Hormonal effects on kallikrein, esterase A2, and their inhibitors in rat urine.

The effects of various hormones on the urinary excretion of kallikrein and esterase A2 were studied in rats. Chronic treatment with antidiuretic hormone had no effect on the excretion of either enzyme. Deoxycorticosterone treatment or a low sodium diet stimulated urinary kallikrein excretion (as is well known), but had no effect on urinary esterase A2. Dexamethasone markedly suppressed the excretion of both kallikrein and esterase A2 and increased the excretion of proteins (inhibitors) that bind to each of these enzymes. It is likely that the suppressive effect of glucocorticoid on urinary kallikrein and esterase A2 activities is due to the increase in urinary binding proteins. There is also a strong correlation between the excretion of kallikrein and esterase A2 in normal untreated rats. Such a correlation might arise from a common effect of glucocorticoid-influenced inhibitors on both enzymes.

Isolation and partial characterization of rat urinary esterase A2.

An enzyme, esterase A2, which hydrolyzes tosyl-arginine methyl ester was isolated from the urine of female, inbred, Dahl-salt-resistant rats using DEAE-Sephadex ion-exchange, aprotinin-agarose affinity and molecular sieve column chromatography. The purest preparation obtained showed four closely migrating bands on polyacrylamide gel electrophoresis. All four bands of the esterase A2 preparation had enzyme activity since all were stainable on zymograms using N-acetyl-L-methionine alpha-naphthyl ester as substrate. Three of these four bands showed decreased electrophoretic mobility following treatment with neuraminidase, indicating that variable sialic acid content accounts for part of the microheterogeneity. The preparation of esterase A2 used was free of rat urinary kallikrein as shown by radioimmunoassay, electrophoretic and isoelectric focusing experiments. The relative kinin-generating ability of rat urinary kallikrein and esterase A2 was highly dependent on the assay used. Using canine plasma as a source of kininogen and the rat uterus to bioassay kinins, esterase A2 was 47% as active as kallikrein; using pure bovine low-molecular-weight kininogen and a radioimmunoassay to measure generated kinins, esterase A2 was only 6% as active as kallikrein. Esterase activity of A2 was activated non-specifically by proteins and detergents. Esterase A2 was 50% inhibited by an 8-fold molar excess of aprotinin and by a 26.5-fold molar excess of soybean trypsin inhibitor, but ovomucoid inhibitor was not inhibitory.

Alteration in immune complex glomerulonephritis by arachidonic acid.

We employed a model of immune complex glomerulonephritis produced in mice by the daily injection of apoferritin to study the effect of treatment with arachidonic acid (AA). Apoferritin injections produced demonstrable glomerular damage by light microscopy associated with deposition of immunoglobulin along peripheral capillary loops. Treatment with AA 100 micrograms daily resulted in significantly less glomerular damage and a shift inthe location of immune complex deposition to the mesangium. The amount of anti-apoferritin antibody was determined by hemagglutination and found to be significantly decreased in mice treated with AA. Separate studies employing this dose of AA revealed that the number of IgM antibody producing cells to SRBC was not altered by AA.

Anomalous response of urinary kallikrein to deoxycorticosterone in Dahl salt-sensitive rats.

Previous evidence shows that salt-sensitive (S) rats have a net increase in plasma mineralocorticoid activity due to 18-hydroxy-11-deoxycorticosterone and decreased urinary kallikrein excretion compared to salt-resistant (R) rats. Since mineralocorticoids stimulate urinary kallikrein excretion, these results are inconsistent. This inconsistency was explained by the fact that, while R rats responded normally to treatment with deoxycorticosterone (DOC) by an increase in urinary kallikrein excretion, S rats showed no change in urinary kallikrein even when treated with 10 mg of DOC/day for 24 days. S and R rats responded identically to DOC with changes muscle electrolytes and relative hypertrophy of the renal distal tubule. Other measures of chronic mineralocorticoid response in S rats beside kallikrein were, therefore, intact. It was found that S rats were capable of responding to Na deficient diet with an increase in urinary kallikrein comparable to R rats. It was argued, therefore, that mineralocorticoid receptor mechanisms and distal-tubular cell responsiveness are intact in S rats. Mild glomerular and tubular scarring was found in S rats and the severity of renal lesions was increased by DOC treatment in S rats. These lesions correlated well with blood pressure and proteinuria. No such lesions were present in control or DOC treated R rats. It was suggested that failure of urinary kallikrein to respond to DOC in S rats may be a secondary phenomenon resulting from renal damage.

