European Journal of Pharmacology 743 (2014) 37–41
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Effects of systemic administration of kynurenic acid and glycine on renal haemodynamics and excretion in normotensive and spontaneously hypertensive rats Bożena Bądzyńska a, Izabela Zakrocka b, Janusz Sadowski a, Waldemar A. Turski b, Elżbieta Kompanowska-Jezierska a,n a b
Department of Renal and Body Fluid Physiology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland Department of Experimental and Clinical Pharmacology, Medical University, Ceramiczna 1, 20-150 Lublin, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 July 2014 Received in revised form 11 September 2014 Accepted 11 September 2014 Available online 28 September 2014
Both NMDA receptor and kynurenic acid (KYNA), a glycine-site NMDA receptor antagonist, are present in the kidney yet their functional role remains unclear. Our aim was to examine effects of intravenous KYNA and glycine on arterial blood pressure (MAP) and renal haemodynamics and excretion in anaesthetized normotensive Sprague–Dawley (S–D) and in spontaneously hypertensive (SHR) rats. Renal blood flow (RBF, renal artery probe) and renal cortical (CBF) and outer- and inner medullary perfusion (laser-Doppler) were measured, along with diuresis (V) and sodium excretion (UNaV). KYNA given alone (150 mg kg 1 iv) or during infusion of glycine at 1 g kg 1 h 1 iv (Gþ K) increased or decreased RBF, respectively, in both S–D and SHR. Neither treatment altered MAP. In both strains glycine alone increased RBF and CBF 50–60% and was clearly diuretic and natriuretic, less so in SHR. KYNA increased UNaV by 4.171.7 μmol min 1and V by 11.174.3 μl min 1 in S–D (Po0.05 for both); the respective increases in SHR were by 1.770.6 μmol min 1 and 4.771.7 μl min 1 (Po0.02 for both). GþK treatment increased UNaV by 5.271.4 μmol min 1 (Po0.01) and V by 29.674.6 μmol min 1 (Po0.001) in S–D, and by 2.770.7 μmol min 1 (Po0.05) and 19.373.5 μl min 1 (Po0.0006) in SHR. In conclusion, KYNA increased renal excretion, apparently by inhibiting tubular reabsorption, whereas glycine substantially increased renal haemodynamics by an illdefined mechanism, with a secondary increase in the excretion. Combined Gþ K treatment could be utilised to combat body fluid retention and possibly alleviate hypertension, without endangering renal perfusion and function. & 2014 Elsevier B.V. All rights reserved.
Keywords: Renal haemodynamics Renal excretion Kynurenic acid Glycine
1. Introduction Kynurenic acid (KYNA), a tryptophan metabolite, is an antagonist of glutamate N-methyl-D-aspartate (NMDA), kainate, αamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and alpha-7 nicotinic acetylcholine receptors (Stone 1993; Hilmas et al. 2001). It also affects G-protein-coupled receptor GPR35 (Wang et al. 2006). Concentration of KYNA in human urine was estimated to be 4–40 μM (Zhao et al. 2011; Crow et al. 2008; Hiratsuka et al. 2012).
Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; CBF, cortical blood flow; D-AP5, D-2-amino-5-phosphonopentanoic acid; GFR, glomerular filtration rate; IMBF, inner medullary blood flow; KYNA, kynurenic acid; MAP, mean arterial pressure; NMDA, N-methyl-D-aspartic acid; OMBF, outer medullary blood flow; RBF, renal blood flow; SHR, spontaneously hypertensive rats; TGF, tubulo-glomerular feedback; UNaV, sodium excretion; V, urine flow n Corresponding author. Tel.: þ 48 22 6086546; fax: þ 48 22 6685532. E-mail address: [email protected] (E. Kompanowska-Jezierska). http://dx.doi.org/10.1016/j.ejphar.2014.09.020 0014-2999/& 2014 Elsevier B.V. All rights reserved.
