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The arcades are long, branched renal tubules which connect deep and mid-cortical nephrons to cortical collecting ducts in the renal cortex. Because they are inaccessible by standard physiological techniques, their functions are poorly understood. In this paper, we demonstrate that the arcades are a site of expression of two proteins, aquaporin-2 (the vasopressin-regulated water channel) and the V2 vasopressin receptor, that are important to regulated water transport in the kidney. Using a peptide-derived polyclonal antibody to aquaporin-2, quantitative ELISA in microdissected segments showed that aquaporin-2 is highly expressed in arcades and that the expression is increased in response to restriction of fluid intake. Immunocytochemistry revealed abundant aquaporin-2 labeling of structures in the cortical labyrinth in a pattern similar to that of the Na(+)-Ca2+ exchanger and kallikrein, marker proteins expressed in arcades but not in cortical collecting ducts. RT-PCR experiments demonstrated substantial aquaporin-2 and V2 receptor mRNA in microdissected arcades. In situ hybridization, using 35S-labeled antisense cRNA probes for the V2 receptor demonstrated strong labeling of both arcades and cortical collecting ducts. Thus, these results indicate that the arcades contain the specific proteins associated with vasopressin-regulated water transport, and may be a heretofore unrecognized site of free water absorption.

Rat Kidney is reported to contain the rK7-kallikrein gene expressed at a high mRNA level. However, there is no direct evidence that the rK7 enzyme is present in kidney or urine. We have analyzed rat kidney and urine for rK7-like enzyme activity, using distinctive properties of purified rK7 enzyme (hydrolysis of specific substrate, inhibition by SBTI). Rat urine and kidney cortex contain substantial levels of rK7-like enzyme activity. Nearly 40% of the enzyme in urine is in an active form while nearly all of the rK7 in kidney cortex is in the inactive form. Treatment with low salt diet and mineralocorticoid increases rK7 in both kidney cortex and urine approximately 2-fold. The percentage of active enzyme in urine increases markedly on this regimen.

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.

We determined kininogen concentration and kininogenase activity in the blood-free, aorta homogenates of 15 week old spontaneously hypertensive rats (SHR) and in their age-matched wistar kyoto (WKY) controls. Active kininogenase in the SHR was only 18% of the WKY controls. Thus, the kininogen concentration was inversely related to kininogenase activity suggesting a local interaction of these components and possible generation of kinins within the vascular wall.

We determined kinin-generating activity (kininogenase) in the thoracic aorta of spontaneously hypertensive rats (SHRs) at age 5 and 15 weeks and in appropriately age-matched Wistar-Kyoto (WKY) rats. Aorta homogenates were incubated with partially purified dog kininogen, and the resulting kinins were extracted with ethanol. The kinins were determined by a sensitive kinin radioimmunoassay (RIA). Kininogenase activity was expressed as mean +/- SEM, picogram kinin generated/mg x protein/h. Active kininogenase in SHR was approximately one-third in 5-week-old and about one-fifth in 15-week-old rats when compared with their normotensive controls. Total kininogenase activity in SHRs was approximately 80% and 58% of the normotensive controls at ages 5 weeks and 15 weeks, respectively. Active enzyme was 14% of the total in 5-week-old SHRs, and it was only 5% of the total in 15-week-old SHRs. It seems unlikely that the changes in kininogenase are secondary to hypertension because blood pressor is only marginally elevated at 5 weeks according to the literature. We hypothesize that genetic hypertensive rats may suffer from an inherent deficiency in the kininogenase activity of the vascular wall. The deficiency may also be in the mechanism of activation of precursor enzyme.

We have studied rat vascular smooth muscle (VSM) cells in culture for the presence of key elements of the glandular kallikrein-kinin system. Direct radioimmunoassay (RIA) using antiserum against rat urinary kallikrein detected a glandular kallikrein-like enzyme (GKLE) in VSM cells and in media. VSM homogenates and culture media had kininogenase activity, generating kinins from dog kininogen. About half of the GKLE was enzymatically inactive which could be activated with trypsin. Kininogenase activity was inhibited completely by aprotinin but only 20% by soybean trypsin inhibitor (SBTI). Trypsin liberated kinins from homogenates and media, demonstrating that VSM cells contain kininogen. Homogenates and media rapidly degrade bradykinin. GKLE, kininogen, and bradykininase activity were all present in VSM cells grown in defined media that contain no serum, thus eliminating any contamination or artefacts from fetal calf serum in standard culture media. Blood vessels of the rat have been reported to contain GKLE. Our observations indicate that GKLE is synthesized by VSM cells, not deposited from plasma. Furthermore, VSM cells synthesize kininogen and bradykininase(s), the other key elements of the glandular kallikrein-kinin system. Thus it is possible that the system functions as an autocoid mechanism that regulates local vascular tone.

