Amiloride inhibits mammalian renal kallikrein and a kallikrein-like enzyme from toad bladder and skin.

Renal kallikrein is localized in luminal plasma membranes of the mammalian distal nephron and gains access to urine from this site. Its activity is regulated, in part, by aldosterone. These facts led us to study the effects of amiloride, a drug known to inhibit sodium reabsorption and potassium secretion at this site, on kallikrein activity. Amiloride inhibited the esterolytic activity of purified rat or human urinary kallikrein or of rat renal cortical cells upon a synthetic substrate (ID50 = 0.12-0.23 mM). Kinetic analyses showed that the enzyme inhibition was noncompetitive and reversible in nature. The kinin-generating activity of kallikrein acting upon kininogen substrates was also inhibited by amiloride, as measured by bioassay in the rat uterus of guinea pig ileum or by radioimmunoassay of liberated kinins (ID50 = 85 microM). No other diuretic drug tested inhibited kallikrein activity, except triamterene, which did so, weakly. In addition, kallikrein-like enzyme activity was discovered in the urinary bladder or skin of Bufo marinus toads and this activity was also inhibited by amiloride. The localization of the enzyme and its inhibition by this drug suggest that further study of relationships amongst the glandular kallikrein-kinen system and renal ion and water transport is warranted.

Abnormal regulation of renal kallikrein in experimental diabetes. Effects of insulin on prokallikrein synthesis and activation.

The effects of streptozotocin (STZ) diabetes and insulin on regulation of renal kallikrein were studied in the rat. 1 and 2 wk after STZ injection, diabetic rats had reduced renal levels and urinary excretion of active kallikrein. Tissue and urinary prokallikrein levels were unchanged, but the rate of renal prokallikrein synthesis relative to total protein synthesis was reduced 30-45% in diabetic rats. Treatment of diabetic rats with insulin prevented or reversed the fall in tissue level and excretion rate of active kallikrein and normalized prokallikrein synthesis rate. To further examine insulin's effects, nondiabetic rats were treated with escalating insulin doses to produce hyperinsulinemia. In these rats, renal active kallikrein increased. Although renal prokallikrein was not increased significantly by hyperinsulinemia, its synthesis was increased. As this was accompanied by proportionally increased total protein synthesis, relative kallikrein synthesis rate was not changed. Excretion of active kallikrein was unchanged, but prokallikrein excretion was markedly reduced. Therefore, increased tissue active kallikrein seen with hyperinsulinemia can be explained not only by increased synthesis but also by retention and increased activation of renal prokallikrein. These studies show that STZ diabetes produces an impairment in renal kallikrein synthesis and suggest that this disease state also impairs renal prokallikrein activation. The findings also suggest that insulin modulates renal kallikrein production, activation, and excretion.

Studies on rat renal cortical cell kallikrein. I. Separation and measurement.

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained. Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83-108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6-10(-2) to 4.6 - 10(-2) alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase units per 10(8) cells. However, cells incubated in a nutrient medium at 37 degrees C for 3-8 h contained no measurable kallikrein activity, whereas the surrounding medium had kallikrein activity which could be significantly increased by aldosterone and decreased by spironolactone.

Studies on rat renal cortical cell kallikrein. II. Identification of kallikrein as an ecto-enzyme.

Suspensions of viable renal cortical cells hydrolyzed a synthetic ester substrate (alpha-N-tosyl-L-arginine methyl ester, Tos-Arg-OMe) and generated kinins from a kininogen substrate. This kallikrein-like esterase activity increased linearly with cell number, or time of exposure to substrate. No radiolabelled substrate or product was found within the cells. Most of the activity appeared to be on cell surfaces as supernatant media had less than 20% of the Tos-Arg-OMe esterase activity on the cell suspensions. Cell surface Tos-Arg-OMe esterase activity was inhibited by aprotinin, benzamidine, pentamidine, and a tris-amidine derivative (alpha,alpha',alpha''-tris(3-amidinophenoxy)mesitylene). Preincubation of cells with phospholipase A2 increased renal cell surface esterase activity up to 76% while only slightly increasing supernatant activity. In contrast, preincubation with deoxycholate caused clearing of suspensions and a marked increase in supernatant esterase activity. Renal cell kininogenase (EC 3.4.21.8) activity was inhibited by preincubation with aprotinin, the tris-amidine derivative, or anti-rat urinary kallikrein antibody. Kallikrein elaborated by renal cells formed a single precipitin line with an antibody to rat urinary kallikrein but the two enzymes were not immunologically identical. We conclude that kallikrein's active sites are facing the external environment of renal cortical cells in suspension with access to substrates, inhibitors, and antibody.

