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Potassium-induced increase in renal kallikrein secretion is attenuated in dissected renal connecting tubules of young spontaneously hypertensive rats
International Immunopharmacology 2 (2002) 1957 – 1964 www.elsevier.com/locate/intimp
Potassium-induced increase in renal kallikrein secretion is attenuated in dissected renal connecting tubules of young spontaneously hypertensive rats Mariko Yamanaka a,b, Izumi Hayashi c,d, Tomoe Fujita c, Seok Ho Cha e, Hitoshi Endou e, Masaaki Higashihara a,b, Masataka Majima c,d,* a Fourth Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan Fourth Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa 228-8555, Japan c Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan d Department of Molecular Pharmacology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa 228-8555, Japan e Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan b
Abstract It is suggested that attenuation of the renal kallikrein – kinin system (KKS) involved the development of hypertension in young spontaneously hypertensive rats (SHR). In the present study, a comparison was made between young SHR and Wistar Kyoto rats (WKY) to examine the ability to secrete renal kallikrein from the microdissected connecting tubules (CNT) by potassium or an ATP-sensitive potassium channel blocker, 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A), both of which are renal kallikrein secretagogues. Maximum effect of potassium on kallikrein secretion was observed 10 min after placing the tubules at concentration of 20 mM. Kallikrein secretion was also increased concentration-dependently by PNU-37883A (0.1, 1, 10, and 100 AM). In the presence of EDTA, NiCl2, verapamil, xestspongin C (an inositol 1,4,5-trisphosphate (IP3) receptor-selective antagonist), or ruthenium red (a ryanodine-sensitive receptor blocker), potassium-induced increase in renal kallikrein secretion was inhibited. Augmentation of renal kallikrein secretion by potassium or PNU-37883A was diminished in SHR compared to WKY. These results indicate that the ability to secrete renal kallikrein by potassium was attenuated in young SHR compared with WKY. Furthermore, it is suggested that the potassium-induced renal kallikrein secretion requires an extracellular Ca2 + entry through Ca2 + channels including L-type Ca2 + channels and Ca2 + release from intracellular Ca2 + stores through IP3 receptor and ryanodine receptor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Renal kallikrein secretion; Microdissected connecting tubules; Potassium; ATP-sensitive potassium channel blocker; Spontaneously hypertensive rats
1. Introduction * Corresponding author. Department of Pharmacology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Tel.: +81-42-778-8822; fax: +81-42778-8467. E-mail address: [email protected] (M. Majima).
There are two forms of kallikrein –kinin system (KKS), plasma KKS and tissue KKS, where bioactive peptides of kinins are generated from a high and low
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molecular weight kininogens by kallikrein, respectively [1]. In the kidney, renal kallikreins are localized in the connecting tubules (CNT) and cortical collecting tubules [2– 4]. They release kinins into urine from kininogens, which were also localized in distal tubules and collecting tubules [4,5]. In the early 1970s, it was reported that patients with essential hypertension excrete less urinary kallikrein than normal healthy subjects [6– 8]. Thereafter, many reports have suggested preventive effect of renal KKS on the development of hypertension [9– 11]. Renal KKS has been considered to play an important role in the homeostasis of sodium and water balance in the kidney [12,13]. Spontaneously hypertensive rats (SHR) are known as an important animal model for essential hypertension. The systolic blood pressure of SHR starts to increase from the age of 4 weeks, and the hypertension develops from the age of 7 –15 weeks. Excretion of urinary kallikrein in 4-week-old SHR has been shown to be significantly lower than that in agematched Wistar Kyoto rats (WKY), the normotensive controls [13,14]. Continuous infusion of a nonpeptide mimic of bradykinin into renal artery of 6-week-old SHR for 6 days significantly reduced mean blood pressure [15], suggesting attenuation of renal KKS activity in young SHR may be involved in facilitation of the development of hypertension. It was known that the supplementation of potassium lowered high blood pressure in animal models and in humans. Several studies indicate that potassium loading can increase the secretion of renal kallikrein. Systemic potassium loading increases urinary kallikrein excretion in both humans [16,17] and animals [18,19], and high concentration of potassium solution increases release of renal kallikrein from rat sliced kidney [20]. In addition, natriuresis induced by potassium loading is inhibited by treatment of rats with bradykinin B2 receptor antagonist [21]. However, variable response of blood pressure to potassium intake was reported in hypertensive patients [22]; thus, hereditary factors were also postulated in a comparative study of urinary potassium and kallikrein excretion using a genetic analysis of monozygotic and dizygotic twins [23]. Maximum likelihood segregation analysis on 769 individuals in 58 Utah pedigrees reported that a urinary kallikrein level was not associated with urinary potassium level in the inferred kallikrein genotype of low homozygotes [24], sug-
gesting a genetic defect is involved in the renal kallikrein in response to potassium. Additionally, a previous report showing accumulation of renal kallikrein granules in the CNT of SHR [25] suggests disordered renal kallikrein secretion in SHR. Therefore, this study was aimed to investigate whether potassium-induced increase in renal kallikrein secretion was attenuated in CNT of young SHR compared with that of WKY. CNT were isolated from both strains of rat by the microdissection technique so that a direct effect of drugs on the increase in renal kallikrein secretion could be examined. We previously reported that 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A), which can block the ATP-sensitive potassium channels on renal tubules selectively, showed an augmentative effect on renal kallikrein secretion without an additive effect on potassium-induced increase in renal kallikrein secretion [26]. Thus, the effect of PNU37883A on renal kallikrein secretion was also compared between SHR and WKY. Furthermore, mechanisms of the potassium-induced increase in renal kallikrein secretion were also studied with respect to extracellular Ca2 + entry and the release from intracellular calcium stores.