To test the hypothesis that renin is circulating in pressor amounts in renal hypertension, two-kidney Goldblatt hypertension was produced in rats. After hypertension had been present for a variable period of time (4 to 21 days), the ischemic kidney was removed, and an infusion of rat renin was started immediately to replace the endogenous renin that the ischemic kidney had been releasing. Since the amount of renin released by the clipped kidney was not known, the amount infused was regulated by a feedback mechanism set to keep the mean blood pressure at the same level as before ipsilateral nephrectomy. Hypertension remission was therefore prevented by this procedure. The PRA at the end of the renin infusion was similar to that prior to the ipsilateral nephrectomy. This finding indicates that the levels of the PRA circulating in renal hypertension are within the pressor range, thus supporting the hypothesis that renin plays a role in the pathogenesis of the acute or semiacute phase of renal hypertension. However, the participation of other factors cannot be ruled out since no correlation was found between PRA and blood pressure.

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.

Renal kallikrein is present in human urine in both an active and an inactive form. Several previous studies have examined the response of active kallikrein excretion to alterations in sodium and water metabolism, but the response of inactive kallikrein has not been evaluated systematically. We have developed a method for determining inactive kallikrein in urine using two assays. Active kallikrein is measured using a kininogenase assay. Total (active plus inactive) kallikrein is measured using a direct RIA. Inactive kallikrein is calculated from the difference between active and total kallikrein excretion. We have used this technique to study the effect of alterations in sodium and water metabolism on kallikrein excretion. Acute volume expansion with saline and moderate acute or chronic increases in water intake did not alter the excretion of either active or inactive kallikrein. Dietary sodium restriction increased the excretion of total kallikrein by 30%; active kallikrein increased 82%, while inactive kallikrein excretion was found to be unchanged. Spironolactone reduced total kallikrein excretion in subjects on a low salt diet. Again, the change in excretion was entirely attributable to the active form of the enzyme. Total kallikrein excretion increased during the first 3 days of fludrocortisone administration in subjects on a high salt intake and then plateaued. Active kallikrein increased progressively throughout the 7-day study period. Inactive kallikrein increased during the first 4 days, then fell to control levels. Thus, mineralocorticoid initially stimulates the formation and/or release into urine of both active and inactive kallikrein. Later, reciprocal changes in active and inactive enzymes occur.

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.

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.

1. Urinary kallikrein, sodium, potassium and water excretion, and plasma renin activity were measured before and during the reversal of experimental hypertension produced by unclamping the renal artery in rats. 2. Kallikrein excretion decreased significantly after unclamping, suggesting that it does not play a significant role in the reversal of hypertension. 3. A decrease in plasma renin activity coupled with a slight increase of sodium excretion was observed, indicating that these might participate in the reversal of hypertension.

A protein-binding radioimmunoassay (RIA) has been developed for human urinary kallikrein (HUK). HUK was purified to apparent homogeneity and rabbit anti-human urinary kallikrein serum was prepared. Radio-iodination of HUK was performed in 20 microgram batches with 1 mCi of 125I-Bolton-Hunter reagent in 0.1 M sodium borate buffer, pH 8.5, at 0 degree C. The 30 min reaction was terminated by adding 0.5 ml of 0.2 M glycine. Radiolabelled HUK was purified on a Sephacryl S-200 column (1x 50 cm) in 0.05 M Tris.HCl plus 0.2% gelatin, pH 7.4 (RIA buffer). 125I-HUK emerged as a single, uniform peak at Ve/Vo of 1.3. The RIA reaction mixture contained 15,000 cpm of 125I-HUK, 0.1 ml of diluted anti-kallikrein serum, 0.1 ml unlabelled kallikrein (1-100 ng) or the unknown sample and RIA buffer to a total of 0.5 ml. The reaction mixture was incubated at 22 degrees C for 18 hr, and 100 microliter to goat-anti-rabbit IgG serum was added to each tube. The tubes were incubated 18 hr at 4 degrees C, centrifuged 4,000 rpm for 30 min and, after decantation, the precipitate was counted for 125 I. The RIA reported here is sufficiently sensitive to detect 1 ng of kallikrein and should offer a useful technique to determine alterations of the kallikrein-kinin system in response to stimuli; both physiologic and pathologic.