It was proven that exogenous KYNA is well absorbed from the gastrointestinal tract in rats and very easily penetrates to the blood stream, reaching high concentration in the kidney (Turski et al. 2009). In animal studies, KYNA administered intrathecally or into the rostral ventrolateral medulla decreased arterial blood pressure (Mills et al. 1990; Bergamaschi et al. 1995; Ito et al. 2000, 2001). It is noteworthy that this effect was more pronounced in hypertensive rats, suggesting that KYNA plays an important role in restoring the cardiovascular system function under pathological conditions. Kidney tissue contains large amounts of KYNA (Pawlak et al. 2003), however, its involvement in controlling renal haemodynamics and excretion is not established. In a frequently quoted work, NMDA receptors were detected in the proximal tubules, and intraperitoneal KYNA caused renal vasoconstriction and attenuated the renal vasodilator response to glycine infusion; no data on renal excretion were reported (Deng et al. 2002). The aim of the present study was to examine effects of KYNA, applied intravenously, on renal haemodynamics and excretion in anaesthetized rats. Since the excretion may be influenced not only by the rate
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of total renal perfusion (renal blood flow, RBF) and glomerular filtration (GFR) but also by changing intrarenal regional circulation, especially in the medulla, medullary perfusion was also measured using laser-Doppler methodology. Since the effects of exogenous KYNA may depend on baseline NMDA receptor activity, we studied the responses either without or with background activation of these receptors with glycine. The long established effect of glycine to increase renal haemodynamics and excretion (Pitts, 1944) was described as renal “functional reserve” which was reported to be lacking in experimental hypertension (De Nicola et al. 1991). Therefore, effects of both glycine and KYNA were examined in parallel studies with normotensive and spontaneously hypertensive rats (SHR).
2.4. Analytical procedures and statistics
2. Materials and methods
3. Results
2.1. Animals
3.1. Effects on haemodynamics
Male Sprague–Dawley rats (280–300 g), and spontaneously hypertensive rats (SHR, 270–280 g) were anaesthetised with intraperitoneal thiopentobarbital (Thiopental, Sandoz-Kundl, Austria), 100 mg kg 1 body weight. The experimental procedures were approved by the extramural IV Local Ethical Committee (Warsaw). 2.2. Surgical procedures and measurements Effects were examined of intravenously applied kynurenic acid (KYNA, Sigma-Aldrich, St. Louis, MO, USA) on blood pressure, renal haemodynamics and renal excretion in rats which were either untreated or pre-treated with glycine (Sigma-Aldrich). The rats were placed on a heated surgery table to maintain rectal temperature at about 37 1C. A polyethylene tube was placed in the trachea to ensure free airways. The left kidney was surgically exposed from a subcostal flank incision immobilised in a plastic holder, similar as that used for micropuncture. The ureter was cannulated for timed urine collection. To maintain plasma volume, during surgical preparation 3% bovine albumin (Applied Chemistry GmbH, Darmstadt, Germany) in Ringer solution was infused i.v. at the volume rate of 3 ml h 1. At the end of surgical preparation and after placement of the probes used for intrarenal measurements, the infusion of albumin was replaced by one which delivered 3 ml h 1 of isotonic saline. Measurements of mean arterial blood pressure (MAP, Stoelting BP Meter, Stoelting, Wood Dale IL, USA), renal excretion, renal artery blood flow (RBF, Transonic noncannulating Flow Probe T106, Transonic, Ithaca, NY, USA) and laserDoppler fluxes (Periflux 4001 system, Perimed, Jarfalla, Sweden) which measured perfusion of the renal cortex (CBF), outer and inner medulla (OMBF and IMBF), were conducted throughout experiments. The details of experimental procedures and measurements were described previously (Bądzyńska and Sadowski 2006). 2.3. Protocols After control measurements and urine collections (C), a bolus of KYNA (K, 150 mg kg 1 iv) dissolved in Ringer solution alkalized to pH 7.8 was given (C þ K test). Preliminary studies have shown that this was the smallest dose which regularly altered renal haemodynamics and excretion. Alternatively, KYNA was given during a background infusion of glycine (G, 1 g kg 1 h 1), within a C þG þK test. The two tests were performed in one experiment, separated by at least one-hour recovery periods. After experiments, the rats were killed with a lethal intravenous dose of sodium thiopenthal and the position of the intrarenal probes was checked at the kidney's cross-section.
Urine volume was determined gravimetrically. Urine sodium concentration was measured by a flame photometer (Jenway PFP7, Essex, UK), and urine osmolality using a cryoscopic osmometer (Osmomat 030, Gonotec GmbH, Berlin, Germany). The differences between mean values were evaluated by repeated measurement analysis of variance (ANOVA) followed by paired Student's t test. For inter-group comparison, the classical oneway ANOVA was used, followed by post-hoc modified t statistics (Wallenstein et al. 1980). Bonferroni correction was used for multiple comparisons. The standard error of the mean (S.E.M.) measured data dispersion, and Pr0.05 was accepted as the significance level.
