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The effect of changing extracellular pH (pHo) on intracellular pH (pHi) in mesenteric arterioles of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats was investigated using the fluorescent indicator seminaphthorhodafluor c-SNARF-1 to measure pHi. Although the pHi of arterioles from WKY varied directly with pHo, that of SHR arterioles varied inversely. This abnormal pHi regulation in SHR was corrected by 50-microM 5-(N-ethylisopropyl)amiloride (EIPA). Vmax of H+ transport in response to changing extracellular Na+ was higher in SHR than in WKY, but Km did not differ. The Hill coefficient for H+ transport with respect to pH; was 1.69 in prehypertensive and 1.56 in hypertensive SHR. These results indicate that the Na+/H+ exchanger is particularly hyperactive in mesenteric arterioles of SHR.

Renal kallikrein is localized in the connecting tubule cells and secreted into the tubular fluid at late distal nephron segments. The present experiments were performed to further test the hypothesis that renal kallikrein reduces bicarbonate secretion of cortical collecting duct (CCD). The effect of orthograde injections of pig pancreatic kallikrein (1 or 3 micrograms/ml) into the renal tubular system was investigated. Urine fractions (Fr) were collected after a 2-min stop flow. Changes in the urine fraction with respect to those in free-flow urine samples (Ff) were related to the respective polyfructosan (Inutest) ratio. Renal kallikrein activity (Fr:Ff kallikrein/ Fr:Ff polyfructosan) increased significantly in the first two urine fractions collected after glandular kallikrein administration (kallikrein, 1 microgram/ml, P < 0.05; kallikrein, 3 micrograms/ml, P < 0.01). HCO3- secretion of collecting ducts was significantly reduced dose dependently by orthograde and also reduced by retrograde pig pancreatic kallikrein administration. Release of kinins into the fractions was not affected by the retrograde kallikrein injection, even though the kallikrein activity increased considerably (2.26 +/- 0.2 vs. 1.55 +/- 0.2, P < 0.05). Adequacy of retrograde injections for delivering substances to the CCD was demonstrated by injecting colloidal mercury and detecting the appearance of this mercury in the renal cortex by transmission electron microscopy. The integrity of the renal tissue after a retrograde ureteral injection was confirmed by scanning electron microscopy. These results confirm and extend previous data (M. Marin-Grez and P. Vallés. Renal Physiol. Biochem. 17: 301-306, 1994; and M. Marin-Grez, P. Vallés, and P. Odigie. J. Physiol. 488: 163-170, 1995) showing that renal kallikrein reduces bicarbonate secretion at the CCD, probably by inhibiting HCO3- transported by a mechanism unrelated to its kininogenase activity. Support for this assessment was obtained in experiments testing the effect of kallikrein on the luminal bicarbonate secretion of a subpopulation of Madin-Darby canine kidney cells capable of extruding the anion. Kallikrein inhibited HCO3-/Cl- exchange, and the degree of inhibition was dose dependent. This inhibition occurred in the absence of kininogen in the bathing solution.

The present experiments were performed to investigate whether renal kallikrein release by isolated perfused rat kidneys correlates with acid-base-related parameters. Kallikrein excretion per millilitre of glomerular filtrate was inversely correlated with perfusate pH (r = -0.49, P < 0.001) and HCO3- concentration (r = -0.46, P < 0.005). A direct relationship between kallikrein excretion per millilitre of glomerular filtrate and urinary Na+/K+ ratio was found (r = 0.59, P < 0.001). Some 86% of the variability (F ratio 110, P < 0.00001) of urinary kallikrein activity was attributable to the perfusate pH and the urinary cation ratio. Therefore, urinary kallikrein activity was highly correlated with perfusate H+ activity corrected by the urinary Na+/K+ ratio (r = 0.92, P < 0.0001). Kallikrein secretion into the distal tubular fluid appears to be regulated by blood H+ activity, and modulated by factors that affect the distal Na+ and K+ handling. The HCO3 - excretion rate was inversely correlated with the urinary kallikrein activity (r = -0.62, P < 0.001). This finding confirms previous data from the author's laboratory showing a kallikrein involvement in the regulation of HCO3- secretion in rats and rabbits. Kallikrein probably transduces the sensing of interstitial fluid H+ activity by the connecting tubule cells into an appropriate translocation of HCO3- or H+ to the tubular lumen by the intercalated cells.

