Narendra B. Oza

Activation of a prorenin-like substance in human plasma by trypsin and by urinary kallikrein.

SUMMARY Inactive plasma renin (a prorenlp-like substance) can be activated by trypsin. There are also endogenous neutral serine proteases in plasma that can activate inactive renin in vitro, but only after protease inhibitors are either destroyed by acidification (the alkaline phase of acid activation) or inactivated by cold (cryoactivation). In the present study we have shown that cold also facilitates trypsin activation. But even at −4°C, as much as 1 rag/ral of trypsin was required to overcome endogenous inhibitors and reproducibly activate inactive plasma renin during a 1-hour incubation at pH 7.4. After partial destruction of plasma protease inhibitors by acidification to pH 33., less trypsin was required for complete activation at pH 7.4:200 Mg/ml at 25°C and 100 Mg/ml at −4°C. In contrast, 10 fig/ml of the renal enzyme urinary kallikrein completely activated inactive renin in previously acidified plasma at 25°C. Maximum activation of inactive plasma renin by either trypsin or renal kallikrein was almost identical. Both enzymes caused activation in plasmas deficient in Hageman factor or Fletcher factor (prekallikrein), suggesting that their ability to activate inactive renin is not mediated by these neutral serine proteases of the intrinsic coagulation system.Using maximum trypsin activation to define “total” renin, we found that among 22 normal subjects and hypertensive patients there was a direct relationship between the proportion of active renin in plasma (active/ total) and the concurrent urinary kallikrein excretion (r = 0.46, p < 0.05). Normotensive white subjects had a higher proportion of active plasma renin than blacks, in whom urinary kallikrein is reported to be low. Altogether, these data suggest that there might be a link between prorenin and renal kallikrein in vivo. Further studies are required to evaluate this possibility and to determine whether prior hydrolysis of inactive renin, for example, by acidification, is required for renal kallikrein to activate inactive renin.

Components of the kallikrein-kinin system in rat urine☆

Using a direct radioimmunoassay and a kininogenase assay, we determined that 68% of rat urinary kallikrein was enzymatically active while 32% was in an inactive form which was activated by trypsin. Inorganic cations, at concentrations found in rat urine, were inhibitory in an amidase assay but appeared to potentiate kininogenase activity of pure rat urinary kallikrein. In random urines, kinin concentration was 4.2 +/- 0.7 ng/ml. Trypsinization of the urines generated 52.9 +/- 25.8 ng kinin/ml, indicating that kininogen was present. The rate of kinin formation in vivo may depend on the availability of kininogen and the concentration of inorganic cations in urine, as well as on other well-recognized factors, such as the kallikrein activity of the urine.

Reduced ratio of active-to-total urinary kallikrein in essential hypertension.

Urinary kallikrein excretion was studied in 34 patients with mild, normal-renin, essential hypertension without evidence of target organ damage and in 23 normotensive controls, using assays that measure both active (kininogenase activity) and total (active plus inactive) kallikrein. There was no significant difference in either active or total kallikrein excretion between the two groups. However, the ratio of active-to-total enzyme was decreased in the hypertensives (0.83 +/- 0.03 units/micrograms) compared to the normotensives (1.00 +/- 0.05 units/micrograms) (p less than 0.002). The active-to-total ratio was inversely related to sodium excretion in both groups, indicating that the proportion of active to inactive enzyme increased in response to reduced sodium intake. We conclude that, although absolute excretion of active and total kallikrein is not decreased, enzyme activity per microgram of total kallikrein excreted is reduced in mild, normal-renin essential hypertension. This abnormality may be due to a defective enzyme, or to a reduced excretion of active relative to inactive kallikrein. The latter could result from the presence of a urinary kallikrein inhibitor or to reduced activation of a proenzyme.