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography.

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0. Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC50 values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [14C]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by 125I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.

Kallikreins and kinins. Molecular characteristics and cellular and tissue responses.

Kallikrein-kininogen-kinin systems are now topics of widespread interest. The long-standing appreciation of their diverse pharmacological properties and biochemical characteristics is being supplemented by modern definitions of their cellular receptors' signal-transduction mechanisms and physiological and pathological roles. The assignment of important homeostatic responsibilities for kinins, including those in autocrine and paracrine signaling for skeletal and cardiac muscle energy metabolism, is now subject to definitive experimental evaluation via the availability of better kallikrein inhibitors, specific kinin receptor antagonists, and techniques of genetic manipulation.

Renal kallikrein and hemodynamic abnormalities of diabetic kidney.

The relationship between renal hemodynamic abnormalities and renal kallikrein activity was studied in streptozocin-induced diabetic rats. Diabetic rats were either not treated with insulin and had plasma glucose levels greater than 400 mg/dl (severely hyperglycemic diabetic [SD]) or were treated with 1.5-1.75 U/day protamine zinc insulin and had glucose levels of 200-300 mg/dl (moderately hyperglycemic diabetic [MD]). In SD rats, kidney tissue level and excretion of active kallikrein were reduced after 3 wk compared with age-matched nondiabetic control rats (tissue, 11.7 +/- 1.9 vs. 20.5 +/- 1.8 ng/mg protein, P less than 0.005; urine, 126 +/- 12 vs. 179 +/- 10 micrograms/24 h, P less than 0.005). Despite increased kidney size, renal plasma flow (RPF) was reduced in SD rats (5.38 +/- 0.23 vs. 6.37 +/- 0.20 ml/min, P less than 0.05). Glomerular filtration rate (GFR) was not significantly lower (2.77 +/- 0.60 vs. 3.02 +/- 0.56 ml/min). In MD rats, kidney tissue level and excretion of active kallikrein were increased after 5 wk compared with age-matched nondiabetic control rats (tissue, 28.4 +/- 1.3 vs. 23.3 +/- 1.7 ng/mg protein, P less than 0.05; urine, 289 +/- 16 vs. 196 +/- 13 micrograms/24 h, P less than 0.001). In MD rats, GFR and RPF were increased (3.80 +/- 0.11 and 8.04 +/- 0.17 ml/min, respectively) compared with control rats (3.22 +/- 0.05 and 7.28 +/- 0.09 ml/min, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary and renal tissue kallikrein in the streptozocin-diabetic rat.