2. Materials and methods 2.1. Materials Male Sprague –Dawley (SD) rats were purchased from SLC Japan (Hamamatsu, Japan). Male SHR and WKY were supplied from Sankyo Lab. Animal Service (Tokyo, Japan). All rats used were 4– 6 weeks old and specific pathogen-free. Rats were given normal rat chow and tap water ad libitum. They were housed at constant humidity (60 F 5%) and temperature (25 F 1 jC) and were kept in a continuous 12-h light– dark cycle. All of the procedures were conducted in accordance with the guiding principles for the care and use of laboratory animals approved by the Animal Care Committee of Kitasato University. The following drugs were used: 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A) was a gift from Pharmacia and Upjohn (Kalamazoo, MI); disodium-ethylenediaminetetraacetate, dihydrate (EDTA-2Na, Doujindo, Kuma-
M. Yamanaka et al. / International Immunopharmacology 2 (2002) 1957–1964
moto, Japan), and nickel chloride (NiCl2, SigmaAldrich, St. Louis, MO) were purchased. Verapamil, xestospongin C, and ruthenium red were purchased from Wako (Osaka, Japan). All other chemicals were the analytical grade of commercial sources. 2.2. Methods 2.2.1. Microdissection of CNT CNT were isolated according to the previous report by Tomita et al. [2] with a slight modification. Briefly, immediately after rats were subjected to cerebral concussion, laparotomy was performed. Two parts of the abdominal aorta, upper and lower, away from the bifurcation of the left renal artery, were ligated. Through the needle placed in the abdominal aorta, kidneys were infused with Hank’s solution containing 1 mM CaCl2, bovine serum albumin (1 mg/ml, Sigma-Aldrich), and collagenase type I (1 mg/ml, Sigma-Aldrich). The circulating blood of the left kidney streamed out of the renal vein. The left kidney was nephrectomized and was sliced to a thickness of approximately 1 mm. Those slices were incubated at 37 jC in the solution described above under the aerobic condition. Those preparations, kept at 4 jC, were observed under a stereomicroscope and microdissection was performed. The obtained single nephron segment, CNT, was used for experiment. 2.2.2. Experimental procedure CNT were dissected from the kidney of SHR, WKY, and SD rats after collagenase digestion under a stereomicroscope. Dissected CNT prepared from the kidney were placed in the solution containing 20 mM KCl, 140 mM NaCl, 1 mM CaCl2, and 10 mM HEPES (pH 7.4) for 0, 10, or 20 min. To examine dose-dependent effects of potassium and PNU37883A, the CNT were incubated in the solution containing KCl (at the concentration of 0, 10, 20, 40, or 70 mM) or PNU-37883A (at the concentration of 0, 0.1, 1, 10, or 100 AM) for 10 min. To maintain isotonicity of the solution, concentration of NaCl was decreased to 70 mM for 70 mM KCl, 100 mM for 40 mM KCl, 120 mM for 20 mM KCl, and 130 mM for 10 mM KCl. For incubation with PNU-37883A, 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl and 1 mM CaCl2 was used. In the examination for effects of Ca2 + channel blockers on K+-induced
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kallikrein secretion, dissected CNT of SD rats were placed in the above buffer and subsequently in saline for 10 min. Then the CNT were incubated with the buffer containing 1 mM EDTA, 1 mM NiCl2, or 100 AM verapamil. Each reagent was dissolved in saline and was spiked in the medium. In the experiment to test effects of IP3 receptor-selective antagonist or ryanodine-sensitive receptor blocker on K+-induced kallikrein secretion, the CNT were placed in the solution containing xestspongin C (0.3, 3, or 30 AM), or ruthenium red (50 AM). Xestspongin C (1.33 mg/ml) or ruthenium red (4.29 mg/ml) was dissolved in 1% ethanol or saline, respectively. A medium containing 1% ethanol alone was used for control experiments of xestspongin C. 2.2.3. Measurement of released kallikrein activity Kallikrein activities in both incubated supernatant and the CNT were measured using a peptidyl fluorogenic substrate for glandular kallikrein, Pro – Phe– Arg – 4-methyl-coumaryl-amide (Pro – Phe – Arg – MCA, Peptide Institute, Minoh, Osaka, Japan). The CNT were solubilized with 5 Al of 5% Triton-X for 10 min at room temperature to release the retained kallikrein in the tubules. No visible nephron contour could be observed under stereomicroscopy in this condition. The solubilized tubules were dispersed into the solution in 100 Al of 10 mM HEPES (pH 7.4) containing 140 mM NaCl and 1 mM CaCl2 and was incubated at 37 jC for 30 min with 1 Al of Pro– Phe – Arg –MCA (10 mM) diluted with 0.05 M Tris – HCl (pH 8.0), 0.1 M NaCl, and 0.01 M CaCl2. After the incubation, 5 Al of 17% acetic acid was added to stop the reaction. Fluorescence intensity of the incubation mixture was measured at 380 nm (excitation) and at 460 nm (emission) by fluorescence spectrophotometer (M850; Hitachi, Tokyo, Japan). Renal kallikrein secretion was expressed as released kallikrein (%), which was defined as the ratio of the activity to total kallikrein activity (the released kallikrein and the kallikrein retained in the CNT). 2.2.4. Data analysis Values are expressed as means F S.E.M. One-way ANOVA or Student’s t-test was used to evaluate statistical significance of difference. Differences with a probability of 5% or less were considered to be significant ( P < 0.05).