The basal mean arterial pressure (MAP) was 120 72 mmHg in Sprague–Dawley rats and 166 75 mmHg in SHR. Administration of glycine, KYNA or glycine þKYNA did not significantly alter MAP (Table 1). A representative tracing of RBF responses to KYNA given alone (C þK) or superimposed on glycine infusion (C þG þK) in an S–D rat and in an SHR is shown in Fig. 1, and averaged haemodynamic data for the two rat groups are given in top panels of Fig. 2. In Sprague–Dawley rats only administration of KYNA slightly increased RBF and CBF (Fig. 2, top panels). Glycine infusion induced a substantial increase in RBF and CBF in both rat strains. Administration of KYNA induced a modest but significant decrease in glycine-enhanced RBF and did not affect glycine-enhanced CBF, similarly in Sprague–Dawley rats and SHR. In Sprague–Dawley rats KYNA infusion did not alter medullary perfusion parameters, OMBF and IMBF, but glycine alone tended to increase them: OMBF from 171714 to 188717 PU, and IMBF from 99710 to 109713 PU (differences not significant). KYNA decreased glycine-enhanced IMBF from 109713 to 94715 PU (Po0.03). In SHR, KYNA alone did not alter the medullary perfusion parameters. Glycine alone significantly increased OMBF, from 164 to 189 712 PU (P o0.007). KYNA decreased glycine-enhanced OMBF from 189 712 to 15478 PU (Po0.03) and IMBF from 72 77 to 6276 PU (Po 0.02). 3.2. Effects on renal excretion The data on renal excretion are presented in the lower panels of Fig. 2. In both Sprague–Dawley rats and SHR administration of KYNA significantly increased sodium excretion (UNaV) and urine flow (V). Glycine infusion induced an increase in UNaV in Sprague– Dawley rats only but it increased V in both rat strains. Administration of KYNA enhanced glycine-induced changes in renal excretion parameters measured in both Sprague–Dawley rats and SHR (Fig. 2). The changes in total solute excretion (UosmV) were roughly parallel to those of UNaV and V (the data are not shown in Fig. 2). Glycine significantly increased UosmV in S–D rats only, from Table 1 Mean arterial pressure (MAP 7 SEM, mmHg) in Sprague–Dawley (SD, n¼8) and spontaneously hypertensive rats (SHR, n ¼9). Comparison of values under control conditions, and after KYNA (K) given alone or injected during glycine infusion (Gþ K).
SD SHR
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Fig. 1. A representative tracing of RBF changes induced by KYNA (150 mg kg 1 iv) given during control saline infusion and by glycine infusion (1 g kg 1 h 1) followed by KYNA at the same dose, in an S–D rat and in an SHR.
7 72 to 18 72 μosmol min 1 (P o0.001). The subsequent increases after KYNA, to 28 73 μosmol min 1 in S–D rats and to 167 2 μosmol min 1 in SHR were both significant (P o0.02 in each case). KYNA given alone increased UosmV from 11 71 to 17 71 μosmol min 1 in S–D rats (P o0.0001), a corresponding slight increase in SHR, from 7 71 to 10 71 μosmol min 1 was not significant.
4. Discussion 4.1. Systemic administration of KYNA and blood pressure We found that acute, systemic administration of KYNA did not alter arterial pressure (MAP) in normotensive Sprague–Dawley rats and SHR. This is in agreement with the findings of Deng et al. (2002) who reported that in normotensive Wistar rats blood pressure was not altered by systemic administration of 5,7dichlorokynurenic acid, an inhibitor of glycine site of NMDA-R, or MK-801, a non-competitive NMDA antagonist. Nevertheless, in a later study Deng and Thomson (2009) did register a MAP decrease after intravenous administration of MK-801 (Deng and Thomson 2009). It should be noted, however, that MK-801 readily penetrates the blood–brain barrier and KYNA does not. Unlike with systemic administration, KYNA administered intrathecally or into the rostral ventrolateral medulla regularly exerted hypotensive action (Mills et al. 1990; Bergamaschi et al. 1995; Ito et al. 2000, 2001). The central antihypertensive effect was repeatedly confirmed and found to be more pronounced in spontaneously hypertensive rats (Mills et al. 1990; Kapoor et al. 1994; Mizutani et al. 2002). Taken together, the results from the literature and our findings indicate that blood pressure-lowering effects of KYNA are mediated by its action on brain structures without any demonstrable direct short-term influence at the periphery. However, the effect of long-lasting systemic administration of KYNA on blood pressure has never been investigated. Therefore, a chronic antihypertensive action of KYNA related to its natriuretic and diuretic effects, as shown in our study, cannot be ruled out.