Developmental patterns of blood pressure and urinary protein, kallikrein, and prostaglandin E2 in Dahl salt-hypertension-susceptible rats.

S and R female rats were raised on a 1% NaCl diet, and excretion rates of urinary protein, kallikrein esterase activity, and PGE2 were measured (1) at 1 1/2 months of age, when both S and R rats were normotensive, (2) at 3 months of age, when S rats were mildly hypertensive and R controls remained normotensive, and (3) at 6 months of age, when S rats were markedly hypertensive relative to the still normotensive R rats. Urinary protein excretion rate in S compared to R rats was slightly elevated at 1 1/2 months of age and greatly elevated at 3 and 6 months of age. Urinary kallikrein was measured by hydrolysis of TAME after separation of kallikrein from nonkallikrein TAME esterases on DEAE-Sephadex minicolumns. Kallikrein TAME esterase activity was the same in 1 1/2-month-old S and R rats but became reduced in S relative to R rats at 3 and 6 months of age, concomitant with the development of hypertension and marked proteinuria. Urinary PGE2 was decreased in S rats as compared to R rats at all ages, and therefore the strain difference in urinary PGE2 preceded the development of strain differences in blood pressure and urinary kallikrein activity. We conclude that (1) reduced excretion of urinary kallikrein TAME esterase activity in S rats is probably secondary to hypertension and severe proteinuria and (2) decreased urinary PGE2 excretion in prehypertensive S rats is compatible with, but does not prove, the presence of a primary defect in intrarenal PGE2 production that could be involved in initiating hypertension.

Evidence for an androgen-dependent urinary arginine esterase in the rat: separation from other urinary arginine esterases including kallikrein.

DEAE-Sephadex chromatography of male rat urine resolved three peaks of arginine esterase activity using the synthetic substrate alpha-N-p-tosyl-L-arginine methyl ester . HCl (Tos-Arg-O-Me). Esterase activity in peak 1 (esterase A1 fraction) was present in sexually mature males, but it was not found in mature females, castrated mature males, or sexually immature males. Urinary esterase A1 activity could be restored in castrated mature males by the administration of testosterone, and it is, therefore, androgen dependent. Esterase A1 activity was undetectable and could not be induced in females by daily doses of testosterone (up to 20 mg/day). The second urinary esterase peak (esterase A2 fraction) and the third peak (urinary kallikrein) were found in both male and female rats. Molecular sieve chromatography of esterase fractions A1, A2, and kallikrein, as determined by Sephacryl S-200 chromatography, gave single peaks of activity. Molecular weights were estimated to be 28,500, 42,000, and 41,500, respectively. Kinin-generating activities of esterases A1, A2, and kallikrein were also determined using dog plasma substrate. Esterase A1 fraction had no kinin-generating activity, while esterase A2 fraction demonstrated significant activity but was 12 times less active than kallikrein per Tos-Arg-O-Me esterase unit. Esterase A2 fraction could not induce direct contraction of the rat uterus, and A2 did not cause a transient decrease in rat blood pressure when injected iv, both of which are criteria for kallikrein activity. It is concluded that the androgen-dependent Tos-Arg-O-Me esterase has properties quite different from both esterase A2 and kallikrein. Esterase A2 appears to be similar to the esterase A described in female rat urine by Nustad and Pierce.

Cecal diverticulitis.

