The Kallikrein-Kinin System in the Regulation of Renal Function Henry Ford Hospital, Hypertension Research Abstract Kallikreins Laboratory Division of

Fig.1 Infantile R. F. and Growth.

The Kallikrein-Kinin System in the Regulation of Renal Function Henry Ford Hospital, Hypertension Research Abstract Kallikreins Laboratory Division of Nephrology, Department of Medicine, Detroit, Michigan 48202 Oscar A. Carretero are enzymes that release kinins, potent vasodilator peptides from plasma substrates called kininogens. In the microdissected nephron, prekallikrein and kallikrein are localized in the granular part of the distal and cortical collecting tubules (connecting tubules). Urinary kinins are formed in this part of the nephron and in the papilla and pelvis of the kidney. The intrarenal concentration of kinins can be affected by factors other than kallikrein, such as: kininogen, kininases, presence of kallikrein inhibitors, concentration of electrolytes, and hydrogen ions. There are important interrelations between the kallikrein-kinin, renin-angiotensinaldosterone, and prostaglandin systems. Kallikrein or other serine protease (s) inhibited by aprotinin appear to play a role within the kidney, controlling renal vascular resistance, salt and water excretion, and renin release. However, the final role of this system Introduction Kallikreins still remains to be elucidated. are enzymes that release kinins, potent vasodilator peptides, from plasma substrate called kininogens. The kidney has all the components of the kallikrein-kinin system, and kinins are formed intrarenally in significant amounts (Fig. 1). The renal kallikrein-kinin system has been postulated to play a regulatory role within the kidney controlling Fig. 1 Mechanisms of kinin generation and destruction in the kidney. Fig. 2 The profile of active and inactive kallikrein along the rabbit nephron. Kininogenase activity is expressed as picograms of kinins per micrograms of tissue per minute of incubation. Results are given as means } S. E. M. Diagram below graph illustrates a schematic rabbit nephron showing subdivision of microdissection. The distribution of various cell types in the distal nephron is depicted by horizontal bars under diagrammatic nephron. Abbreviations are: GL=glomerulus ; PCT=proximal convoluted tubule; CAL=cortical thick ascending limb; DCTb=bright portion of distal convoluted tubule; DCTg=granular portion of distal convoluted tubule; CCTg=granular portion of cortical collecting tubule; CCT1=light portion of cortical collecting tubule; MCT =medullary collecting tubule. salt and water excretion, renal vascular resistance and renin release, and consequently the regulation of blood pressure, however, the final role of this