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3. Results 3.1. Time course of the potassium-induced increase in renal kallikrein secretion from dissected CNT In order to examine time course of change in renal kallikrein secretion by potassium, one piece of tubule was incubated with buffer containing 20 mM KCl for 0, 10, and 20 min. Renal kallikrein secretion increased significantly to approximately fourfold within 10 min after the incubation. The amount of released kallikrein by high potassium did not increase more at a 20-min period (Fig. 1). 3.2. Concentration-dependent increase in renal kallikrein secretion by potassium ion and PNU37883A Several concentrations of potassium and PNU37883A were examined whether the renal kallikrein secretion increased with a concentration-dependent manner. Renal kallikrein secretion increased at a concentration of 20 mM KCl with a peak and then tended to decrease at the concentrations of 40 and 70 mM (Fig. 2a). PNU-37883A increased renal kallikrein secretion in a concentration-dependent manner, and
Fig. 2. Concentration-dependent increase in renal kallikrein secretion by KCl and an ATP-sensitive potassium channel blocker. Effects of a high potassium solution at a concentration of 10, 20, 40, or 70 mM on renal kallikrein secretion were tested. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer as described in Fig. 1 in the absence (0), or in the presence of KCl (10 to 70 mM) (a) or PNU-37883A (0.1 to 100 AM) (b) for 10 min. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Fisher’s PLSD and Scheffe´ test. *P < 0.05, significantly different from the value in the absence of the agents.
significant increase was observed at concentrations of 10 and 100 AM (Fig. 2b). Fig. 1. Time course of renal kallikrein release from dissected CNT induced by KCl. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) containing 20 mM KCl, 120 mM NaCl, and 1 mM CaCl2 for 0, 10, or 20 min, kallikrein activities in the buffer and in the tubule were measured by using the fluorogenic substrate, Pro – Phe – Arg – MCA. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Scheffe´ test. *P < 0.05, significantly different from the value at 0 min.
3.3. Effects of EDTA, NiCl2, verapamil, xestospongin C, and ruthenium red on the potassium-induced increase in kallikrein secretion from CNT In order to examine whether extracellular Ca2 + influx was involved in the potassium-induced renal kallikrein secretion, the tubule was incubated with a solution containing EDTA, NiCl2, or verapamil followed by stimulation with 20 mM of KCl. As shown
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in Fig. 3, 20 mM of KCl increased the kallikrein release sixfold. Treatment of CNT with EDTA (1 mM), NiCl2 (1 mM), or verapamil (100 AM) significantly suppressed the augmentation of kallikrein release by 20 mM of KCl. In order to examine whether Ca2 + release from intracellular Ca2 + stores is involved in the potassium-induced renal kallikrein secretion, the tubules were treated with xestospongin C or ruthenium red in the presence of 20 mM KCl. As shown in Fig. 4a, the augmented renal kallikrein release by potassium was significantly suppressed by 3 and 30 AM of xestospongin C in a dose-dependent manner. Ruthenium red (50 AM) also showed a significant suppression of the augmented secretion (Fig. 4b). 3.4. Comparison of the potassium- or PNU-37883Ainduced increases in renal kallikrein secretion between young SHR and WKY As shown in Fig. 5a, renal kallikrein secretion from CNT of WKY rat was increased by stimulation with KCl with a peak at the concentration of 20 mM and no further increase was observed at concentrations of 40 and 70 mM, same as in SD rats. Released kallikrein
Fig. 3. Effects of EDTA, NiCl2, and verapamil on the potassiuminduced increase in renal kallikrein secretion. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl, and 1 mM CaCl2 in the absence or presence of EDTA (1 mM), NiCl2 (1 mM), or verapamil (100 AM) for 10 min. Treatment with saline was as the vehicle. Left open column indicates the value without stimulation by 20 mM KCl. Subsequently, the tubule was further incubated with 10 mM HEPES (pH 7.4) buffer containing 20 mM KCl, 120 mM NaCl, 1 mM CaCl2 for 10 min. Each agent was dissolved in saline. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Scheffe´ test. *P < 0.05, significantly different from value in the vehicle-treated group.
Fig. 4. Suppression of KCl-induced renal kallikrein by IP3 receptorselective antagonist and ryanodine receptor channel blocker. Effects of xestospongin C on the potassium-induced increase in renal kallikrein secretion were observed. CNT dissected from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl, and 1 mM CaCl2 without or with xestospongin C (0.3, 3, and 30 AM) (a) or ruthenium red (50 AM) (b) for 10 min. Subsequently, the tubule was further incubated with 10 mM HEPES (pH 7.4) buffer containing 20 mM KCl, 120 mM NaCl, and 1 mM CaCl2 for 10 min. Xestspongin C and ruthenium red were dissolved in 1% ethanol and saline, respectively. Values are means F S.E.M. Data were analyzed by one-way analysis of variance followed by post hoc Fisher’s PLSD. *P < 0.05, significantly different from the values in the vehicle-treated group. Treatments of CNT with 1% ethanol or saline were as the vehicles for xestspongin C and ruthenium red, respectively. Left open column in each panel indicates the value in the absence of 20 mM KCl. Numbers in parentheses indicate numbers of CNT used.
level in SHR was similar to that in WKY when the tubule was incubated with buffer without KCl. The secretion from CNT of SHR was increased by addition of 10 –70 mM of KCl. However, the levels at concentrations of 20 and 40 mM of KCl in SHR rats were significantly less than those in WKY. PNU-37883A at a concentration of 0.1AM provided a maximum effect on renal kallikrein secretion from CNT of WKY as shown in Fig. 5b. The augmentation in SHR was less than those in WKY. Significant augmentation of the secretion by PNU-37883A at the concentrations of 0.1, 1, and 10 AM from CNT was also observed in SHR. However, significant differences of the augmen-
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Fig. 5. Comparison of renal kallikrein secretion between young SHR and WKY rats by KCl or an ATP-sensitive potassium channel blocker. Dissected CNT prepared from the kidney of SHR and WKY were incubated with 10 mM HEPES (pH 7.4) buffer as described in Fig. 1 in the absence (0), or in the presence of KCl (10, 20, 40, and 70 mM) (a) or PNU-37883A (0.1, 1, 10, and 100 AM) (b) for 10 min. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. *P < 0.05 and #P < 0.05, significantly different from the value in the absence of the agents in WKY and SHR rats, respectively. yP < 0.05, significantly different of values between WKY and SHR rats. In the experiments using SHR and WKY, Student’s t-test was used for the comparison of renal kallikrein secretion between SHR and WKY at each concentration of potassium or PNU-37883A. One-way ANOVA determined to evaluate the significance levels between the control and each concentration of drug treatment with potassium or PNU-37883A.
tation were observed between WKY and SHR at the concentrations of 1 and 100 AM of PNU-37883A.