The renal kallikrein-kinin system is thought to participate in blood pressure regulation and displays abnormalities in human hypertension, as well as in many animal models of hypertension. Urinary excretion and tissue levels of renal kallikrein were measured in streptozocin (STZ)-diabetic rats in relation to blood pressure, glycemia, and insulin treatment. In study 1, STZ-diabetic rats with marked hyperglycemia showed reduced kallikrein-like esterase excretion, compared with control rats, when first measured after 7 days of diabetes (9.9 +/- 2.5 versus 17.5 +/- 2.4 EU/24 h, P less than 0.05). This difference increased with time and, after 210 days, urinary esterase excretion in diabetic and control rats was 6.7 +/- 2.1 and 39.0 +/- 6.0 EU/24 h, respectively (P less than 0.001). Urine kallikrein, measured by radioimmunoassay, was similarly reduced in diabetic rats (40.4 +/- 8.0 versus 88.0 +/- 6.5 micrograms/24 h, at 30 days, P less than 0.001). At 120 days, systolic blood pressures were elevated in diabetic rats (P less than 0.05), and at 180 days over 60% of the diabetic rats had pressures above the highest pressures of control rats. In study 2, STZ-diabetic rats were treated with insulin for 2 wk (2 U NPH at 0800 h, or 2 U NPH at 0800 and 1600 h). In the single-dose group, with hyperglycemia similar to that of diabetic rats in study 1, kallikrein excretion was reduced as early as day 2, compared with nondiabetic rats (56.0 +/- 6.1 versus 109 +/- 9.4 micrograms/24 h, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of testosterone, thyroxine, and cortisol on rat submandibular gland versus renal kallikrein.

The effects of 17 alpha-methyltestosterone (T alpha), T4, or cortisol (F) on tissue kallikrein in the rat submandibular gland and renal cortex were measured. Castrated male or normal female Sprague-Dawley rats were treated with T alpha or T4 for 2 weeks. In addition, F, T alpha, or T4 was given to adrenalectomized female rats for 2 weeks. In the submandibular glands of male rats, castration resulted in a significant reduction of both kallikrein-like alpha-N-tosyl-L-arginine methyl ester (Tos-Arg-OMe) esterase activity and immunoreactive kallikrein content. Treatment with T alpha or administration of T4 significantly increased Tos-Arg-OMe esterase activity and immunoreactive kallikrein over that in the castrated rats receiving vehicle. Both the Tos-Arg-OMe esterase activity and kallikrein content of the submandibular glands of normal female rats were increased significantly by T alpha or T4. However, T alpha or T4 either significantly reduced or had no effect on renal Tos-Arg-OMe esterase activity or kallikrein content in castrated male or normal female rats. Adrenalectomy had no effect on submandibular gland Tos-Arg-OMe esterase activity and kallikrein content in the female rat, but F or T alpha significantly increased submandibular gland Tos-Arg-OMe esterase activity and kallikrein content. These adrenalectomized female rats showed a marked increase in renal cortical Tos-Arg-OMe esterase activity and kallikrein content, and F, T alpha, or T4 markedly decreased both esterase activity and immunoreactivity. These data show that T alpha, T4, and F modulate both kallikrein-like activity and quantity, but in a generally opposite way in the rat submandibular gland vs. the kidney.

Tissue kallikrein in rat brain and pituitary: regional distribution and estrogen induction in the anterior pituitary.

We have detected tissue kallikrein and kallikrein mRNA in various brain regions with a kallikrein direct RIA and with nucleic acid hybridization using a kallikrein cDNA probe. In the direct RIA, rat urinary kallikrein-like activity was found in the pituitary and pineal glands, hypothalamus, cerebral cortex, cerebellum, and brain stem. Pituitary and pineal gland kallikrein concentrations were significantly higher than those in other regions. Only in pituitary was there a significant difference in tissue kallikrein concentration according to sex, with glands from female rats showing levels 4-fold higher than those from male rats. Kallikrein mRNAs were detected in all of the regions and were about 4-fold higher in female than in male pituitary gland. Northern blot analyses show sex dimorphism of pituitary kallikrein mRNA, similar in size to submandibular gland and kidney mRNA. In castrated male rats, whole pituitary kallikrein content was reduced to 50% of the control value and increased 1.7-fold with testosterone replacement and 18-fold with 17 beta-estradiol treatment. Neither T4 nor cortisol affected whole pituitary kallikrein levels in the castrated male rat, but testosterone decreased pituitary kallikrein in normal female rats by 35%. When anterior pituitary or neurointermediate lobe extracts were separately examined, immunoreactive kallikrein was 10.2- and 1.3-fold higher respectively, in female than in male rat lobes. Estradiol benzoate (30 micrograms/kg) administration increased kallikrein levels 90- and 22-fold, respectively, in the anterior pituitary of gonadectomized male and female rats, while it increased by only 40-50% kallikrein levels in the male and female neurointermediate lobe. In dot blot analysis, kallikrein mRNA levels were increased 5-fold by 17 beta-estradiol in the whole pituitary of castrated male rats. In the cytoplasmic dot hybridization analysis, estradiol benzoate treatment increased kallikrein mRNA levels 54-fold in the anterior pituitary of ovariectomized rats. The data show that a tissue kallikrein indistinguishable thus far from a urinary kallikrein is widely distributed in brain and pituitary and that levels of enzyme and mRNA are comparable in certain central sites. Kallikrein levels in the anterior and neurointermediate pituitaries are differentially regulated by estrogen.