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was seen to slightly reduce the glycine-induced increase in RBF. Our results agree in part with those of Deng et al. (2002) who reported that a post-glycine increase in RBF was prevented by 5,7dichlorokynurenic acid . The same blocking effect was observed with MK-801, another NMDA receptor antagonist. On the other hand, Yang et al. (2008) observed that NMDA, a NMDA receptor agonist, did not affect renal blood flow. The mechanism of renal hyperaemic action of glycine appears complex, including both an ill-defined direct vasorelaxant effect and an indirect action mediated by stimulation of NMDA-R localised in the proximal tubules (Deng et al. 2002). Stimulation of these NMDA receptors by glycine increases proximal reabsorption of salt and water, and reduces the delivery of fluid to the distal tubular macula densa receptor area. This inhibits the usual vasoconstrictor signalling to the preglomerular arterioles, a typical response within the tubulo-glomerular feedback mechanism (TGF) and, ultimately, RBF and GFR are increased. However, more recent studies failed to confirm the stimulatory action of glycine on proximal reabsorption (Deng and Thomson 2009). Irrespective of the prevailing mechanism (primary vascular or tubular) of glycineinduced renal hyperaemia, we found that renal haemodynamic response was not abolished or reduced in SHR. Glycine-induced changes in the perfusion of the renal medulla were parallel and most probably secondary to substantial changes in total RBF. Surprisingly, the renal haemodynamics increased also after KYNA, an inhibitor of co-agonistic glycine site on NMDA receptor. It can be assumed that under baseline conditions there was no tonic vasodilator influence of NMDA receptor, hence their inhibition by KYNA did not result in vasoconstriction. Remarkably, modest vasoconstriction did occur when KYNA was given after infusion of glycine which must have activated NMDA receptor. Nevertheless, the reason why renal haemodynamics actually increased after KYNA (given alone) remains obscure. The reduction by KYNA of glycine-induced renal hyperaemia, as shown by Deng et al. (2002) and in this study, supports NMDA receptor dependent control of renal vascular tone. One can reason that intrarenal KYNA might buffer activation of NMDA receptors induced by plasma glycine level which might be elevated after a high-protein meal. Under physiological conditions this mechanism would moderate a possible excessive mobilisation of the “renal functional reserve” as described by Pitts (1944), and help maintain minute-to-minute body fluid balance. However, the exact nature of such a balancing mechanism is unknown. There is no clear-cut evidence on the presence of active NMDA receptor in the renal vasculature. One can speculate that inhibition of proximal epithelial NMDA receptor by KYNA altered TGF activity in the direction opposite to that described for glycine (see above), leading to a decrease GFR and RBF. Indeed, MK-801, an NMDA channel blocker, was reported to decrease proximal reabsorption (Deng and Thomson, 2009). It should be remembered, however, that the responses to KYNA and MK-801 need not be quite similar: the nature of antagonistic action of the two compounds is different and, in contrast to KYNA, MK-801 readily penetrates the blood–brain barrier and may thereby have additional central effects, also in the kidney. 4.3. Glycine and KYNA effects on renal excretion
4.2. Glycine and KYNA effects on renal haemodynamics We found systemic administration of KYNA slightly increased total RBF in S–D rats, without changing perfusion of the renal medulla. Remarkably, glycine acted in the same direction and the glycine-induced increase in renal haemodynamics was even more substantial; this action in the same direction was unexpected because KYNA is an NMDA antagonist acting on glycine site. Some antagonistic effect of the two agents was observed when KYNA
We found that KYNA invariably increased renal excretion in both normotensive Sprague–Dawley strain and in SHR. This finding is in accordance with the observation that in Wistar rats renal artery infusion of D-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist, increased renal excretion and GFR and blocked the decreases thereof induced by NMDA agonist (Yang et al. 2008). Both observations strongly suggest a receptor mediated event. On the other hand, our finding that both in
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RBF 18
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Fig. 2. Total renal blood flow (RBF) and cortical perfusion (CBF, laser-Doppler flux), and renal excretion of sodium (UNaV) and urine flow (V). Data for the left kidney of normotensive Sprague–Dawley rats and SHR, showing effects of KYNA (K), in animals receiving either control saline infusion (C, blank columns) or under background glycine infusion (G, striped columns). In S–D group n¼ 8; in SHR n¼ 9 * different from the appropriate control value at P o0.05 or less, # statistically different from G þ K, † statistically different from K.