4. Discussion Several reports suggest that high potassium intake increase urinary kallikrein excretion as a result of augmentation of secretion of kallikrein or the synthesis induced by glucocorticoids, prostaglandins, or even directly [16,19]. It is possible that potassium supplementation increase the release of aldosterone,
and that this increased aldosterone may upregulate kallikrein synthesis in the CNT. In the present study, a microdissected single nephron of CNT, where renal kallikrein is located, was used to examine renal kallikrein secretion, so that endocrine influences on renal kallikrein secretion from CNT could be ruled out. As a result, a direct effect of potassium to increase renal kallikrein secretion was demonstrated by using CNT. Prolonged incubation of CNT with 20 mM KCl or high concentrations of KCl (40 and 70 mM) resulted in decrease in the potassium-induced augmentation of renal kallikrein secretion. This may be caused by toxic effect of potassium. It was also shown that PNU-37883A, an ATP-sensitive potassium channel blocker, has a direct augmentative effect on renal kallikrein secretion. CNT are known to participate, at least in part, in the secretion of potassium in the distal nephrons. Furthermore, it is known that excess amount of potassium was mainly excreted from distal tubules. Therefore, the concentration of potassium around CNT should become high when high potassium is loaded into the body [17]. An ATPsensitive potassium channel is one of potassium channels, which is known to modulate sodium, potassium, and chloride reabsorption. It also controls potassium secretion on the thick ascending limb of the loop of Henle, distal tubules, and CNT [27]. The above results suggest that CNT not only regulate electrolyte transport but also secrete kallikrein in association with potassium channels. Indeed, the distal nephrons maintain the membrane potential of the tubular cells and membrane of the tubule cells are depolarized in the presence of high potassium in the collecting duct [28,29]. Thus, it is likely that potassium and PNU-37883A may directly stimulate renal kallikrein secretion through changes in membrane potential of CNT. We previously reported that the site of action of potassium and PNU-37883A was the source since no additive effect of those two secretagogues was observed in terms of release of kallikrein [26]. Attenuated responses of CNT to secrete renal kallikrein by potassium or PNU-37883A were demonstrated in SHR compared with WKY. These results indicate that SHR has some disordered mechanisms that may contribute to weaken to secrete renal kallikrein by potassium and PNU-37883A. In case of the islet cells, it has been reported that the exocytosis of
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insulin induced by an ATP-sensitive potassium channel blocker is accompanied with increase in intracellular Ca2 + concentration, resulting from gating of voltage-activated Ca2 + channels by cell depolarization. L-type channel cDNAs have been identified in insulin-secreting clonal h cells [30,31]. The present study indicates that the potassium-induced increase in renal kallikrein secretion was partially inhibited by verapamil, which blocks L-type voltage-activated Ca2 + channel. EDTA or NiCl2 inhibited it much more. Xestspongin C, an IP3 receptor-selective antagonist, or ruthenium red, a ryanodine-sensitive receptor blocker, significantly suppressed the kallikrein secretion. These results suggest that the attenuated response of renal kallikrein secretion by potassium in SHR may be involved in mechanisms associated with extracellular Ca2 + entry via Ca2 + channels including L-type voltage-activated Ca2 + channels or with Ca2 + release from the store sites, which have IP3 receptor channels and ryanodine-sensitive receptor. It is hypothesized that high potassium may increase intracellular Ca2 + levels following membrane depolarization of the CNT, triggering a signal transduction of renal kallikrein secretion. Secretion of renal kallikrein will probably occur by means of exocytosis, where the vesicle membrane containing secretory granules fuses with the plasma membrane. Immunoelectric micrographic study has shown that kallikrein granules were present beneath the plasma membrane of the CNT cells [30]. In general, it is well known that exocytosis needs an increase in intracellular Ca2 + concentration [30,31]. Further study to compare Ca2 + mobilization in the cells of the CNT between SHR and WKY should be required to explore mechanisms for attenuated renal kallikrein secretion by potassium in SHR. In conclusion, functionally suppressed secretion of renal kallikrein from CNT would be involved in the development of hypertension in the early stage. Results from the present study may also provide a new approach to enhance the renal KKS, leading to a hypothesis on the preventive role of renal KKS in the early phase of hypertension. On this basis, novel types of antihypertensive drugs to enhance the renal KKS would be proposed. The present study indicated that the potassium-induced renal kallikrein secretion was diminished in the CNT of young SHR. Considering a previous report where children of hypertensive families excrete less kallikrein than those of normotensive
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families [27], the present results will direct to identify a genetic factor responsible for the development of essential hypertension.
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[24] Hunt SC, Hasstedt SJ, Wu LL, Williams RR. Agene – environment interaction between inferred kallikrein genotype and potassium. Hypertension 1993;22:161 – 8. [25] Majima M, Adachi K, Ohno T, Ogino M, Saito M, et al. Failure of the oxytocin-induced increase in secretion of urinary kallikrein in young spontaneously hypertensive rats. Jpn J Pharmacol 1996;71(1):11 – 9. [26] Fujita T, Hayashi I, Kumagai Y, Inamura N, Majima M. Early increases in renal kallikrein secretion on administration of potassium or ATP-sensitive potassium channel blockers in rats. Br J Pharmacol 1999;128:1275 – 83. [27] Wang T, Wang WH, Klein-Robbenhaar G, Giebisch G. Effects of a novel KATP channel blockers on renal tubule function and K channel activity. J Pharmacol Exp Ther 1995;273: 1382 – 9. [28] Stokes JB. Cellular K+ permeation across the cortical collecting tubule: effects of Na+ – K+ pump inhibition and membrane depolarization. Am J Physiol 1984;246(4 Pt 2): F467 – 75. [29] Frindt G, Palmer LG. Low-conductance K channels in apical membrane of rat cortical collecting tubule. Am J Physiol 1989;256(1 Pt 2):F143 – 51. [30] Boyd III AE, Aguilar-Bryan L, Bryan J, Kunze DL, Moss L, et al. Sulfonylurea signal transduction. Recent Prog Horm Res 1991;47:299 – 316. [31] Ashcroft FM. Mechanisms of the glycemic effects of sulfonylureas. Horm Metab Res 1996;28:456 – 63.