A new direct radioimmunoassay of rat urinary kininogen

A protein-binding radioimmunoassay (RIA) of rat low molecular weight (LMW) kininogen with the following characteristics has been developed: sensitivity, 2.5 ng/tube; inter-assay coefficient of variation, 12.4% (N = 28); and intra-assay coefficient of variation, 9.4% (N = 11). The new assay correlated (r = 1) with the determination of kinin equivalence of kininogen after trypsinization. The cross-reactivity of rabbit anti-rat LMW kininogen antibody was 2.5% with bovine LMW kininogen, 5.8% with rat plasma high molecular weight (HMW) kininogen, and none with kinin. Although the antibody appears to partially recognize des-kinin-kininogen, the low degree of cross-reactivity and the extremely low levels of kinin-free-kininogen allow accurate determination of total LMW kininogen in rat urines. The LMW kininogen formed 20% kinins with salivary kallikrein when compared with trypsin, suggesting that the preparation consists of both K- and T-kininogens (K = kallikrein susceptible; T = trypsin susceptible). The newly developed protein-binding RIA recognizes LMW kininogen of rat urine which consists of both K- and T-kininogens.

Regulation of Renal rK1-Kallikrein in Spontaneously Hypertensive Rats

Urinary and renal rK1-kallikrein was studied in spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). It was demonstrated that the antiserum used for kallikrein radioimmunoassay (RIA) reacts with rK1- but not with rK7-protein. The specificity of the kininogenase assay was tested: rK7 had only 8% of the activity of rK1. Urinary kallikrein excretion by RIA was reduced by about two thirds in 5-week-old SHR compared WKY (11.5 versus 37.1 micrograms/24 h). On the contrary, the kidney content of rK1-kallikrein by RIA was increased by 40% in these rats (11.6 versus 8.4 ng/mg protein). The increase in kidney rK1 was confirmed by kininogenase assays. The same pattern of reduced urinary and increased renal rK1-kallikrein was observed in 8-week-old SHR rats. Kidney rK1-kallikrein mRNA tended to be lower (0.10 > p > 0.05) in SHR compared to WKY rats, suggesting that the increased kidney rK1 content is not due to increased rK1 synthesis. We hypothesize that the combination of high kidney content and low urinary excretion may be due to a defective mechanism for secretion of rK1 into the urine by tubular epithelial cells.

Vasopressin Stimulates Urinary Kallikrein Excretion in the Isolated Erythrocyte-Perfused Rat Kidney

We have found that arginine vasopressin (AVP) (10 pg/ml) stimulates urinary kallikrein in the isolated erythrocyte perfused rat kidney. (In this model, perfusate flow rate approximates blood flow rates in vivo and morphology is normal.) Urinary kallikrein excretion rose from 6.9 +/- 0.8 to 14.9 +/- 2.4 ng/min 20 min after the addition of AVP to the perfusate, and then fell towards baseline levels over the next 30 min. 1-Desamino-8-D-AVP (8 pg/ml) caused a comparable increase in kallikrein excretion. Prostaglandin synthesis inhibition with indomethacin did not alter the stimulatory effect of AVP on kallikrein excretion. Parathyroid hormone 1-34 (144 ng/ml) and calcitonin (102 ng/ml) also increased urinary kallikrein. Kallikrein excretion rose from 9.1 +/- 2.0 to 24 +/- 4.5 ng/min in response to calcitonin and from 8.3 +/- 1.6 to 43.7 +/- 3.4 ng/min following the addition of parathyroid hormone to the perfusate. Kallikrein was found to accumulate in the perfusate in a linear fashion. Based on the slope of the relationship between perfusate kallikrein and time, the rate of release of kallikrein into the perfusate was estimated to be 0.79 ng/min in control kidneys. The rate of release of kallikrein into the perfusate in kidneys treated with AVP was the same (0.74 ng/min). Thus while kallikrein is released into the perfusate, this process is not influenced by AVP. In conclusion, AVP stimulates release of kallikrein into the urine (but not the perfusate) independently of systemic events. The effect of AVP is not mediated by prostaglandins. This effect of AVP is mediated via stimulation of the V2 receptor and also occurs in response to two other hormones (calcitonin and parathyroid hormone) that are known to stimulate adenyl cyclase in the rat distal nephron.

A Micro-Kininogenase Assay for Studies of Kallikrein in Renal Micropuncture/Microperfusion

We report the development of a micro-kininogenase assay suitable in studying the dynamics of kallikrein at intranephron segmental level. The detection limit is 119 fg or 2.6 attomoles of kallikrein. Activation of microquantities of kallikrein is possible with the use of Triton X-100. Because of its extremely high sensitivity and reproducibility the assay is likely to also prove useful in physiological studies in which only very small amounts of kallikrein containing samples can be obtained.