B. Bądzyńska et al. / European Journal of Pharmacology 743 (2014) 37–41
normotensive and hypertensive rats a glycine-induced increase in renal excretion was substantially augmented by subsequent administration of KYNA suggests that the baseline activity of NMDA receptor is not a crucial determinant of KYNA-induced natriuresis and diuresis. The additive effects of glycine and KYNA, both enhancing renal excretion, can be explained by involvement of two different mechanisms, primarily glomerular in the case of glycine and primarily tubular for KYNA. Our observations may have a therapeutic potential: combined systemic application of both drugs would deplete body fluids and help lower elevated arterial blood pressure. Owing to glycine the risk is eliminated of kidney ischaemia and likely deterioration of its function, which are common side-effects of standard diuretic therapy. Notably, natriuretic and diuretic effects of the combined treatment were visible also in SHR. Conflict of interest None declared Acknowledgements. The study was funded in part by the Foundation for Polish Science. References Bądzyńska, B., Sadowski, J., 2006. Renal hemodynamic responses to intrarenal infusion of acetylcholine: comparison with effects of PGE2 and NO donor. Kidney Int. 69, 1774–1779. Bergamaschi, C., Campos, R.R., Schor, N., Lopes, O.U., 1995. Role of the rostral ventrolateral medulla in maintenance of blood pressure in rats with Goldblatt hypertension. Hypertension 26, 1117–1120. Crow, B., Bishop, M., Paliakov, E., Norton, D., George, J., Bralley, J.A., 2008. Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry. Biomed. Chromatogr. 22, 1346–1353. De Nicola, L., Blantz, R.C., Gabbai, F.B., 1991. Renal functional reserve in treated and untreated hypertensive rats. Kidney Int. 40, 406–412. Deng, A., Thomson, S.C., 2009. Renal NMDA receptors independently stimulate proximal reabsorption and glomerular filtration. Am. J. Physiol. Renal Physiol. 296, F976–F982. http://dx.doi.org/10.1152/ajprenal.90391.2008.
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Deng, A., Valdivielso, J.M., Munger, K.A., Blantz, R.C., Thomson, S.C., 2002. Vasodilatory N-methyl-D-aspartate receptors are constitutively expressed in rat kidney. J. Am. Soc. Nephrol. 13, 1381–1384. Hilmas, C., Pereira, E.F., Alkondon, M., Rassoulpour, A., Schwarcz, R., Albuquerque, E.X., 2001. The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J. Neurosci. 21, 7463–7473. Hiratsuka, C., Fukuwatari, T., Shibata, K., 2012. Fate of dietary tryptophan in young Japanese women. Int. J. Tryptophan Res. 5, 33–47. Ito, S., Komatsu, K., Tsukamoto, K., Sved, A.F., 2000. Excitatory amino acids in the rostral ventrolateral medulla support blood pressure in spontaneously hypertensive rats. Hypertension 35, 413–417. Ito, S., Komatsu, K., Tsukamoto, K., Sved, A.F., 2001. Tonic excitatory input to the rostral ventrolateral medulla in Dahl salt-sensitive rats. Hypertension 37, 687–691. Kapoor, V., Kapoor, R., Chalmers, J., 1994. Kynurenic acid, an endogenous glutamate antagonist, in SHR and WKY rats: possible role in central blood pressure regulation. Clin. Exp. Pharmacol. Physiol. 21, 891–896. Mills, E., Minson, J., Drolet, G., Chalmers, J., 1990. Effect of intrathecal amino acid receptor antagonists on basal blood pressure and pressor responses to brainstem stimulation in normotensive and hypertensive rats. J. Cardiovasc. Pharmacol. 15, 877–883. Mizutani, K., Sugimoto, K., Okuda, T., Katsuya, T., Miyata, T., Tanabe, T., Higaki, J., Ogihara, T., Yamori, Y., Tsujita, Y., Tago, N., Iwai, N., 2002. Kynureninase is a novel candidate gene for hypertension in spontaneously hypertensive rats. Hypertens. Res. 25, 135–140. Pawlak, D., Tankiewicz, A., Matys, T., Buczko, W., 2003. Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure. J. Physiol. Pharmacol. 54, 175–189. Pitts, R.F., 1944. The effect of infusing glycine and varying the dietary protein intake on renal hemodynamics in the dog. Am. J. Physiol. 142, 355–365. Stone, T.W., 1993. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45, 309–379. Turski, M.P., Turska, M., Zgrajka, W., Kuc, D., Turski, W.A., 2009. Presence of kynurenic acid in food and honeybee products. Amino Acids 36, 75–80. Wang, J., Simonavicius, N., Wu, X., Swaminath, G., Reagan, J., Tian, H., Ling, L., 2006. Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. J. Biol. Chem. 281, 22021–22028. Wallenstein, S., Zucker, C.L., Fleiss, J.L., 1980. Some statistical methods useful in circulation research. Circ. Res. 47 (1), 1–9. Yang, C.C., Chien, C.T., Wu, M.H., Ma, M.C., Chen, C.F., 2008. NMDA receptor blocker ameliorates ischemia‐reperfusion‐induced renal dysfunction in rat kidneys. Am. J. Physiol. Renal Physiol. 294, F1433–F1440. http://dx.doi.org/10.1152/ ajprenal.00481.2007. Zhao, J., Chen, H., Ni, P., Xu, B., Luo, X., Zhan, Y., Gao, P., Zhu, D., 2011. Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 2720–2725.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Effects of systemic administration of kynurenic acid and glycine on renal haemodynamics and excretion in normotensive and spontaneously hypertensive rats Bożena Bądzyńska a, Izabela Zakrocka b, Janusz Sadowski a, Waldemar A. Turski b, Elżbieta Kompanowska-Jezierska a,n a b