Potassium-induced increase in renal kallikrein secretion is attenuated in dissected renal connecting tubules of young spontaneously hypertensive rats Mariko Yamanaka a,b, Izumi Hayashi c,d, Tomoe Fujita c, Seok Ho Cha e, Hitoshi Endou e, Masaaki Higashihara a,b, Masataka Majima c,d,* a Fourth Department of Internal Medicine, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan Fourth Department of Internal Medicine, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa 228-8555, Japan c Department of Pharmacology, Kitasato University School of Medicine, Sagamihara, Kanagawa 228-8555, Japan d Department of Molecular Pharmacology, Kitasato University Graduate School of Medical Sciences, Sagamihara, Kanagawa 228-8555, Japan e Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan b
Abstract It is suggested that attenuation of the renal kallikrein – kinin system (KKS) involved the development of hypertension in young spontaneously hypertensive rats (SHR). In the present study, a comparison was made between young SHR and Wistar Kyoto rats (WKY) to examine the ability to secrete renal kallikrein from the microdissected connecting tubules (CNT) by potassium or an ATP-sensitive potassium channel blocker, 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A), both of which are renal kallikrein secretagogues. Maximum effect of potassium on kallikrein secretion was observed 10 min after placing the tubules at concentration of 20 mM. Kallikrein secretion was also increased concentration-dependently by PNU-37883A (0.1, 1, 10, and 100 AM). In the presence of EDTA, NiCl2, verapamil, xestspongin C (an inositol 1,4,5-trisphosphate (IP3) receptor-selective antagonist), or ruthenium red (a ryanodine-sensitive receptor blocker), potassium-induced increase in renal kallikrein secretion was inhibited. Augmentation of renal kallikrein secretion by potassium or PNU-37883A was diminished in SHR compared to WKY. These results indicate that the ability to secrete renal kallikrein by potassium was attenuated in young SHR compared with WKY. Furthermore, it is suggested that the potassium-induced renal kallikrein secretion requires an extracellular Ca2 + entry through Ca2 + channels including L-type Ca2 + channels and Ca2 + release from intracellular Ca2 + stores through IP3 receptor and ryanodine receptor. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Renal kallikrein secretion; Microdissected connecting tubules; Potassium; ATP-sensitive potassium channel blocker; Spontaneously hypertensive rats
1. Introduction * Corresponding author. Department of Pharmacology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Tel.: +81-42-778-8822; fax: +81-42778-8467. E-mail address: [email protected] (M. Majima).
There are two forms of kallikrein –kinin system (KKS), plasma KKS and tissue KKS, where bioactive peptides of kinins are generated from a high and low
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molecular weight kininogens by kallikrein, respectively [1]. In the kidney, renal kallikreins are localized in the connecting tubules (CNT) and cortical collecting tubules [2– 4]. They release kinins into urine from kininogens, which were also localized in distal tubules and collecting tubules [4,5]. In the early 1970s, it was reported that patients with essential hypertension excrete less urinary kallikrein than normal healthy subjects [6– 8]. Thereafter, many reports have suggested preventive effect of renal KKS on the development of hypertension [9– 11]. Renal KKS has been considered to play an important role in the homeostasis of sodium and water balance in the kidney [12,13]. Spontaneously hypertensive rats (SHR) are known as an important animal model for essential hypertension. The systolic blood pressure of SHR starts to increase from the age of 4 weeks, and the hypertension develops from the age of 7 –15 weeks. Excretion of urinary kallikrein in 4-week-old SHR has been shown to be significantly lower than that in agematched Wistar Kyoto rats (WKY), the normotensive controls [13,14]. Continuous infusion of a nonpeptide mimic of bradykinin into renal artery of 6-week-old SHR for 6 days significantly reduced mean blood pressure [15], suggesting attenuation of renal KKS activity in young SHR may be involved in facilitation of the development of hypertension. It was known that the supplementation of potassium lowered high blood pressure in animal models and in humans. Several studies indicate that potassium loading can increase the secretion of renal kallikrein. Systemic potassium loading increases urinary kallikrein excretion in both humans [16,17] and animals [18,19], and high concentration of potassium solution increases release of renal kallikrein from rat sliced kidney [20]. In addition, natriuresis induced by potassium loading is inhibited by treatment of rats with bradykinin B2 receptor antagonist [21]. However, variable response of blood pressure to potassium intake was reported in hypertensive patients [22]; thus, hereditary factors were also postulated in a comparative study of urinary potassium and kallikrein excretion using a genetic analysis of monozygotic and dizygotic twins [23]. Maximum likelihood segregation analysis on 769 individuals in 58 Utah pedigrees reported that a urinary kallikrein level was not associated with urinary potassium level in the inferred kallikrein genotype of low homozygotes [24], sug-
gesting a genetic defect is involved in the renal kallikrein in response to potassium. Additionally, a previous report showing accumulation of renal kallikrein granules in the CNT of SHR [25] suggests disordered renal kallikrein secretion in SHR. Therefore, this study was aimed to investigate whether potassium-induced increase in renal kallikrein secretion was attenuated in CNT of young SHR compared with that of WKY. CNT were isolated from both strains of rat by the microdissection technique so that a direct effect of drugs on the increase in renal kallikrein secretion could be examined. We previously reported that 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A), which can block the ATP-sensitive potassium channels on renal tubules selectively, showed an augmentative effect on renal kallikrein secretion without an additive effect on potassium-induced increase in renal kallikrein secretion [26]. Thus, the effect of PNU37883A on renal kallikrein secretion was also compared between SHR and WKY. Furthermore, mechanisms of the potassium-induced increase in renal kallikrein secretion were also studied with respect to extracellular Ca2 + entry and the release from intracellular calcium stores.