system still remains to be elucidated. Localization of the kallikrein-kinin system in the nephron. Over 90% of the kallikrein in the kidney is found in the cortex, decreasing from the outer to the inner cortex, with very little kallikrein in the medulla and papilla. Isolated glomeruli have a small amount of kallikrein activity compared to the kallikrein concentration in the total cortex. We recently studied the localization of both active and inactive kallikrein in the microdissected rabbit nephron and found that both forms of the enzyme were localized in the granular portion of the distal and cortical collecting tubules. This segment of the nephron contains more than 85% of the active and inactive kallikrein found in the total microdissected nephron. Only very small amounts of active and inactive kallikein were found in other segments, including the bright portion of the distal segment which has the macula densa (Fig. 2). The granular portions of the distal convoluted and cortical collecting tubules form a single nephron segment called the connecting tubule, which has two types of cells; the connecting tubule cells, and the intercalated cells. Since the connecting tubule cells and kallikrein localize only in the granular part of the distal and collecting segment, and the intercalated cells are thoroughly distributed from the granular distal tubule to the medullary collecting tubule, it is reasonable to assume that kallikrein is syhthesized in the connecting cells. The discrete localization of kallikrein suggests a specific role of renal kallikrein in association with these nephron segments. These studies suggest that the kinins present in the urine are released in the kidney itself when bradykinin is infused into the renal artery, less than 0.2% appears in the urine and more than 90% is inactivated in the vascular compartment of the kidney by kininases. There are two main types of kininases : kininase I and II. Kininase II has been studied more extensively than kininase I, partly because kininase II, in addition to hydrolyzing kinins, converts angiotensin I to angiotensin II: (angiotensin I converting enzyme). The brushh border of the proximal tubule is rich in kininase II, which prevents filtered kinins from reaching the distal nephron. Carone et al. have shown that when labeled bradykinin was injected into the proximal tubule it was almost completely destroyed. However, when this peptide was injected into the distal tubule, it appeared almost intact in the urine. This evidence, plus the fact that renal kallikrein iss secreted into the urine at the level of the distal tubule, seems to indicate that urinary kinins may be formed in the distal part of the nephron. We have confirmed this possibility by using the stop-flow technique and found that kinins are formed in the distal part of the nephron, with the highest concentration located in the final segment of the nephron or even in the renal papilla and pelvis. No evidence of kinin formation was found in the fraction representing the proximal nephron. The origin of the kininogen (substrate) neededd for the formation of kinins in the lumen of the mephron is not known. The plasma is very rich in kininogen and a small amount of plasma kininogen reaching the distal nephron could account for the kinins found in the urine. In human urine, there is a significant amount of immunoreactive kininogen, however kininogen is found in small amounts when measured by its capability to generate kinins. It could be that most of the kininogen has already been consumed by the urinary kallikrein. It has been reported that urinary kinins were absent in a subject with a congenital deficiency of plasma low and high molecular weight kininogen. On the other hand, in a patient without plasma highh molecular weight kinininogen but with near normal low molecular weight kininogen (Fitzgerald trait), we found that the urine contained normal amounts of kinins (unpulished observation). This indicates that kinins in the urine are formed from low molecular weight kininogen and that they are not

formed by plasma kallikrein, since this enzyme releases kinins only from high molecular weight kininogen. Recently, Proud et al. using antibody to low molecular weight kininogen and immunohistochemical techniques, have localized kininogen in cells of distal and collecting tubules. In rabbit renal tissue, we have been unable to demonstrate the presence of kininogen (K. Omata, A. G. Scicli, and O. A. Carretero, unpublished results). The presence of an inhibitor of glandular kallikrein has also been demonstrated in the rat kidney tubules. It is feasible that this inhibitor could play a role in the regulation of intrarenal formation of kinins. Factors that control the intrarenal formation of kinins are not well defined. We have found no correlotion between kallikrein and kinin excretion in urine collected directly from the ureter of the rat14~. Further, we found that acidification of the urine by sodium sulfate infusion, decreased kinin excretion while it increased kallikrein excretion. It is possible that the lower ph within the distal nephron decreases the kininogenase activity of renal kallikrein since the optimum ph of kallikrein is 8.5, On the other hand, alkalinization of normally acidic urine of the rat by infusion of sodium bicarbonate did not produce a change in kinin or kallikrein excretion. It is possible that renal kininase activity increased since their optimum ph is above 7.0; and therefore alkalinization of the urine could result in an increase in both formation and destruction of kinins. It has recently been reported that the concentration of electrolytes also affects the rate of kinin formation. Thus, the intrarenal concentration of kinins may depend on their rates of formation and destruction (Fig. 1). In turn, these rates may depend on the intrarenal concentration of kallikrein, kininogen, kininases, kallikrein inhibitors, electrolytes and hydrogen ions. Relationship Among the Kallikrein-Kinin, Renin- Angiotensin-Aldosterone, Prostaglandin and Vasopressin Systems Plasma and glandular kallikrein in vitro converts inactive renin to active renin, and it has been suggested that renal and plasma kallikrein activate renin in vivo. Suzuki reported that urinary kallikein stimulates renin release from superf used isolated kidney slices. Neither bradykinin in high doses nor trypsin were able to release renin under these experimental conditions, indicating that kallikrein acts directly on the renal tissue to release renin. However, recently, investigators from the same group, (P. Mulrow, personal communication) have been unable to confirm that kallikrein stimulates renin release in the kidney slices preparation. Using isolated superfused glomeruli we have been able to show that both kallikrein and bradykinin stimulate renin release. The affect of bradykinin in a molar basis was more potent that of isoproterenol. In vivo, bradykinin infused into the renal artery of dogs stimulates the release of renin. Recently, Abe et al. reported that aprotinin infused in the renal artery of dogs inhibits the renin release stimulated by a converting enzyme inhibitor. This study may suggest that the release of renin was stimulated either by renal kallikrein directly, or by kinins released by this enzyme since aprotinin hae frequently been used to inhibit kallikrein. However, aprotinin inhibits dog glandular kallikrein poorly or not at all. Furthermore, since aprotinin is a polyvalent inhibitor of serine proteases, its effect on renin release in the dog may be mediated by inhibition of other enzymes. Inhibition of renin release by aprotinin has also been reported in humans and ratsk. These findings indicate that kallikrein or other serine proteases may participate in the control of renin release. The angiotensin I converting enzyme (kininase II) further links the kallkrein-kinin and renin-angiotensin systems. Converting enzyme is found in high concentrations on the endothelial cell surface of the vascular bed of the lung and has the concurrent functions of converting angiotensin I to II and des- (140)