The effect of altering the acid-base status on urinary kallikrein excretion of barbiturate-anaesthetized rats was investigated. Alkalosis was induced in a group of rats by intravenous (i.v.) infusion of NaOH at 0.45 mmol x h(-1) for 30 min. Acidosis was induced in two groups of rats by i.v. infusion of HCl at 1.5 mmol x h(-1) for 30 min (uncompensated acidosis) or 0.15 mmol x h(-1) for 3 h (compensated acidosis), respectively. Time controls received 0.45 mmol x h(-1) NaCl. Rats with alkalosis excreted less kallikrein than their controls (P < 0.05). Rats with uncompensated acidosis excreted more active kallikrein (P < 0.05), whereas rats with compensated acidosis excreted similar amounts when compared with their respective controls. In rats with uncompensated acid-base derangements, the urinary kallikrein excreted per millilitre of glomerular filtrate was correlated with blood H+ activity (r = 0.99, P < 0.01). Arterial blood pressure, haematocrit, glomerular filtration rate, urine flow rate and Na+ and K+ excretions of experimental and control animals did not differ. Thus, renal kallikrein secretion into the tubular fluid appears to be regulated by blood proton activity. This, along with our previous demonstration that kallikrein inhibits HCO3- secretion into the tubular lumen (Renal Physiol 17:301-306, 1994; J Physiol (Lond) 488:163-170, 1995), indicates that this enzyme is part of a feedback loop regulating acid-base balance.

To investigate whether or not MDCK cells may be used as a model for beta-intercalated cells, we studied: (1) The effect of luminal [Cl-]0 changes on pHi measured by video-imaging micro-fluorometry, (2) the influence of the inhibitor 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) on anion-exchange activity, and (3) the effect of acetazolamide on intracellular pH-indicator (c-SNAFL-2) accumulation and anion-exchange activity. At least three different modes of fluorescence accumulation were found in confluent monolayers: cells with high, low or undetectable fluorescence. Highly fluorescent cells responded to a rise of [Cl-]0 (30-140 mM) with a proportional decrease of pHi (7.6-6.4). Acetazolamide (10(-4) M) completely blocked the acidifying effects of the increased [Cl-]0, indicating that HCO3- is the intracellular ion exchanged for extra-cellular Cl-. Acetazolamide caused a reduction of SNAFL-2 fluorescence suggesting that carbonic anhydrase activity contributes to indicator accumulation. The high DIDS concentration (50 microM) required to prevent intracellular acidification suggests that the exchanger involved is identical to that present in beta-intercalated cells. All cells of non-confluent monolayers were highly fluorescent and expressed Cl-/ HCO3(-)-exchanger activity. In conclusion, highly fluorescent MDCK cells in confluent monolayers have a luminal DIDS inhibitable, carbonic anhydrase dependent Cl-/HCO3(-)-exchanger, and may therefore be used as a model for beta-intercalated cells.

Lithium salts are widely used agents for the prophylactic treatment of affective disorders. Lithium salts may be associated with distal nephron dysfunction. Kallikrein is a protease which is generated by the distal nephron. We used an amidolytic assay of chromatographically purified enzyme to determine the urinary excretion rate of active kallikrein in relation to lithium treatment. All plasma lithium concentrations were within the therapeutic range (0.4 to 0.9 mmol/liter). In 15 patients the urinary excretion rate of active kallikrein was 267.4 +/- 65.6 mU/24 hrs before lithium treatment, and fell to 117.8 +/- 39.6 mU/24 hrs (P less than 0.05) on day 14 of lithium treatment. This reduction was associated with a decrease of immunoreactive kallikrein in the same urines by 66%. In another 15 patients who had undergone lithium therapy for an average period of 5.6 years, the urinary excretion rate of active kallikrein was 86.1 +/- 14.5 mU/24 hrs, while 21 age-matched healthy controls had an excretion rate of 364.1 +/- 58.4 mU/24 hrs (P less than 0.05). Measurements of immunoreactive kallikrein in the same urine samples demonstrated a reduction of kallikrein after long-term lithium treatment by 78%. These observations could not be attributed to changes in creatinine clearance, renal sodium or potassium excretion rates or plasma concentrations of aldosterone and vasopressin. Addition of lithium to the urine in vitro had no demonstrable effect on kallikrein measurement by amidolytic assay. We conclude that lithium in therapeutic plasma concentrations may directly suppress the secretion of kallikrein by renal connecting tubule cells.

