Juan Roblero

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.

Response of the Circulatory System to Bradykinin and Related Peptides: A Specific Kinin Assay

The complex response of the mammalian circulatory system to bradykinin and related peptides is made up both of direct actions of the peptides as well as responses of the organism which tend to neutralize the actions of the kinins. The direct vasomotor actions of bradykinin are arteriolar dilation, venular constriction, and increased capillary permeability. These cause the observed sharp fall in blood pressure. Catecholamine release, tachyphylaxis, and increased cardiac output partially neutralize the direct effects, and a very efficient multienzyme kinin metabolizing system rapidly removes bradykinin from the circulation. To study these effects we have used synthetic peptides related in structure to the kinins, in addition to certain drugs. The peptides were synthesized by the solid phase method (Stewart and Young, 1969). An adequate understanding of the factors involved has made it possible to develop a rat blood pressure assay which is specific for each of the known mammalian kinins.

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.

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.

Blunting Effect of Pepsanurin Introduced in the Duodenum on the Atrial Natriuretic Peptide Diuretic Action in Rats

Abstract Pepsanurin (PU) is a peptide(s) obtained by pepsin hydrolysis of human plasma or its globulin fraction. We have reported that the accelerated renal excretory rate induced by atrial natriuretic peptide (ANP) can be considerably blunted by PU either in the intact rat or in the isolated perfused rat kidney. We explored whether or not PU can be part of a signaling mechanism originated in the digestive tract, involved in the regulation of water and electrolyte homeostasis. PU obtained either from human (0.5 ml) or rat plasma (0.25–0.5 ml) administered into the duodenal lumen of rats, counteracted significantly the diuretic-saluretic action of a 0.5- μg bolus of ANP, reproducing qualitatively the effect of its intraperitoneal administration. Human PU reduced the ANP-stimulated renal excretion by 67-90% for Na (P < 0.001) and by 35–54% for water (P < 0.02–P < 0.001); the inhibition induced by rat PU was 45–96% for Na (P < 0.05–P < 0.01) and 35–65% for water (P < 0.05–P < 0.01). Rat PU (0.5 ml) abolished the rise of glomerular filtration rate induced by ANP without affecting fractional Na excretion. All the samples tested decreased K excretion, but in some experiments, the difference did not reach statistical significance. Contrary to the effect of PU, the introduction in the duodenum of (i) isotonic glucose solution, (ii) hydrolysate of bovine serum albumin, or (iii) hydrolysate of casein prepared after the same procedure yielding PU from plasma failed to produce an inhibition of the ANP stimulation of renal excretory rate. In addition, human plasma incubated at 37°C for 24 to 48 hr, prior to pepsin digestion, did not yield PU, which indicates that PU is generated from a substrate sensitive to endogenous enzymes and/or that its stability is vulnerable to endogenous enzymes.

Inhibitory effect of renin extracts upon urinary kallikrein excretion.

Summary Intraperitoneal injections of 1–5 I.U. of renin purified extracts, obtained either from hog or rat kidneys, in hyperhydrated rats receiving distilled water or 0.4% NaCl (5% body weight) produce not only a striking increase in the sodium excretion rate but a very significant decrease in kallikrein excretion as well. In the urine excreted in the first hour after renin administration kallikrein practically disappeared in the urine; with higher doses the inhibitory effect was very marked and lasted up to 120 minutes. In the same rats under a second hyperhydration, nonassociated with renin injection, kallikrein tends to return to control levels.