troying kinins. There is evidence that 90% of the kinins administered in the venous site of the systemic circulation are destroyed by one passage through the lung, thus most of the kinins formed in tissues which might enter the vascular compartment would be destroyed before they reach the peripheral circulation. This suggests that the biological effects of kinins take place within the confines of the organ where they are released. Mineralocorticoid administration increases urinary kallikrein excretion and renal tissue kallikrein concentration, while spironolactone, an aldosterone antagonist, decreases it. In dogs, this increase in urinary kallikrein excretion by the administration of deoxycorticorticosterone acetate (DOCA) was found only after the dogs escaped from the sodiumretaining effects of DOCA. Thus, at the present time it is not clear whether aldosterone stimulates renal kallikrein secretion, either directly or through an alteration of water and electrolyte metabolism. Angiotensin has also been reported to stimulate renal kallikrein excretion. Kinins infused into the renal artery stimulate the synthesis of prostaglandins probably prostaglandin E2 (PGE2) in the collecting duct and renal medulla, and prostacyclin (PGI2) in the arterioles. This effect of kinins is produced by an increased release of arachidonic acid as a consequence of the activation of phospholipase A2. Furthermore, part of the renal vasodilator and natriuretic effect of kinins is mediated through the release of prostaglandins and can be inhibited by prostaglandin synthesis inhibitors such as indomethacin and meclofenamate. However, these findings are not universal and some investigators have been unable to demonstrate that the renal vasodilator effects of kinins are mediated by prostaglandins. On the other hand, prostaglandins have been reported to stimulate, while prostaglandin synthesis inhibitors suppress, renal kallikrein release. The interactions between the kallikrein-kinin system, the renin-angiotensin-aldosterone, and the Fig. 3 Interactions among the kallikrein-kinin, renin-angiotensin-aldosterone, and prostaglandin systems. prostaglandin systems are depicted in Fig. 3. These interactions may play an important physiological role. It could be speculated that increased activity in the renin-angiotensin system would produce both a peripheral and renal vasoconstriction that could impair renal blood flow. However, angiotensin II and aldosterone stimulate the release of renal kallikrein and prostaglandin, which could produce local vasodilation and maintain renal blood flow even in the presence of high concentrations of angiotensin II. Antidiuretic hormone (ADH) stimulates the release of kallikrein and the intrarenal formation of kinins. Further, kinins antagonize the effect of ADH in the toad bladder and in the kidney. Thus, it is possible that kinins antagonize or modulate the effects of ADH in the kidney either directly or through the release of prostaglandins. In conclusion, there are numerous complex interactions between the kallikrein-kinin, renin-angiotensin-aldosterone, ADH, and prostaglandin systems. Many are not completely understood and some of them may be of no physiological importance. Physiological role of the renal kallikrein-kinin system Although some of the actions of renal kallikrein, such as the activation of inactive renin, may be due to its direct catalytic effect, most of its effect seems to be mediated by kinin release. The infusion of kinins inro the renal artery results in an increased blood-flow diuresis and natriuresis without changing the glomerular filtration rate. Like most vasodilator drugs, kinins produce a greater increase in blood