We have used free-flow micropuncture to study the tubular locus at which kallikrein enters the urine. Kallikrein was measured by a newly developed, very sensitive assay for kininogenase activity; active kallikrein was measured directly by this assay and total kallikrein after activation of inactive kallikrein. Kallikrein was readily detected in all of 17, late distal tubular fluid-samples. In contrast, kallikrein was too low to detect in 15 of 17 proximal or in 11 of 14 early distal tubular fluid samples. Calculations indicate that less than 10% of urinary kallikrein could have derived from filtration or from proximal secretion of kallikrein. We conclude that urinary kallikrein enters the urine via secretion in the distal tubule. Filtration or proximal secretion of kallikrein does not contribute significantly to urinary kallikrein excretion.

The excretion of kallikrein in urine varies, but the pathophysiologic implications are not clear. To help clarify the role of the urinary kallikrein-kinin system, we have begun to define components of the system as they occur in urine. To minimize artifacts which may arise through extensive purification procedures, we studied urinary protein concentrates prepared by ultrafiltration. The concentrates were separated by chromatography on Sephacryl. Urine contains abundant kininase activity, but in strongly inhibited forms. Kininase II is separable into at least two forms. Another major kininase can hydrolyze benzoyl-Pro-Phe-Arg and is inhibited by arginine but not by BPP9a or SQ 14,225. Its molecular weight is approximately 63,000. A third kininase, not inhibited by BPP9a, is excluded from Sephacryl. Human urine appears to contain only one kallikrein-like enzyme (MW 45,000). In addition, urine contains a protein (MW approximately 80,000) which reacts with trypsin to release bradykinin and which inhibits the hydrolysis of Pro-Phe-Arg-[3H]anilide by urinary kallikrein. Thus, in addition to kallikrein and kinins, urine contains kininogen and at least three kininase enzymes. Urinary ultrafiltrate contains an inhibitory substance (approximately MW 400).

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.

The kallikrein-kinin system has been thought to participate in the pathogenesis of anaphylaxis. Kallikrein, released from lungs, has been postulated to contribute to cardiovascular collapse. Further to test the hypothesis, we examined for the occurrence of a kallikrein-like enzyme in guinea pig lungs and examined for release of such an enzyme by isolated, perfused lungs of guinea pig sensitized to and challenged with egg albumin. In addition, we treated guinea pigs with the bradykinin potentiating agents, BPP9a and SQ 14,225. In parallel experiments, we examined for effects of non-steroidal anti-inflammatory agents on the supposition that prostaglandin-related substances may mediate or modulate actions of kinins during anaphylaxis. A plasma kallikrein-like enzyme was found in lung homogenates and occurred in concentrations greater than that of plasma itself. Similarly, a store of kininogen occurs in lungs. However, using a sensitive radioassay for kallikrein-like enzymes, we were unable to confirm that antigenic challenge of sensitized lungs causes the release of enzyme into pulmonary venous effluent. Further, we were unable to modify the acute course of anaphylaxis by pretreatment of guinea pigs with bradykinin potentiating agents. However, indomethacin and aspirin at 20-40 mg/kg were found to greatly increase the severity of pulmonary anaphylaxis in terms of increased resistance to ventilation and increased numbers of lung hemorrhages. Paradoxically, aspirin or sodium salicylate at 80-100 mg/kg prevents the characteristic rise of insufflation pressure and the formation of lung hemorrhages.

We have developed a sensitive, highly selective assay for human urinary kallikrein (HUK) that uses Pro-Phe-Arg-[3H]benzyl-amide as substrate. The substrate was prepared from Pro-Phe-Arg-3-iodo-benzylamide by dehalogenation in 3H2 gas. HUK is measured by its ability to release [3H]benzylamine. The pH optimum is 9.5. Urokinase, plasmin and thrombin do not interfere. The assay can measure as little as 5 ng of HUK in a 15 min incubation at 37 degrees C. Typically, we use 50 microliter of dialyzed urine for HUK assays. Reactions are terminated by adding 0.1 M NaOH, and reaction product is separated from substrate by partitioning with an equal volume of toluene. A sample of the toluene phase is submitted for liquid scintillation counting. As judged by separations obtained on molecular sieve chromatography (Sephacryl), only one urinary enzyme possesses the ability to hydrolyze our substrate. The enzyme MW 45,000, is inhibited by Trasylol but not by soya bean trypsin inhibitor (SBTI). It is reactive with and is inhibited by antibodies prepared against pure HUK.