Department of Renal and Body Fluid Physiology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland Department of Experimental and Clinical Pharmacology, Medical University, Ceramiczna 1, 20-150 Lublin, Poland
art ic l e i nf o
a b s t r a c t
Article history: Received 2 July 2014 Received in revised form 11 September 2014 Accepted 11 September 2014 Available online 28 September 2014
Both NMDA receptor and kynurenic acid (KYNA), a glycine-site NMDA receptor antagonist, are present in the kidney yet their functional role remains unclear. Our aim was to examine effects of intravenous KYNA and glycine on arterial blood pressure (MAP) and renal haemodynamics and excretion in anaesthetized normotensive Sprague–Dawley (S–D) and in spontaneously hypertensive (SHR) rats. Renal blood flow (RBF, renal artery probe) and renal cortical (CBF) and outer- and inner medullary perfusion (laser-Doppler) were measured, along with diuresis (V) and sodium excretion (UNaV). KYNA given alone (150 mg kg 1 iv) or during infusion of glycine at 1 g kg 1 h 1 iv (Gþ K) increased or decreased RBF, respectively, in both S–D and SHR. Neither treatment altered MAP. In both strains glycine alone increased RBF and CBF 50–60% and was clearly diuretic and natriuretic, less so in SHR. KYNA increased UNaV by 4.171.7 μmol min 1and V by 11.174.3 μl min 1 in S–D (Po0.05 for both); the respective increases in SHR were by 1.770.6 μmol min 1 and 4.771.7 μl min 1 (Po0.02 for both). GþK treatment increased UNaV by 5.271.4 μmol min 1 (Po0.01) and V by 29.674.6 μmol min 1 (Po0.001) in S–D, and by 2.770.7 μmol min 1 (Po0.05) and 19.373.5 μl min 1 (Po0.0006) in SHR. In conclusion, KYNA increased renal excretion, apparently by inhibiting tubular reabsorption, whereas glycine substantially increased renal haemodynamics by an illdefined mechanism, with a secondary increase in the excretion. Combined Gþ K treatment could be utilised to combat body fluid retention and possibly alleviate hypertension, without endangering renal perfusion and function. & 2014 Elsevier B.V. All rights reserved.
Keywords: Renal haemodynamics Renal excretion Kynurenic acid Glycine
1. Introduction Kynurenic acid (KYNA), a tryptophan metabolite, is an antagonist of glutamate N-methyl-D-aspartate (NMDA), kainate, αamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and alpha-7 nicotinic acetylcholine receptors (Stone 1993; Hilmas et al. 2001). It also affects G-protein-coupled receptor GPR35 (Wang et al. 2006). Concentration of KYNA in human urine was estimated to be 4–40 μM (Zhao et al. 2011; Crow et al. 2008; Hiratsuka et al. 2012).
Abbreviations: AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; CBF, cortical blood flow; D-AP5, D-2-amino-5-phosphonopentanoic acid; GFR, glomerular filtration rate; IMBF, inner medullary blood flow; KYNA, kynurenic acid; MAP, mean arterial pressure; NMDA, N-methyl-D-aspartic acid; OMBF, outer medullary blood flow; RBF, renal blood flow; SHR, spontaneously hypertensive rats; TGF, tubulo-glomerular feedback; UNaV, sodium excretion; V, urine flow n Corresponding author. Tel.: þ 48 22 6086546; fax: þ 48 22 6685532. E-mail address: [email protected] (E. Kompanowska-Jezierska). http://dx.doi.org/10.1016/j.ejphar.2014.09.020 0014-2999/& 2014 Elsevier B.V. All rights reserved.
It was proven that exogenous KYNA is well absorbed from the gastrointestinal tract in rats and very easily penetrates to the blood stream, reaching high concentration in the kidney (Turski et al. 2009). In animal studies, KYNA administered intrathecally or into the rostral ventrolateral medulla decreased arterial blood pressure (Mills et al. 1990; Bergamaschi et al. 1995; Ito et al. 2000, 2001). It is noteworthy that this effect was more pronounced in hypertensive rats, suggesting that KYNA plays an important role in restoring the cardiovascular system function under pathological conditions. Kidney tissue contains large amounts of KYNA (Pawlak et al. 2003), however, its involvement in controlling renal haemodynamics and excretion is not established. In a frequently quoted work, NMDA receptors were detected in the proximal tubules, and intraperitoneal KYNA caused renal vasoconstriction and attenuated the renal vasodilator response to glycine infusion; no data on renal excretion were reported (Deng et al. 2002). The aim of the present study was to examine effects of KYNA, applied intravenously, on renal haemodynamics and excretion in anaesthetized rats. Since the excretion may be influenced not only by the rate
38
B. Bądzyńska et al. / European Journal of Pharmacology 743 (2014) 37–41
of total renal perfusion (renal blood flow, RBF) and glomerular filtration (GFR) but also by changing intrarenal regional circulation, especially in the medulla, medullary perfusion was also measured using laser-Doppler methodology. Since the effects of exogenous KYNA may depend on baseline NMDA receptor activity, we studied the responses either without or with background activation of these receptors with glycine. The long established effect of glycine to increase renal haemodynamics and excretion (Pitts, 1944) was described as renal “functional reserve” which was reported to be lacking in experimental hypertension (De Nicola et al. 1991). Therefore, effects of both glycine and KYNA were examined in parallel studies with normotensive and spontaneously hypertensive rats (SHR).