Theodore Cooper Memorial Lecture. Kallikreins and kinins. Some unanswered questions about system characteristics and roles in human disease.

Kinins can affect many aspects of cellular function, but their roles in human homeostatic mechanisms and disease are just beginning to be understood. In this brief review, some of the interesting new observations about kallikrein-kinin system characteristics, roles in cell behavior, and aberrancy in diseases of relevance to readers interested in hypertension will be discussed. Along the way, questions raised by these observations will be posed. They show that we still have much to learn about the contributions of kinins to human cardiovascular diseases but now have in addition both a strong rationale for asking them and the tools to make them operational.

Effects of dietary potassium and race on urinary excretion of kallikrein and aldosterone in man.

Urinary excretion of kallikrein by 16 normal and 8 hypertensive subjects was studied at three levels of dietary potassium: 85 meq/day for 5 days, 185 meq/day for 7 days, and 25 meq/day for 10 days. Excretion of kallikrein varied directly with potassium intake and paralleled excretion of aldosterone in both normotensive and hypertensive subjects. Mean levels of excretion of kallikrein at 85, 185, and 25 meq intake of potassium were 10.8, 19.1, and 5.8 esterase U/day (EU/day), respectively, for the normotensive subjects and 8.8, 13.9, and 6.1 EU/day for the hypertensive subjects. Mean levels of excretion of kallikrein were significantly higher in white that in black subjects among normals and hypertensives [13.0 vs. 5.9 EU/day for normals (P less than 0.05) and 13.7 vs. 4.0 EU/day for hypertensives (P less than 0.05) on the 85 meq/day diet]. The parallel changes in excretion of kallikrein and aldosterone support the hypothesis that changes in effective levels of aldosterone induce changes in the excretion of kallikrein. Because of racial differences in excretion of kallikrein, matched groups should be used for comparisons of the kallikrein system in disease states.

The radioimmunoassay of human urinary kallikrein and comparisons with kallikrein activity measurements.

Human urinary kallikrein was purified to homogeneity, and an antiserum to it was raised in rabbits. A RIA was devised which uses this rabbit antiserum (Keq = 2.75 x 10(11) M-1) in a final dilution of 1:2,500,000 and the purified kallikrein labeled with 125I using a lactoperoxidase method. Assay sensitivity is 8 pg kallikrein. Thus far, the assay is specific for human and perhaps monkey urinary kallikrein. Correlations between this assay of immunoreactive kallikrein and the alpha-N-Tosyl-L-arginine-[3H]methylester (Tos-Arg-OMe) activity method or a kininogenase assay were highly significant (r = 0.94 and 0.96, respectively) and show that each assay measures human urinary kallikrein comparably. Low or high dietary sodium intakes, maneuvers known to change human urinary Tos-Arg-OMe esterase excretion, change immunoreactive kallikrein to an equivalent degree. Normal black children, already known to excrete significantly less Tos-Arg-OMe esterase than white children, excrete similarly reduced amounts of immunoreactive kallikrein. Kallikrein excretion in children with cystic fibrosis of the pancreas was not different from that in normal children. The data show that a specific and sensitive direct RIA for human urinary kallikrein has been developed and that both the Tos-Arg-OMe esterase and kininogenase assays measure human urinary kallikrein activity specifically, at least in the described circumstances.

