H. R. Croxatto

Urinary Kallikrein Excretion in a Spontaneously Hypertensive Strain of Rats

Summary Concentration and 24-hr excretion of urinary kallikrein in spontaneous hypertensive Wistar strain rats of both sexes obtained by selected inbreeding (25th generation) are significantly decreased as compared with the excretion in normotensive inbred rats (24th generation) descending from common ancestors. Apparently in these hypertensive rats there is an abnormal capacity of the kidneys to produce or release kallikrein, but more studies will be necessary to correlate this findings with blood pressure increase.

Release of renal kallikrein to the perfusate by isolated rat kidney.

The addition of furosemide to the fluid used to perfuse isolated rat kidney increases the kallikrein activity found in the perfusion fluid. The experiments favour the concept that furosemide activates a kallikrein precursor or/and the synthesis and release of kallikrein in the kidneys.

Inhibition of Atrial Natriuretic Peptide Excretory Action by Bradykinin

We examined whether the excretory effect of atrial natriuretic peptide could be antagonized by intravenously administered bradykinin or by elevated endogenous kinin levels attained during converting enzyme inhibition. Urinary volume and sodium and potassium excretion were determined every 20 minutes in female, anesthetized Sprague-Dawley rats (weight, 0.19 to 0.22 kg) infused with 10 microL/min isotonic glucose. In some experiments, urinary cGMP content was measured by radioimmunoassay. Two intravenous boluses of 209 pmol (0.5 micrograms) atrial natriuretic peptide were given before and after the injection of test substances, and the response ratio was used to quantify inhibition. Single injections of 94.3 or 142 pmol (100 or 150 ng) bradykinin, 3 minutes prior to atrial natriuretic peptide, inhibited the excretion of water, sodium, and potassium by 70%, 75%, and 50%, respectively. Larger (236 to 472 pmol) or smaller (23.6 to 47.2 pmol) bradykinin doses were ineffective. None of the bradykinin doses tested affected basal urinary output, systemic pressure, or the modest depressor effect of atrial natriuretic peptide. The anti-atrial natriuretic peptide effect of bradykinin was completely prevented by the kinin receptor antagonist Hoe 140. Converting enzyme inhibition with ramipril (96 nmol IV) also blunted atrial natriuretic peptide diuresis and natriuresis by 70% and reduced urinary cGMP excretion by 50%. These effects of ramipril were mediated by endogenous kinin accumulation, since they were abolished by pretreatment with Hoe 140. It is concluded that intrarenal kinins modulate the renal actions of atrial natriuretic peptide, and at a precise concentration bradykinin strongly antagonizes atrial natriuretic peptide by preventing its transduction mechanism.

Urinary kallikrein excretion in congestive heart failure

Abstract The renal kallikrein-kinin system has been implicated in the control of local renal blood flow, salt and water excretion and blood pressure regulation.1 Renal kallikrein is synthesized in the connecting tubule cells of the rat and human nephron. From here it may be secreted to the surrounding blood vessels or directly into the lumen of the tubule or to both.2 Kallikrein releases kinins from plasma substrates called kininogens. Kinins are potent vasodilators and have natriuretic and diuretic properties.1 Congestive heart failure (CHF) is characterized by an abnormal sodium and water retention and increased vascular resistance. The role of other vasoactive systems such as renin-angiotensin, prostaglandins and cathecolamines has been extensively studied. However, there is no information regarding participation of the renal kallikrein-kinin system in CHF. We determined urinary kallikrein in patients with CHF in different functional classes and we analyzed its relation with indexes of renal function and with other vasoactive systems such as plasma renin activity and circulating cathecolamines.

A Peptide Released by Pepsin From Kininogen Domain 1 Is a Potent Blocker of ANP-Mediated Diuresis-Natriuresis in the Rat

A 20-amino acid peptide, KYEIKEGDCPVQSGKTWQDC (PU-D1), released by pepsin hydrolysis of LMW kininogen domain 1 was tested for its ability to antagonize the diuretic and natriuretic effect of ANP(103-125) in anesthetized rats. A single dose of 10.8 or 21.6 pmol (25 or 50 ng) PU-D1 given intravenously or into the duodenal lumen suppressed the diuresis-natriuresis induced by 209 pmol (500 ng) ANP by 43% to 59% and 69% to 96%, respectively. None of the doses tested (2.16 to 432 pmol, 5 ng to 1 microg) modified systemic blood pressure. Strikingly, a single IV dose of 10.8 pmol PU-D1 blocked the action of ANP for more than 3 hours. ANP blockade by PU-D1 was annulled completely by the bradykinin (BK) B2 receptor inhibitor Hoe 140. On a molar basis, PU-D1 is more effective than BK and kinins of 15, 16, and 18 amino acids for blocking the ANP-mediated diuresis-natriuresis. As with BK and other kinins, the inhibitory effect of Pu-D1 on ANP is obtained only within a small range of picomol doses. A single dose of 2.16 or 4.32 pmol PU-D1 or 47 pmol (50 ng) BK is ineffective against ANP if injected alone. However, when both substances are administered concomitantly at these subthreshold doses, they totally suppress ANP-induced diuresis-natriuresis. These results raise the question of whether PU-D1, released from kininogen domain 1, either alone or associated with BK, may interact with ANP in the regulation of urinary water and electrolyte excretion in physiological and pathological conditions.