Dihydralazine (0.1 mg/kg), injected intravenously into male Sprague-Dawley rats, caused a decrease in mean arterial blood pressure and an increase in renal plasma flow, while urine volume remained unchanged. Dihydralazine had no effect on kallikrein excretion in the urine and on kallikrein activity in the renal cortex. No correlation was found between renal kallikrein and either renal plasma flow or mean arterial blood pressure. The excretion of kinins in the urine rose markedly after the administration of dihydralazine; no correlation between urinary kinins and urinary or renal kallikrein was observed. Dihydralazine had no influence on the kininogen content of blood-free renal cortex. The enzymatic activity of kininase II in renal cortex was not impaired by dihydralazine. It is suggested that the increased formation of kinins within the kidney could be involved in the vasodilating and blood pressure lowering effect of dihydralazine.

1. Rats were made hypertensive by ligating the aorta between the origins of both renal arteries. Sham-operated animals served as controls. Urinary and renal kallikrein activities, as well as plasma and renal renin activities, were measured 8 and 90 days after surgery. 2. Blood pressure was 155 +/- 6 mmHg on day 8 after aortic ligature and 142 +/- 6 mmHg on day 90; in controls pressures were 107 +/- 3 and 110 +/- 5 mmHg respectively. 3. Eight days after aortic ligature, kallikrein activity in the ischaemic kidneys was about 6.5 times, and in the non-ischaemic kidneys almost 2 times, that in controls. After 90 days the kallikrein activity was reduced to one-half of that in the controls in the ischaemic kidneys and it was normal in the contralateral. 4. The urinary kallikrein excretion of hypertensive rats was about one-third of that of the controls at both 8 and 90 days after aortic ligature. 5. The plasma renin activity in hypertensive rats was approximately seven times that in control animal 8 days after aortic ligature and did not differ from the control value after 90 days. Renin activity in the kidneys showed the same pattern as in other models of renovascular hypertension: elevation in the ischaemic kidney and reduction in the non-ischaemic one.

The effect of adrenal steroids (mineralo- and glucocorticoids) as well as that of the adrenocorticotrophic peptide tetracosactide (beta 1-24 corticotropin) on the renal kallikrein activity and on the urinary kallikrein excretion of rats was investigated. After the animals had been adapted to metabolic cages, they were injected with deoxycorticosterone acetate (15 mg/kg day), corticosterone (40 mg/kg day), both steroids combined or the vehicle (sesame oil). Additional groups of rats received tetracosactide (0.05, 0.1 or 0.2 mg/day) or the vehicle (100 microliter of 38 X 10(-3) M ZnCl2). After four days of treatment the urinary kallikrein excretion was higher in deoxycorticosterone-treated rats than in their controls. This increase was prevented when corticosterone was administered simultaneously. The renal kallikrein activity of corticosterone as well as that of deoxycorticosterone plus corticosterone-treated rats was subnormal. A dose-related reduction of both the renal kallikrein activity and the urinary kallikrein excretion was observed 2 days after starting the tetracosactide administration. It may be concluded that a stimulation of the endogenous release of glucocorticoids in the rat reduces the renal kallikrein activity and that glucocorticoids can prevent the stimulating effect of mineralocorticoids.

