from the rat kidney cortex

Activation of purified urinary inactive kallikrein by an extract from the rat kidney cortex was investigated. The extract produced a dose-dependent activation of the inactive kallikrein and the optimum pH for this activation was 5.0. Marked depression of the activation was observed when the extract was pre-incubated with E-64, p-CMB and iodeacetate, but not with DFP, PMSF or pepstatin A. The molecular weight of the inactive kallikrein (Mr 44,000) was reduced to 38,000 by treatment with the extract, this molecular weight value being identical with that of urinary active kallikrein. These results indicate that the rat kidney cortex contains a protease catalyzing conversion of urinary inactive kallikrein into its active form, and that the protease has properties compatible with those of a thiol protease, but not of trypsin which has been used as a tool for the activation of urinary inactive kallikrein. The thiol protease is probably one of regulators of the kallikrein-kinin system in the kidney.

Renal inactive kallikrein as the possible origin of urinary inactive kallikrein in the rat.

An inactive kallikrein, which could be activated with trypsin was isolated from the rat kidney cortex using diethylaminoethyl (DEAE)-cellulose chromatography. The inactive kallikrein had no vasodilator action, whereas the injection of trypsin-activated form of this enzyme into the femoral artery of dogs resulted in a marked increase in the arterial blood flow. Apparent molecular weight of the inactive kallikrein was estimated to be 4.4 X 10(4) by gel filtration, and this enzyme was converted to the renal active kallikrein (M.W. 3.8 X 10(4] by trypsin. The inactive kallikrein is immunologically identical with the trypsin-activated form of inactive kallikrein and active kallikrein. There were no significant differences in the chromatographic behavior on a DEAE-cellulose column, Km value for prolyl-phenylalanylarginine-4-methylcoumaryl-7-amide hydrolysis and profile of inhibition by trypsin inhibitors between the active kallikrein and the trypsin-activated form of inactive kallikrein. The above properties of the renal inactive kallikrein were similar to those of inactive kallikrein found in the urine. These results suggest that the inactive kallikrein in the rat kidney would be proteolytically converted to its active enzyme and that a part of the inactive kallikrein would be excreted into urine in a form itself.

High-performance liquid chromatography with a continous-flow enzyme detector for the demonstration of the conversion of rat urinary inactive kallikrein into its active form

High-performance liquid chromatography with a continous-flow enzyme detector for the demonstration of the conversion of rat urinary inactive kallikrein into its active form