A Fragment of Human Kininogen Containing Bradykinin Blunts the Diuretic Effect of Atrial Natriuretic Peptide

Abstract A synthetic 15 amioacids kinin, named PU-15, is able to block the diuretic natriuretic action of Atrial Natriuretic Peptide (ANP). The structure of PU-15 is Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Ser-Arg-lso, having the aminoacid sequence of a fragment of human kininogens. The increase in the urinary excretion of sodium, potassium, and water, elicited by a bolus of 0.5 μg of ANP in anesthetized rats, is blocked by PU-15 (100-150 ng) given either intravenously 3 min before ANP injection, or injected intraperitoneally or in the duodenal lumen, 40 min before ANP. This ANP blockade, which mimics the action of pepsanurin, is only obtained with doses of PU-15 in a narrow range around 100 picomol/rat, and do not modify blood pressure. Larger doses, 2- to 8-fold the effective dose, either do not change the response to ANP or raise the excretion of sodium and water. The administration of HOE-140, a bradykinin B2 receptor blocker, prior to PU-15, completely abolishes the anti-ANP action of PU-15. These findings lend support to the proposal that kinins released from the intestinal tract during prandial period can modulate renal excretory function.

Adrenergic Receptor Blockers on Pressor Activity of Incubated Human Plasma

Summary In normal, nephrectomized or adrenalectomized rats the injection of azamethonium (ganglionic blocking agent) increases the pressor effect of incubated plasma or its active fraction (VA) and also that produced by angiotensin and bradykinin. However the pressor activity of incubated plasma or VA given by a single injection or infusion is: (a) not modified by adrenalectomy, and (b) not inhibited by the previous injections of α or β adrenergic receptor blocking agents, such as phentolamine and propranolol, respectively. These results rule out that the pressor effect is produced by the liberation of catecholamines. Since phentolamine alone or after azamethonium reduces the pressor effects of angiotensin and markedly inhibits or abolishes the pressor action of bradykinin, the conclusion can be drawn that the pressor effect of plasma and VA is not due to the release of angiotensin or bradykinin. The results of the gel filtration and electrophoresis of incubated plasma are in agreement with the view that VA is an albumin or an albumin bound molecule.

Vasoactive Substances in Blood and Urine of DCA Hypertensive Rats

The effect of intravenous injection of rat anephrotensin on rats treated with DCA for periods of up to 30 weeks was studied. It was found that in these animals, anephrotensin raises blood pressure, whereas in controls it acts as a depressor. Blood serum of DCA-treated rats yielded greater anephrotensin activity after acid incubation than did that of controls. Treated animals excreted, through the kidneys, more anephrotensin-like substance than controls. The pressor effect of the homogenized kidneys of these animals was less than that of controls; this is attributed to decrease in renin. Higher anephrotensin yield and the pressor response to anephrotensin were found to occur previous to the onset of hypertension; this suggests a causal relationship with the hypertension-provoking mechanism. The observations point to a similarity between DCA-treated and nephrectomized rats.

Kinin B2 Receptors Mediate Blockade of Atrial Natriuretic Peptide Natriuresis Induced by Glucose or Feeding in Fasted Rats

We have shown previously that the kininogen-derived peptides bradykinin, prokinins, and PU-D1, given intravenously or into the duodenal lumen, block the atrial natriuretic peptide (ANP)-induced diuretic-natriuretic effect in fasting, anesthetized rats infused with isotonic glucose. HOE-140, an inhibitor of bradykinin B2 receptors, completely suppresses this ANP blockade. When intravenous glucose infusion is omitted, the above-described inhibition of ANP does not take place. Therefore, to clarify the role of glucose and/or feeding in this phenomenon, we used fasted, anesthetized rats to test how the ANP excretory response was affected by (1) short-term feeding before anesthesia, (2) 1 mL of isotonic glucose introduced into the stomach, and (3) the interaction of HOE-140 with these treatments. In addition, we tested the effects of 1 mL of intragastric glucose administration and HOE-140 on urinary excretion in awake rats. In anesthetized rats, both glucose administration and feeding significantly inhibited the diuretic-natriuretic effect of ANP for up to 90 minutes. Similarly, intragastric glucose delayed spontaneous sodium and water excretion for 90 minutes in awake rats. In all 3 cases, pretreatment with HOE-140 (2.5 microg IV) fully prevented the inhibition of ANP excretory action, ruling out osmotic effects as the cause of reduced diuresis. These results indicate that the presence of glucose in the digestive tract triggers an inhibitory effect on ANP renal actions that requires activation of kinin B2 receptors, providing strong support to our hypothesis that during the early prandial period, gastrointestinal signals elicit a transient blockade of renal excretion with a mechanism involving the kallikrein-kinin system.

Kallikrein—Kinin and Renin—Angiotensin Systems in Renovascular Hypertension in Rats

Summary Plasma renin activity (PRA) and the levels of urinary kallikrein (UK) were studied simultaneously in Goldblatt one- and two-kidney hypertensive rats (1KG and 2KG) 5, 10, and 15 weeks after clamping the left renal artery. Increase in PRA was statistically significant in 2KG rats (P < 0.0005 on the 5th and 10th week, and P < 0.002 on the 15th week). PRA was not significantly different to that of controls in 1KG rats. Contrariwise, the urinary kallikrein levels were significantly lower in 1KG rats (P <0.002) in relation to values found in normotensive rats. In 2KG rats, urinary kallikrein levels became significantly lower than those in controls only 15 weeks after surgery. Special acknowledgment to Prof. M. L. Forcelledo for her criticism and for the great help in performing the AI R.I.A. procedure. This work was supported by D.I.U.C. 308/77 and P.N.U.D.-U.N.E.S.C.O., RLA 76/006 Grants.