2.4. Analytical procedures and statistics
2. Materials and methods
3. Results
2.1. Animals
3.1. Effects on haemodynamics
Male Sprague–Dawley rats (280–300 g), and spontaneously hypertensive rats (SHR, 270–280 g) were anaesthetised with intraperitoneal thiopentobarbital (Thiopental, Sandoz-Kundl, Austria), 100 mg kg 1 body weight. The experimental procedures were approved by the extramural IV Local Ethical Committee (Warsaw). 2.2. Surgical procedures and measurements Effects were examined of intravenously applied kynurenic acid (KYNA, Sigma-Aldrich, St. Louis, MO, USA) on blood pressure, renal haemodynamics and renal excretion in rats which were either untreated or pre-treated with glycine (Sigma-Aldrich). The rats were placed on a heated surgery table to maintain rectal temperature at about 37 1C. A polyethylene tube was placed in the trachea to ensure free airways. The left kidney was surgically exposed from a subcostal flank incision immobilised in a plastic holder, similar as that used for micropuncture. The ureter was cannulated for timed urine collection. To maintain plasma volume, during surgical preparation 3% bovine albumin (Applied Chemistry GmbH, Darmstadt, Germany) in Ringer solution was infused i.v. at the volume rate of 3 ml h 1. At the end of surgical preparation and after placement of the probes used for intrarenal measurements, the infusion of albumin was replaced by one which delivered 3 ml h 1 of isotonic saline. Measurements of mean arterial blood pressure (MAP, Stoelting BP Meter, Stoelting, Wood Dale IL, USA), renal excretion, renal artery blood flow (RBF, Transonic noncannulating Flow Probe T106, Transonic, Ithaca, NY, USA) and laserDoppler fluxes (Periflux 4001 system, Perimed, Jarfalla, Sweden) which measured perfusion of the renal cortex (CBF), outer and inner medulla (OMBF and IMBF), were conducted throughout experiments. The details of experimental procedures and measurements were described previously (Bądzyńska and Sadowski 2006). 2.3. Protocols After control measurements and urine collections (C), a bolus of KYNA (K, 150 mg kg 1 iv) dissolved in Ringer solution alkalized to pH 7.8 was given (C þ K test). Preliminary studies have shown that this was the smallest dose which regularly altered renal haemodynamics and excretion. Alternatively, KYNA was given during a background infusion of glycine (G, 1 g kg 1 h 1), within a C þG þK test. The two tests were performed in one experiment, separated by at least one-hour recovery periods. After experiments, the rats were killed with a lethal intravenous dose of sodium thiopenthal and the position of the intrarenal probes was checked at the kidney's cross-section.
Urine volume was determined gravimetrically. Urine sodium concentration was measured by a flame photometer (Jenway PFP7, Essex, UK), and urine osmolality using a cryoscopic osmometer (Osmomat 030, Gonotec GmbH, Berlin, Germany). The differences between mean values were evaluated by repeated measurement analysis of variance (ANOVA) followed by paired Student's t test. For inter-group comparison, the classical oneway ANOVA was used, followed by post-hoc modified t statistics (Wallenstein et al. 1980). Bonferroni correction was used for multiple comparisons. The standard error of the mean (S.E.M.) measured data dispersion, and Pr0.05 was accepted as the significance level.