The first protein-binding assay is described for the direct determination of kallikrein in rat urine. Isolation of urinary kallikrein and preparation of its specific antibody have been previously described. Outlined here are the methods for 3H-labelling of kallikrein, for isolation of 3H-labelled immunoreactive enzyme, and for separation of free and antibody-bound kallikrein with the aid of double antibody. Assay and equilibrium conditions were characterized and a protocol is presented for the measurement of kallikrein concentration. The direct assay is sensitive, accurate and it correlates well (r = 0.81) with a functional assay. The assay may hold promise to determine catalytically active and inactive enzyme in urine, tissues and biological fluids.

We studied the effect of ions on the ability of purified human urinary kallikrein to cleave its natural substrate (kininogen) as well as two synthetic substrates, tosylarginine [3H]methyl ester and Pro-Phe-Arg-[3H]benzylamide. The kininogenase activity of kallikrein is markedly dependent upon the concentration of cations in vitro. Kininogenase activity is very low when measured in a low electrolyte buffer. The addition of cations to the reaction mixture increases activity by up to 27-fold. Maximum activity is achieved with 100 mM sodium, 100 mM potassium, or 20 mM magnesium. The activity is stable at higher concentrations of cation. Renal kallikrein is believed to act within distal tubular fluid in vivo. The concentration of cations in this fluid varies widely in response to alterations in salt and water metabolism. Thus, the relationship of kininogenase activity to the concentration of cations demonstrated in vitro may be relevant to the activity of kallikrein at its presumed site of action in the kidney. In separate experiments, we evaluated the effect of ions on the amidase and esterase activities of kallikrein which are the basis of several assays in routine use for physiological studies. In contrast to their stimulatory effect on kininogenase activity, cations inhibit amidase and to a lesser extent esterase activity. Additional studies indicate that urinary cations probably account entirely for the well known ability of normal urine to inhibit the amidase and esterase activities of kallikrein.

The distribution of kininogenase activity in the dog kidney was studied. It was found that kininogenase decreased from the outer to the inner cortex and that the medulla and papilla have very little kininogenase activity. Kininogenase activity was also measured in isolated glomeruli devoid of juxtaglomerular apparatus and in the total cortex of rat kidney. In the isolated glomeruli, the kininogenase activity per milligram of protein was only 15% of that found in the total cortex. It was concluded that the main localization of these enzymes is not in the glomeruli, but somewhere else in the cortical segment of the nephron.

Two human urinary kallikreins (fractions A-1 and A-2) were purified to apparently homogeneous forms. The two kallikreins were separated by affinity chromatography using Trasylol (aprotinin) covalently bound to Sepharose. The kallikreins were eluted with a pH gradient (pH 9.5-3.0). Fraction A-1 was eluted between pH 6.2 and 4.2 and fraction A-2 was eluted between pH 4.2 and 3.1. Final purification was obtained by chromatography on Sephacryl SS-200. Antibodies prepared against fraction A-2 were also reactive with fraction A-1; thus it may be possible to measure both by radioimmunoassay using the same antibody preparation.

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.

In clinical cases of cholera Vibrio cholerae grows, multiplies and undergoes lysis in the intestine. Although there are numerous works on the enzymatic make-up of V. cholerae, there is hardly any literature on the effect of different gastrointestinal enzymes on the toxin in bringing about the signs and symptoms of the disease. According to one school of thought, cholera toxin plays an important role in the pathogenesis of the disease.The effects of gastrointestinal enzymatic extracts of rabbit and pure gastrointestinal enzymes on the toxin were studied. The resultant degraded products were tested for their ability to produce cholera in young rabbits.When the toxin was treated with adult-rabbit enzymatic extract, it was degraded and the choleragenic property was lost, but this was not true of toxin treated with young-rabbit enzymatic extract. This might explain the resistance of the adult rabbit to the infection. The choleragenic property of the toxin was also lost when it was treated with lipase of phosphorylase but not with trypsin or pepsin. The lipopolysaccharide portion of the toxin was shown to be important from the point of view of pathogenesis.