In male Sprague-Dawley rats the influence of salt loading (1% NaCl), deoxycorticosterone acetate (2 X 15 mg/kg/day resp. 250 mg/kg sc), corticosterone (2 X 20 mg/kg/day sc) and adrenocorticosterone (0.5 mg/kg/day tetracosactid sc) on the activity of renal kallikrein and renal renin activity was investigated. Salt loading lowered renal kallikrein activity, deoxycorticosterone stimulated its activity and in combination they had no effect on renal kallikrein activity. The time course of kallikrein stimulation by deoxycorticosterone showed no relationship to the escape phenomenon of the kidney from the sodium retaining effect of the mineralocorticoid hormone. Reduction of endogenous mineralocorticoid hormones by adrenalectomy caused a marked reduction of urinary and renal kallikrein activity. Corticosterone suppressed the activity of the renal kallikrein-kinin system at the same time as the reduction in urinary aldosterone excretion. Adrenocorticotrophin caused the same decrease in the activity of renal kallikrein as corticosterone. Urinary aldosterone excretion, however, was significantly stimulated. Thus, the known positive correlation between kallikrein and aldosterone was missing. In all experiments the urinary excretion of kallikrein correlated highly with the kallikrein activity measured in renal cortical tissue. However, no correlation was found between kallikrein and urine volume or urinary excretion of sodium and potassium. In our experiments no relationship between the activity of the renin-angiotensin system and that of the renal kallikrein-kinin system was observed. Furthermore, no clear relationship was found between systemic blood pressure and the activity of the renal kallikrein-kinin system.

The present experiments were undertaken to assess the effect of an atrial extract (ANF) and of the synthetic atriopeptin II (APII) on filtration pressure of rat kidneys. Continuous recordings of stop flow pressure (SFP) were made to obtain an index of the change of glomerular capillary pressure produced by atrial peptides and its time course. Short-term infusion of ANF or APII increased SFP from 40.6 +/- 0.99 to 50.7 +/- 1.42 mm Hg (p less than 0.001) and from 44.0 +/- 1.28 to 52.7 +/- 1.75 mm Hg (p less than 0.001) respectively. The maximum response was achieved promptly. Return of SFP to control was slow: 20 minutes after termination of the infusion SFP was still elevated by 4.9 +/- 1.27 mm Hg (p less than 0.01). Tubule and stellate vessel pressures increased less than 2mm Hg, changes that were not significant. Arterial pressure fell 6 mm Hg (p less than 0.05). When arterial pressure was reduced by an aortic clamp to 85-90 mmHg prior to administration of APII the response of SFP was markedly blunted (from a mean increase of 9.0 +/- 1.07 mm Hg to 4.5 +/- 0.53 mm Hg). The increase of SFP probably reflects an increase of glomerular capillary pressure. The finding suggests that atrial peptides increase glomerular filtration rate at least in part by increasing filtration pressure.

Rat urine, even at a 1:10 final dilution in Tyrode's solution, stimulates contraction of the ureteral musculature in vitro. This effect can be ascribed to the presence of kallikrein or a kallikrein-like enzyme in urine. Isometric contractions of ureters were prevented by previous addition of aprotinin to the organ bath. Urine also lost its activity after inactivation of enzymes by heat or acid treatment.

In rats diuresis was produced by 1. furosemide (5 mg/kg/s.c.), 2. triamterene (50 mg/kg/p.o.), 3. mannitol (250 microliters, 30%) and 4. change (+ 35 mm Hg) in perfusion pressure in the isolated perfused rat kidney. In all experiments a prolonged increase in diuresis and natriuresis was effected. Urinary potassium excretion was markedly enhanced by furosemide and change in perfusion pressure, moderately increased by mannitol, and significantly depressed by triamterene. Urinary kallikrein excretion, however, showed in all of the experiments a biphasic course with an initial increase and secondary decrease. The increase of kallikrein excretion was observed only in the periods when urine flow started to increase. Thus, no correlation was found between kallikrein excretion and either urine volume or urinary sodium excretion. Kallikrein excretion correlated with potassium excretion in urine after furosemide, triamterene, and mannitol, however, it did not after increase in perfusion pressure. In renal cortical tissue kallikrein activity was reduced after all experiments, independently of the mechanisms, diuresis was induced.