flow in the inner cortex than in the outer. Unlike other vasodilators, kinins do not decrease proximal sodium and water reabsorption in outer cortical nephrons available for micropuncture. Accordingly, the natriuretic effect of kinins is either due to inhibition of sodium reabsorption in the distal part of the nephron or to changes in deep nephron reabsorption. Kinins may affect sodium reabsorption as the result of a direct effect on the transport of sodium in the nephron, a vasodilator effect with changes in the interstitial fluid pressure, changes in the osmotic gradient of the renal medulla, or a combination of all these effects. Changes in the osmotic gradient of the renal medulla could explain the decrease in urinary osmolality and vasopressinresistant diuresis caused by kinins. Kinins administered into the renal artery probably do not mimic the effects produced by kinins formed intrarenally by endogenous kallikrein, since their sites of action of concentration could be different. Kauker has shown that kinins administered directly into the late proximal tubule increase sodium excretion, suggesting that kinins in the lumen of the nephron have natriuretic effects. The role of endogenously generated kinins in the regulation of renal blood flow and water and electrolyte excretion has been studied by using angiotensin converting enzyme inhibitors (CEI). These inhibitors increase the concentration of endogenous kinins by inhibiting kininase II. After CEI administration, renal blood flow increases in the juxtaedullary cortex with a simultaneous increase in the fractional excretion of sodium. However, the use of CEI does not allow for differentiation between the potentiation of kinin activity and the inhibition of the conversion of angiotensin I to angiotensin II. Administration of aprotinin, an inhibitor of kallikrein and other serine proteases, to volume-expanded rats decreased GFR, renal blood flow, urinary volume, sodium, potassium, and PGE2 excretion. Furthermore, administration of antibodies against kinins to saline-infused rats resulted in a decrease in sodium excretion. These findings suggest that intrarenally released kinins cause natriuretis, diuresis, and release of prostaglandins. Recently, Johnston et al. have shown that aprotinin increases renal vascular resistance under conditions of dietary sodium restriction or with reduced renal perfusion pressure. While aprotinin may not be acting specifically against kallikrein, their results support a role for a serine protease (s) in the regulation of renal blood flow. Using the isolated perfused that kallikrein kidney, it has been demonstrated is secreted not only into the nephron, but also in the interstitial and vascular space of the kidney. However, we have recently found that the difference in arteriovenous concentration of immunoreactive glandular kallikrein is negative, suggesting that the kidney not only releases, but also depurates immunoreactive glandular kallikrein from plasma. Locally formed kinins could participate in the regulation of renal blood flow distribution. It has recently been reported that when kinins are applied to the serosa of the intestine, the transport of chloride is stimulated from the serosal to the mucosa. Whether this effect also occurs in the nephron is not known, but it suggests that kinins may be important in the regulation of electrolytes. In summary, the renal kallikrein-kinin system in the kidney is localized in the granular part of the distal and collecting tubules (connecting tubule). This system appears to be interrelated to the reninangiotensin-aldosterone and prostaglandin systems. Kallikrein or other serine protease (s) inhibited by aprotinin appear to play a role within the kidney' controlling salt and water excretion, renal vascular resistance, and renin release. However, the final role of this system still remains to be elucidated. References 1) Frey, K. E., H. Kraut, E. Werle : Kallikrein Padutin. edited by R. Vogel, Munchen : Ferdinand Enke Verlag Stuttgart, 1977. 2) Carretero, O. A., A. G. Scicli : Possible of kinins (142)

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Fig. 1 Binding characteristics of 125I-Tyr8- bradykinin to renal cortex and medulla.

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