2. Materials and methods 2.1. Materials Male Sprague –Dawley (SD) rats were purchased from SLC Japan (Hamamatsu, Japan). Male SHR and WKY were supplied from Sankyo Lab. Animal Service (Tokyo, Japan). All rats used were 4– 6 weeks old and specific pathogen-free. Rats were given normal rat chow and tap water ad libitum. They were housed at constant humidity (60 F 5%) and temperature (25 F 1 jC) and were kept in a continuous 12-h light– dark cycle. All of the procedures were conducted in accordance with the guiding principles for the care and use of laboratory animals approved by the Animal Care Committee of Kitasato University. The following drugs were used: 4-morpholinecarboximidine-N-1-adamantyl-NV-cyclohexylhydrochloride (PNU-37883A) was a gift from Pharmacia and Upjohn (Kalamazoo, MI); disodium-ethylenediaminetetraacetate, dihydrate (EDTA-2Na, Doujindo, Kuma-
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moto, Japan), and nickel chloride (NiCl2, SigmaAldrich, St. Louis, MO) were purchased. Verapamil, xestospongin C, and ruthenium red were purchased from Wako (Osaka, Japan). All other chemicals were the analytical grade of commercial sources. 2.2. Methods 2.2.1. Microdissection of CNT CNT were isolated according to the previous report by Tomita et al. [2] with a slight modification. Briefly, immediately after rats were subjected to cerebral concussion, laparotomy was performed. Two parts of the abdominal aorta, upper and lower, away from the bifurcation of the left renal artery, were ligated. Through the needle placed in the abdominal aorta, kidneys were infused with Hank’s solution containing 1 mM CaCl2, bovine serum albumin (1 mg/ml, Sigma-Aldrich), and collagenase type I (1 mg/ml, Sigma-Aldrich). The circulating blood of the left kidney streamed out of the renal vein. The left kidney was nephrectomized and was sliced to a thickness of approximately 1 mm. Those slices were incubated at 37 jC in the solution described above under the aerobic condition. Those preparations, kept at 4 jC, were observed under a stereomicroscope and microdissection was performed. The obtained single nephron segment, CNT, was used for experiment. 2.2.2. Experimental procedure CNT were dissected from the kidney of SHR, WKY, and SD rats after collagenase digestion under a stereomicroscope. Dissected CNT prepared from the kidney were placed in the solution containing 20 mM KCl, 140 mM NaCl, 1 mM CaCl2, and 10 mM HEPES (pH 7.4) for 0, 10, or 20 min. To examine dose-dependent effects of potassium and PNU37883A, the CNT were incubated in the solution containing KCl (at the concentration of 0, 10, 20, 40, or 70 mM) or PNU-37883A (at the concentration of 0, 0.1, 1, 10, or 100 AM) for 10 min. To maintain isotonicity of the solution, concentration of NaCl was decreased to 70 mM for 70 mM KCl, 100 mM for 40 mM KCl, 120 mM for 20 mM KCl, and 130 mM for 10 mM KCl. For incubation with PNU-37883A, 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl and 1 mM CaCl2 was used. In the examination for effects of Ca2 + channel blockers on K+-induced
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kallikrein secretion, dissected CNT of SD rats were placed in the above buffer and subsequently in saline for 10 min. Then the CNT were incubated with the buffer containing 1 mM EDTA, 1 mM NiCl2, or 100 AM verapamil. Each reagent was dissolved in saline and was spiked in the medium. In the experiment to test effects of IP3 receptor-selective antagonist or ryanodine-sensitive receptor blocker on K+-induced kallikrein secretion, the CNT were placed in the solution containing xestspongin C (0.3, 3, or 30 AM), or ruthenium red (50 AM). Xestspongin C (1.33 mg/ml) or ruthenium red (4.29 mg/ml) was dissolved in 1% ethanol or saline, respectively. A medium containing 1% ethanol alone was used for control experiments of xestspongin C. 2.2.3. Measurement of released kallikrein activity Kallikrein activities in both incubated supernatant and the CNT were measured using a peptidyl fluorogenic substrate for glandular kallikrein, Pro – Phe– Arg – 4-methyl-coumaryl-amide (Pro – Phe – Arg – MCA, Peptide Institute, Minoh, Osaka, Japan). The CNT were solubilized with 5 Al of 5% Triton-X for 10 min at room temperature to release the retained kallikrein in the tubules. No visible nephron contour could be observed under stereomicroscopy in this condition. The solubilized tubules were dispersed into the solution in 100 Al of 10 mM HEPES (pH 7.4) containing 140 mM NaCl and 1 mM CaCl2 and was incubated at 37 jC for 30 min with 1 Al of Pro– Phe – Arg –MCA (10 mM) diluted with 0.05 M Tris – HCl (pH 8.0), 0.1 M NaCl, and 0.01 M CaCl2. After the incubation, 5 Al of 17% acetic acid was added to stop the reaction. Fluorescence intensity of the incubation mixture was measured at 380 nm (excitation) and at 460 nm (emission) by fluorescence spectrophotometer (M850; Hitachi, Tokyo, Japan). Renal kallikrein secretion was expressed as released kallikrein (%), which was defined as the ratio of the activity to total kallikrein activity (the released kallikrein and the kallikrein retained in the CNT). 2.2.4. Data analysis Values are expressed as means F S.E.M. One-way ANOVA or Student’s t-test was used to evaluate statistical significance of difference. Differences with a probability of 5% or less were considered to be significant ( P < 0.05).