The localization of renin, converting enzyme (CE) and angiotensin II (ANG II) in the kidneys of rats and mice was investigated with immunocytochemical methods. According to the presence and specific intrarenal localization of these components of the renin-angiotensin-system (RAS) our results suggest that in addition to the well known systemic effects of the RAS, there are interactions of its components inside the kidney. These interactions may lead to the generation of an extra portion of ANG II in the renal blood stream with its target cells determined by the localization of CE at the luminal side of well defined endothelial areas. These intrarenal-intravasal reactions may or may not reinforce the action of "systemic" ANG II, generated prerenally. In addition, the existence of true intrarenal-interstitial interactions, with the different components and actions of this intrarenal RAS restricted entirely to the kidney is suggested by our results, particularly the demonstration of ANG II within epitheloid cells and the dissociation of systemic renin and ANG II from their local concentrations in renal hypertensive rats.

The effect of atrial natriuretic peptide (ANP) on renal kallikrein release was investigated in barbiturate anesthetized rats. ANP injections in a pharmacological dose (1 microgram i.v.) increased kallikrein excretion for about 15 min. The effect of ANP on kallikrein excretion was abolished when amiloride (2.5 mg i.v.) was administered prior to the injection of the peptide. Intravenous infusion of ANP (1 or 5 micrograms/h) or of isotonic saline (2 or 4 ml/h) did not alter urinary kallikrein output. When the ANP infusion was discontinued, the excretory rate of the enzyme tended to decrease. This decrease was prevented by the administration of 0.1 mg/h desoxycorticosterone-glucoside. A transient increase in kallikrein excretion, coincident with the appearance of diuresis, was observed during ANP infusion in the mineralocorticoid-treated animals. The rate of kallikrein release into the incubation medium by rat kidney slices was not affected by the presence of ANP (10(-9)M). These results can be interpreted as effects following an increased distal sodium delivery rather than a direct effect of ANP on connecting tubule cells (CNT cells).

Kallikrein activity in urine and in the renal cortex of the isolated rat kidney, perfused at constant pressure, decreased, while renal perfusate flow, glomerular filtration rate, excretion of sodium and of potassium and urine flow remained unchanged. Abrupt changes in perfusion pressure resulted in corresponding changes in renal perfusate flow, glomerular filtration rate, excretion of sodium and of potassium and urine flow. Urinary kallikrein increased little and transiently only, each time perfusion pressure was elevated. At the end of the experiments, a marked decrease in kallikrein activity in the renal tissue was found. From these results, it might be concluded that the excretion of kallikrein from the isolated perfused rat kidney resulted in a marked depletion of kallikrein stores in the kidney, probably due to an insufficient kallikrein synthesis.

The activity of the renal kallikrein-kinin system was investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI); Long-Evans rats (LE) were taken as controls. In the rats with DI, urinary kallikrein excretion was lower (P less than 0.05) than in the LE rats. However, when related to total renal mass or to body weight, there was no difference between the two strains. Kallikrein activity in the renal cortex was similar in the Brattleboro and the LE rats. Antidiuretic hormone (vasopressin tannate) in a dose of 100 mU given once daily for 3 days had no effect on urinary kallikrein excretion in either of the strains. Water deprivation for 24 h resulted, also in both strains, in a similar reduction in urinary kallikrein excretion. The renal kallikrein-kinin system of LE rats and that of DI rats does not principally differ in basic activity, nor in response to the administration of vasopressin, nor to water deprivation.

Atrial natriuretic peptide (ANP) can be extracted from rat hearts, and is found to increase fluid excretion by the kidneys when injected into test animals. The mechanism of ANP action is still unclear. ANP may reduce sodium reabsorption in the renal tubules, but it is also known that it increases the rate of glomerular filtration in the kidney, and relaxes preparations of smooth muscle, including one made from arteries that supply the kidney. To clarify its mode of action, we have studied directly the effects of semi-purified and synthetic ANP on blood vessels in the kidney of anaesthetized rats. We found that ANP causes a vasodilatation of the blood vessels which supply the glomeruli and a vasoconstriction of the arterioles which drain them. This substantiates the finding that increased filtration pressure participates in the natriuretic response.