Bradykinin B2 receptor modulates renal prostaglandin E2 and nitric oxide.

Bradykinin and lys-bradykinin generated intrarenally appear to be important renal paracrine hormones. However, the renal effects of endogenously generated bradykinin are still not clearly defined. In this study, we measured acute changes in renal excretory and hemodynamic functions and renal cortical interstitial fluid levels of bradykinin, prostaglandin E2, and cGMP in response to an acute intrarenal arterial infusion of the bradykinin B2 receptor antagonist Hoe 140 (icatibant), cyclooxygenase inhibitor indomethacin, or nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) given individually or combined in uninephrectomized, conscious dogs (n=10) in low sodium balance. Icatibant caused a significant decrease in urine flow, urinary sodium excretion, and renal plasma flow rate (each P<.001). Glomerular filtration rate did not change during icatibant administration. Icatibant produced an unexpected large increase in renal interstitial fluid bradykinin (P<.0001) while decreasing renal interstitial fluid prostaglandin E2 and cGMP (each P<.001). Both indomethacin and L-NMMA when given individually caused significant antidiuresis and antinatriuresis and decreased renal blood flow (each P<.001). Glomerular filtration rate decreased during L-NMMA administration (P<.001) and did not change during indomethacin administration. Combined administration of icatibant and indomethacin or L-NMMA caused significant decreases in renal excretory and hemodynamic functions, which were not different from changes observed with icatibant alone. The failure of icatibant to change renal function after inhibition of cyclooxygenase and nitric oxide synthase activity suggests that the effects of kinin B2 receptor are mediated by intrarenal prostaglandin E2 and nitric oxide generation. The increase in renal interstitial fluid bradykinin during icatibant requires further study of possible alterations in kinin synthesis, degradation, or clearance as a result of B2 receptor blockade.

Cerebrospinal fluid kinins and cardiovascular function. Effects of cerebroventricular melittin.

Biochemical and histochemical studies have identified components of the kallikrein-kinin system in the brain. The present study was designed to determine whether kinins could be detected in cerebrospinal fluid (CSF) and if these levels could be altered. Endogenous CSF samples were taken from the cisterna magna of pentobarbital-anesthetized mongrel dogs (n = 11) and were shown to contain 13 +/- 3 pg/ml (mean +/- SE) of immunoreactive kinin ( ikinin ) measured by RIA. The ( ikinin ) samples showed complete parallelism to standard synthetic bradykinin. Ventriculocisternal perfusion of the anesthetized dog brain with artificial CSF alone at a rate of 0.191 ml/min for 240 minutes had little or no effect on basal levels of CSF ( ikinin ), mean arterial pressure (MAP), or heart rate (HR). Melittin (20 microM), an activator of membrane-bound kallikrein, added to the perfusion system for 60 minutes caused a significant and sustained elevation in CSF ( ikinin ) levels from 19 pg/ml up to a maximum of 194 pg/ml (p less than 0.01). This change was accompanied by a prolonged increase in MAP of up to 22 mm Hg (p less than 0.01) and a transient increase in HR of 14 bpm (p less than 0.05). Melittin (2 microM) had no significant effect on CSF ( ikinin ) levels or MAP, but resulted in a sustained increase in HR of 17 to 25 bpm (p less than 0.01). The cardiovascular responses to centrally administered melittin (20 microM) were attenuated by concomitant administration of aprotinin (2000 KIU/ml). This study establishes the existence of ( ikinin ) in the CSF, shows that such levels can be manipulated, and suggests that central kinins may be involved in the modulation of cardiovascular function.

Characterization of monoclonal and polyclonal antibodies to human tissue kallikrein.