Cecal diverticula may be solitary or an extension of leftsided diverticulosis. They may be true or false. Diverticulitis in the cecal area is usually found at the time of surgery for presumed appendicitis. If the diagnosis is obvious grossly, a simple diverticulectomy may be performed. Most cases in our series were of the "hidden" variety. In these cases, a right hemicolectomy was performed because the lesion could not be distinguished from cecal carcinoma.

Effects of dexamethasone on excretion of urinary kallikrein and urinary protein in Dahl salt-sensitive and salt-resistant rats.

The effect of glucocorticoid treatment on urinary kallikrein excretion was assessed in Dahl salt-hypertension susceptible (S) and salt-hypertension resistant (R) rats. A single dose of dexamethasone (100 micrograms) caused a marked water diuresis and a slight decrease in urinary kallikrein excretion in both S and R rats. A single dose of dexamethasone also caused the S rat to excrete massive amounts of protein into the urine, almost 3-fold higher than S rats treated with oil; the effect on R rat urinary protein was similar, but less severe. Daily administration of dexamethasone (100 micrograms/day) for 7 days caused marked suppression of urinary kallikrein excretion in both S and R rats. Increased urinary protein following chronic treatment was still evident in the dexamethasone-treated S rats but not in the dexamethasone-treated R rats. Chronic glucocorticoid treatment probably inhibits urinary kallikrein activity by suppressing pituitary and adrenal function which would remove the stimulatory effect of aldosterone on urinary kallikrein excretion. There was no evidence for a stimulatory role of glucocorticoids on urinary kallikrein.

A qualitative difference in plasma renin activity in Dahl rats susceptible or resistant to salt-induced hypertension.

Plasma renin activity (PRA) was studied in the rats bred by Dahl for susceptibility (S-strain) or resistance (R-strain) to salt (NaCl) induced hypertension. The pH curves for PRA had different shapes. The difference in shape of the pH curves was reflected in the ratio of PRA pH 8/PRA pH 6.5. This ratio was shown to be characteristic of the strain and to be independent of changes in absolute PRA level induced by variation in dietary NaCl. The ratio of PRA pH 8/PRA pH 6.5 was also different between strains in weanling as well as adult rats. The underlying cause for the strain difference in the effect of pH on PRA is unknown, but may involve molecular differences between strains in either renin or renin substrate.

Total and kallikrein arginine esterase activities in the urine of salt-hypertensive susceptible and resistant rats.

Urinary enzymes that hydrolyze the artificial substrate alpha-N-p-tosyl-L-arginine methyl ester (TAME) were studied in Dahl salt-sensitive (S) and salt-resistant (R) rats. Total urinary TAME esterase activity (kallikrein and non-kallikrein) showed a marked increase with dialysis against water, but only in hypertensive S rats with proteinuria. This phenomenon suggests the presence of dialyzable TAME esterase inhibitor(s) in urine following renal damage, but these data do not define what urinary esterases might be affected. Partially purified urinary kallikrein exhibited a ratio of kininogenase to esterase activity which was equal for S and R rats. Thus, the marked discrepancy between kininogenase and esterase activities reported by Carretero et al. with S and R whole urine is not a function of the S and R kallikrein molecules but is probably related to interfering substances in the whole urine. Urinary kallikrein excretion was measured on individual rat samples by TAME esterase activity following dialysis and separation from non-kallikrein TAME esterase(s) using DEAE-Sephadex minicolumns. S rats had lower urinary kallikrein excretion that R when the S rats were hypertensive and showed marked proteinuria. Young S and R rats raised on low salt showed similar blood pressures and similar kallikrein excretion. High salt (8% NaCl) diet decreased kallikrein excretion in both S and R, but the decrease was greater in the S rats which became hypertensive and had increased urine protein excretion. These data suggest that the lower urinary kallikrein excretion in S rats relative to R rats is probably a consequence of hypertension and renal damage rather than a primary cause of hypertension.