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3. Results 3.1. Time course of the potassium-induced increase in renal kallikrein secretion from dissected CNT In order to examine time course of change in renal kallikrein secretion by potassium, one piece of tubule was incubated with buffer containing 20 mM KCl for 0, 10, and 20 min. Renal kallikrein secretion increased significantly to approximately fourfold within 10 min after the incubation. The amount of released kallikrein by high potassium did not increase more at a 20-min period (Fig. 1). 3.2. Concentration-dependent increase in renal kallikrein secretion by potassium ion and PNU37883A Several concentrations of potassium and PNU37883A were examined whether the renal kallikrein secretion increased with a concentration-dependent manner. Renal kallikrein secretion increased at a concentration of 20 mM KCl with a peak and then tended to decrease at the concentrations of 40 and 70 mM (Fig. 2a). PNU-37883A increased renal kallikrein secretion in a concentration-dependent manner, and
Fig. 2. Concentration-dependent increase in renal kallikrein secretion by KCl and an ATP-sensitive potassium channel blocker. Effects of a high potassium solution at a concentration of 10, 20, 40, or 70 mM on renal kallikrein secretion were tested. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer as described in Fig. 1 in the absence (0), or in the presence of KCl (10 to 70 mM) (a) or PNU-37883A (0.1 to 100 AM) (b) for 10 min. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Fisher’s PLSD and Scheffe´ test. *P < 0.05, significantly different from the value in the absence of the agents.
significant increase was observed at concentrations of 10 and 100 AM (Fig. 2b). Fig. 1. Time course of renal kallikrein release from dissected CNT induced by KCl. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) containing 20 mM KCl, 120 mM NaCl, and 1 mM CaCl2 for 0, 10, or 20 min, kallikrein activities in the buffer and in the tubule were measured by using the fluorogenic substrate, Pro – Phe – Arg – MCA. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Scheffe´ test. *P < 0.05, significantly different from the value at 0 min.
3.3. Effects of EDTA, NiCl2, verapamil, xestospongin C, and ruthenium red on the potassium-induced increase in kallikrein secretion from CNT In order to examine whether extracellular Ca2 + influx was involved in the potassium-induced renal kallikrein secretion, the tubule was incubated with a solution containing EDTA, NiCl2, or verapamil followed by stimulation with 20 mM of KCl. As shown
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in Fig. 3, 20 mM of KCl increased the kallikrein release sixfold. Treatment of CNT with EDTA (1 mM), NiCl2 (1 mM), or verapamil (100 AM) significantly suppressed the augmentation of kallikrein release by 20 mM of KCl. In order to examine whether Ca2 + release from intracellular Ca2 + stores is involved in the potassium-induced renal kallikrein secretion, the tubules were treated with xestospongin C or ruthenium red in the presence of 20 mM KCl. As shown in Fig. 4a, the augmented renal kallikrein release by potassium was significantly suppressed by 3 and 30 AM of xestospongin C in a dose-dependent manner. Ruthenium red (50 AM) also showed a significant suppression of the augmented secretion (Fig. 4b). 3.4. Comparison of the potassium- or PNU-37883Ainduced increases in renal kallikrein secretion between young SHR and WKY As shown in Fig. 5a, renal kallikrein secretion from CNT of WKY rat was increased by stimulation with KCl with a peak at the concentration of 20 mM and no further increase was observed at concentrations of 40 and 70 mM, same as in SD rats. Released kallikrein
Fig. 3. Effects of EDTA, NiCl2, and verapamil on the potassiuminduced increase in renal kallikrein secretion. Dissected CNT prepared from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl, and 1 mM CaCl2 in the absence or presence of EDTA (1 mM), NiCl2 (1 mM), or verapamil (100 AM) for 10 min. Treatment with saline was as the vehicle. Left open column indicates the value without stimulation by 20 mM KCl. Subsequently, the tubule was further incubated with 10 mM HEPES (pH 7.4) buffer containing 20 mM KCl, 120 mM NaCl, 1 mM CaCl2 for 10 min. Each agent was dissolved in saline. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. Data were analyzed by one-way analysis of variance followed by post hoc Scheffe´ test. *P < 0.05, significantly different from value in the vehicle-treated group.
Fig. 4. Suppression of KCl-induced renal kallikrein by IP3 receptorselective antagonist and ryanodine receptor channel blocker. Effects of xestospongin C on the potassium-induced increase in renal kallikrein secretion were observed. CNT dissected from kidney of SD rats were incubated with 10 mM HEPES (pH 7.4) buffer containing 140 mM NaCl, and 1 mM CaCl2 without or with xestospongin C (0.3, 3, and 30 AM) (a) or ruthenium red (50 AM) (b) for 10 min. Subsequently, the tubule was further incubated with 10 mM HEPES (pH 7.4) buffer containing 20 mM KCl, 120 mM NaCl, and 1 mM CaCl2 for 10 min. Xestspongin C and ruthenium red were dissolved in 1% ethanol and saline, respectively. Values are means F S.E.M. Data were analyzed by one-way analysis of variance followed by post hoc Fisher’s PLSD. *P < 0.05, significantly different from the values in the vehicle-treated group. Treatments of CNT with 1% ethanol or saline were as the vehicles for xestspongin C and ruthenium red, respectively. Left open column in each panel indicates the value in the absence of 20 mM KCl. Numbers in parentheses indicate numbers of CNT used.