Monoclonal antibodies to purified human urinary kallikrein have been developed. Selection of antibody producing clones was based on 125I-kallikrein binding activity of hybridoma media in both radioimmunoassay and enzyme-linked immunosorbent assay. Three clones (2 IgG1, 1 IgG2b) were subcloned, characterized, and compared with the polyclonal antiserum generated in rabbits immunized with the purified kallikrein. With radioimmunoassay, mouse ascitic fluids or rabbit antisera dilutions showing 50% binding to 125I-kallikrein were 1:1.2 X 10(6) (E7A9), 1:1.2 X 10(5) (H6A6), 1:8.0 X 10(4) (E12H1), and 1:1.4 X 10(6) (the rabbit antisera). With enzyme-linked immunosorbent assay, mouse ascitic fluids from clones E7A9 and H6A6 showed half-maximal absorbance at dilutions of 1:2.1 X 10(5) and 1:1.0 X 10(5) respectively, and the polyclonal antiserum showed half-maximal absorbance at a dilution of 1:2.0 X 10(4). These monoclonal antibodies showed no cross-reactivity with rat tissue kallikrein, rat urinary plasminogen activator, or dog pancreatic kallikrein, while the polyclonal antiserum showed some cross-reactivity. The binding of monoclonal or polyclonal antibodies to 125I-human urinary kallikrein was not affected by human plasma kallikrein, thrombin, or urokinase in a competitive radioimmunoassay. By using purified human urinary kallikrein immobilized to agarose, antibodies produced by clones E7A9 and H6A6 and in the rabbit antisera were purified to homogeneity. Each of these affinity-purified antibodies inhibited the esterase activity, and two of the three inhibited the kininogenase activity, of human urinary kallikrein. A sandwich immunosorbent assay was developed to measure this kallikrein using monoclonal antibody from the clone E7A9 in conjunction with the polyclonal antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat renal interstitial bradykinin, prostaglandin E2, and cyclic guanosine 3',5'-monophosphate. Effects of altered sodium intake.

Kinins generated intrarenally probably affect renal function by altering levels of various mediators and messengers, including prostaglandin E2 (PGE2) and cyclic guanosine 3',5'-monophosphate (cGMP). Using a microdialysis technique, we monitored levels of cortical and medullary renal interstitial fluid kinins, PGE2, and cGMP after 5 days of 0.15% (low), 0.28% (normal), or 4.0% (high) sodium intake. Samples were collected from anesthetized rats (n = 5 for each diet). During normal sodium intake, renal interstitial fluid kinin, PGE2, and cGMP levels in dialysate leaving the cortex were 113 +/- 8 pg/min, 1.23 +/- 0.11 pg/min, and 0.05 +/- 0.004 pmol/min, respectively. In the fluid leaving the medulla, the levels were 93.0 +/- 17 pg/min, 2.28 +/- 0.14 pg/min, and 0.08 +/- 0.005 pmol/min, respectively. In rats consuming a low sodium diet, renal cortical interstitial fluid kinin and cortical and medullary PGE2 and cGMP appearance rates were significantly increased (P < .01). Rats consuming a high sodium diet showed renal cortical and medullary kinin levels that were decreased 100-fold (P < .01), whereas PGE2 and cGMP were increased (P < .01) compared with levels in rats with normal sodium intake. Renal interstitial fluid kinin is extremely sensitive to dietary sodium, but changes in interstitial fluid PGE2 and cGMP are not always directionally similar, suggesting different regulations of these substances in response to sodium intake.

Brain kallikrein-kinin system abnormalities in spontaneously hypertensive rats.

The objective of the present study was to determine whether the brain kallikrein-kinin system differs between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) and if so, whether any detected differences occur before the development of hypertension in SHR. We measured cerebrospinal fluid levels of various components of the system in adult and young prehypertensive SHR and WKY. Cerebrospinal fluid kinin concentration and appearance rate were higher in SHR. Cerebrospinal fluid active kallikrein level and kininogenase activity were also higher in adult SHR. In addition, cerebrospinal fluid kinin concentration and appearance rate were higher in prehypertensive, 5- to 6-week-old SHR compared with age-matched WKY. However, no differences in cerebrospinal fluid kallikrein or kininogenase activity were observed between the two strains of young rats. Cerebrospinal fluid kinin concentration was higher in young versus adult rats of the same strain. In WKY, cerebrospinal fluid kallikrein also decreased with age although cerebrospinal fluid kallikrein concentration did not decrease in young and adult SHR. Together, these data suggest that there is a hyperactive kallikrein-kinin system in the brain of SHR that may contribute to the hypertensive state in this animal model.