level in SHR was similar to that in WKY when the tubule was incubated with buffer without KCl. The secretion from CNT of SHR was increased by addition of 10 –70 mM of KCl. However, the levels at concentrations of 20 and 40 mM of KCl in SHR rats were significantly less than those in WKY. PNU-37883A at a concentration of 0.1AM provided a maximum effect on renal kallikrein secretion from CNT of WKY as shown in Fig. 5b. The augmentation in SHR was less than those in WKY. Significant augmentation of the secretion by PNU-37883A at the concentrations of 0.1, 1, and 10 AM from CNT was also observed in SHR. However, significant differences of the augmen-
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Fig. 5. Comparison of renal kallikrein secretion between young SHR and WKY rats by KCl or an ATP-sensitive potassium channel blocker. Dissected CNT prepared from the kidney of SHR and WKY were incubated with 10 mM HEPES (pH 7.4) buffer as described in Fig. 1 in the absence (0), or in the presence of KCl (10, 20, 40, and 70 mM) (a) or PNU-37883A (0.1, 1, 10, and 100 AM) (b) for 10 min. Values are means F S.E.M. Numbers in parentheses indicate numbers of CNT used. *P < 0.05 and #P < 0.05, significantly different from the value in the absence of the agents in WKY and SHR rats, respectively. yP < 0.05, significantly different of values between WKY and SHR rats. In the experiments using SHR and WKY, Student’s t-test was used for the comparison of renal kallikrein secretion between SHR and WKY at each concentration of potassium or PNU-37883A. One-way ANOVA determined to evaluate the significance levels between the control and each concentration of drug treatment with potassium or PNU-37883A.
tation were observed between WKY and SHR at the concentrations of 1 and 100 AM of PNU-37883A.
4. Discussion Several reports suggest that high potassium intake increase urinary kallikrein excretion as a result of augmentation of secretion of kallikrein or the synthesis induced by glucocorticoids, prostaglandins, or even directly [16,19]. It is possible that potassium supplementation increase the release of aldosterone,
and that this increased aldosterone may upregulate kallikrein synthesis in the CNT. In the present study, a microdissected single nephron of CNT, where renal kallikrein is located, was used to examine renal kallikrein secretion, so that endocrine influences on renal kallikrein secretion from CNT could be ruled out. As a result, a direct effect of potassium to increase renal kallikrein secretion was demonstrated by using CNT. Prolonged incubation of CNT with 20 mM KCl or high concentrations of KCl (40 and 70 mM) resulted in decrease in the potassium-induced augmentation of renal kallikrein secretion. This may be caused by toxic effect of potassium. It was also shown that PNU-37883A, an ATP-sensitive potassium channel blocker, has a direct augmentative effect on renal kallikrein secretion. CNT are known to participate, at least in part, in the secretion of potassium in the distal nephrons. Furthermore, it is known that excess amount of potassium was mainly excreted from distal tubules. Therefore, the concentration of potassium around CNT should become high when high potassium is loaded into the body [17]. An ATPsensitive potassium channel is one of potassium channels, which is known to modulate sodium, potassium, and chloride reabsorption. It also controls potassium secretion on the thick ascending limb of the loop of Henle, distal tubules, and CNT [27]. The above results suggest that CNT not only regulate electrolyte transport but also secrete kallikrein in association with potassium channels. Indeed, the distal nephrons maintain the membrane potential of the tubular cells and membrane of the tubule cells are depolarized in the presence of high potassium in the collecting duct [28,29]. Thus, it is likely that potassium and PNU-37883A may directly stimulate renal kallikrein secretion through changes in membrane potential of CNT. We previously reported that the site of action of potassium and PNU-37883A was the source since no additive effect of those two secretagogues was observed in terms of release of kallikrein [26]. Attenuated responses of CNT to secrete renal kallikrein by potassium or PNU-37883A were demonstrated in SHR compared with WKY. These results indicate that SHR has some disordered mechanisms that may contribute to weaken to secrete renal kallikrein by potassium and PNU-37883A. In case of the islet cells, it has been reported that the exocytosis of
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insulin induced by an ATP-sensitive potassium channel blocker is accompanied with increase in intracellular Ca2 + concentration, resulting from gating of voltage-activated Ca2 + channels by cell depolarization. L-type channel cDNAs have been identified in insulin-secreting clonal h cells [30,31]. The present study indicates that the potassium-induced increase in renal kallikrein secretion was partially inhibited by verapamil, which blocks L-type voltage-activated Ca2 + channel. EDTA or NiCl2 inhibited it much more. Xestspongin C, an IP3 receptor-selective antagonist, or ruthenium red, a ryanodine-sensitive receptor blocker, significantly suppressed the kallikrein secretion. These results suggest that the attenuated response of renal kallikrein secretion by potassium in SHR may be involved in mechanisms associated with extracellular Ca2 + entry via Ca2 + channels including L-type voltage-activated Ca2 + channels or with Ca2 + release from the store sites, which have IP3 receptor channels and ryanodine-sensitive receptor. It is hypothesized that high potassium may increase intracellular Ca2 + levels following membrane depolarization of the CNT, triggering a signal transduction of renal kallikrein secretion. Secretion of renal kallikrein will probably occur by means of exocytosis, where the vesicle membrane containing secretory granules fuses with the plasma membrane. Immunoelectric micrographic study has shown that kallikrein granules were present beneath the plasma membrane of the CNT cells [30]. In general, it is well known that exocytosis needs an increase in intracellular Ca2 + concentration [30,31]. Further study to compare Ca2 + mobilization in the cells of the CNT between SHR and WKY should be required to explore mechanisms for attenuated renal kallikrein secretion by potassium in SHR. In conclusion, functionally suppressed secretion of renal kallikrein from CNT would be involved in the development of hypertension in the early stage. Results from the present study may also provide a new approach to enhance the renal KKS, leading to a hypothesis on the preventive role of renal KKS in the early phase of hypertension. On this basis, novel types of antihypertensive drugs to enhance the renal KKS would be proposed. The present study indicated that the potassium-induced renal kallikrein secretion was diminished in the CNT of young SHR. Considering a previous report where children of hypertensive families excrete less kallikrein than those of normotensive
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families [27], the present results will direct to identify a genetic factor responsible for the development of essential hypertension.
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