Human plasma inactive renin: purification and activation by proteases.

A new affinity chromatographic procedure was devised to purify inactive renin by using a selective hydrophobic interaction of inactive renin to octyl-Sepharose. Additional extensive purification was accomplished by immunoaffinity chromatography on antihuman renin immunoglobulin G-Sepharose. A trace amount of active renin was removed by chromatography on pepstatin-Sepharose. Human plasma inactive renin purified by this method was free from protease inhibitors and permitted the investigation of protease-mediated activation without the acid treatment which was used previously to remove inhibitors. Human plasma kallikrein, human plasmin, cathepsin B1, and arginine esteropeptidases associated with mouse epidermis growth factor and nerve growth factor were effective activators. Human urinary kallikrein, hog pancreatic kallikrein, and rat urinary esterase A were inefficient activators of low potency. Thrombin, factor Xa, factor XIIa, and urokinase did not activate inactive renin. The in vitro activation of 56,000-dalton inactive renin by these proteases was not accompanied by a recognizable reduction in molecular weight. Activation required plasma albumin, presumably as a protecting substance. These results suggest that human inactive renin can be activated by a minimum change in its molecular size.

Urinary kallikrein excretion in insulin-dependent diabetes mellitus and its relationship to glycemic control.

The renal kallikrein-kinin system is thought to be involved in vasoregulatory and epithelial ion-transporting processes. Renal kallikrein has not been studied in patients with diabetes mellitus, a disease in which abnormalities of renal hemodynamics and electrolyte handling occur. The urinary excretion of this kallikrein was measured in 20 type I diabetic patients and 10 normal subjects. On a 120-meq Na diet, daily kallikrein excretion, determined by both esterase activity and direct RIA, in 12 poorly controlled diabetic patients [hemoglobin A1c (HbA1c) = 14.2 +/- 0.5% (mean +/- SEM)] was significantly greater (P less than 0.05) than excretion in 8 diabetic patients in good to moderately good control (HbA1c = 9.4 +/- 0.5%) or in 10 normal subjects. In these groups, urinary esterase activities were 9.4 +/- 1.0, 6.1 +/- 1.4, and 6.7 +/- 0.5 esterase units/24 h, respectively. Corresponding excretion values of immunoreactive kallikrein were 171 +/- 14, 118 +/- 26, and 123 +/- 11 micrograms/24 h. Creatinine clearances were similar in the three groups. Urinary kallikrein was also measured in 8 diabetic and 8 normal subjects during 7 subsequent days of 10 meq Na intake. It increased less in diabetic patients than in normal subjects during Na depletion (P less than 0.02). The increase in urinary kallikrein in the diabetic patients was inversely related to their HbA1c levels (r = 0.88; P less than 0.01). The effect of glycemic control on urinary kallikrein excretion was determined in nine diabetic patients. Initial glycemic control was achieved using an artificial endocrine pancreas (Biostator) and was maintained by continuous sc insulin infusion with a portable pump. Before glycemic control, urinary kallikrein was 190 +/- 30 micrograms/24 h (by RIA). After 8-12 days of glycemic control, excretion fell to 144 +/- 23 micrograms/24 h (P less than 0.02). The abnormalities in kallikrein excretion in diabetic patients were not correlated with differences in water, electrolyte, protein, glucose, or aldosterone excretion in any of the studies. These results show that kallikrein excretion was increased in patients with poorly controlled insulin-dependent diabetes, and excretion rose less in diabetic subjects with low Na intake than in normal subjects. Strict glycemic control decreased urinary kallikrein excretion. These findings suggest that the renal kallikrein-kinin system is functioning abnormally in diabetes mellitus.