The basal mean arterial pressure (MAP) was 120 72 mmHg in Sprague–Dawley rats and 166 75 mmHg in SHR. Administration of glycine, KYNA or glycine þKYNA did not significantly alter MAP (Table 1). A representative tracing of RBF responses to KYNA given alone (C þK) or superimposed on glycine infusion (C þG þK) in an S–D rat and in an SHR is shown in Fig. 1, and averaged haemodynamic data for the two rat groups are given in top panels of Fig. 2. In Sprague–Dawley rats only administration of KYNA slightly increased RBF and CBF (Fig. 2, top panels). Glycine infusion induced a substantial increase in RBF and CBF in both rat strains. Administration of KYNA induced a modest but significant decrease in glycine-enhanced RBF and did not affect glycine-enhanced CBF, similarly in Sprague–Dawley rats and SHR. In Sprague–Dawley rats KYNA infusion did not alter medullary perfusion parameters, OMBF and IMBF, but glycine alone tended to increase them: OMBF from 171714 to 188717 PU, and IMBF from 99710 to 109713 PU (differences not significant). KYNA decreased glycine-enhanced IMBF from 109713 to 94715 PU (Po0.03). In SHR, KYNA alone did not alter the medullary perfusion parameters. Glycine alone significantly increased OMBF, from 164 to 189 712 PU (P o0.007). KYNA decreased glycine-enhanced OMBF from 189 712 to 15478 PU (Po0.03) and IMBF from 72 77 to 6276 PU (Po 0.02). 3.2. Effects on renal excretion The data on renal excretion are presented in the lower panels of Fig. 2. In both Sprague–Dawley rats and SHR administration of KYNA significantly increased sodium excretion (UNaV) and urine flow (V). Glycine infusion induced an increase in UNaV in Sprague– Dawley rats only but it increased V in both rat strains. Administration of KYNA enhanced glycine-induced changes in renal excretion parameters measured in both Sprague–Dawley rats and SHR (Fig. 2). The changes in total solute excretion (UosmV) were roughly parallel to those of UNaV and V (the data are not shown in Fig. 2). Glycine significantly increased UosmV in S–D rats only, from Table 1 Mean arterial pressure (MAP 7 SEM, mmHg) in Sprague–Dawley (SD, n¼8) and spontaneously hypertensive rats (SHR, n ¼9). Comparison of values under control conditions, and after KYNA (K) given alone or injected during glycine infusion (Gþ K).
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Fig. 1. A representative tracing of RBF changes induced by KYNA (150 mg kg 1 iv) given during control saline infusion and by glycine infusion (1 g kg 1 h 1) followed by KYNA at the same dose, in an S–D rat and in an SHR.
7 72 to 18 72 μosmol min 1 (P o0.001). The subsequent increases after KYNA, to 28 73 μosmol min 1 in S–D rats and to 167 2 μosmol min 1 in SHR were both significant (P o0.02 in each case). KYNA given alone increased UosmV from 11 71 to 17 71 μosmol min 1 in S–D rats (P o0.0001), a corresponding slight increase in SHR, from 7 71 to 10 71 μosmol min 1 was not significant.
4. Discussion 4.1. Systemic administration of KYNA and blood pressure We found that acute, systemic administration of KYNA did not alter arterial pressure (MAP) in normotensive Sprague–Dawley rats and SHR. This is in agreement with the findings of Deng et al. (2002) who reported that in normotensive Wistar rats blood pressure was not altered by systemic administration of 5,7dichlorokynurenic acid, an inhibitor of glycine site of NMDA-R, or MK-801, a non-competitive NMDA antagonist. Nevertheless, in a later study Deng and Thomson (2009) did register a MAP decrease after intravenous administration of MK-801 (Deng and Thomson 2009). It should be noted, however, that MK-801 readily penetrates the blood–brain barrier and KYNA does not. Unlike with systemic administration, KYNA administered intrathecally or into the rostral ventrolateral medulla regularly exerted hypotensive action (Mills et al. 1990; Bergamaschi et al. 1995; Ito et al. 2000, 2001). The central antihypertensive effect was repeatedly confirmed and found to be more pronounced in spontaneously hypertensive rats (Mills et al. 1990; Kapoor et al. 1994; Mizutani et al. 2002). Taken together, the results from the literature and our findings indicate that blood pressure-lowering effects of KYNA are mediated by its action on brain structures without any demonstrable direct short-term influence at the periphery. However, the effect of long-lasting systemic administration of KYNA on blood pressure has never been investigated. Therefore, a chronic antihypertensive action of KYNA related to its natriuretic and diuretic effects, as shown in our study, cannot be ruled out.
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was seen to slightly reduce the glycine-induced increase in RBF. Our results agree in part with those of Deng et al. (2002) who reported that a post-glycine increase in RBF was prevented by 5,7dichlorokynurenic acid . The same blocking effect was observed with MK-801, another NMDA receptor antagonist. On the other hand, Yang et al. (2008) observed that NMDA, a NMDA receptor agonist, did not affect renal blood flow. The mechanism of renal hyperaemic action of glycine appears complex, including both an ill-defined direct vasorelaxant effect and an indirect action mediated by stimulation of NMDA-R localised in the proximal tubules (Deng et al. 2002). Stimulation of these NMDA receptors by glycine increases proximal reabsorption of salt and water, and reduces the delivery of fluid to the distal tubular macula densa receptor area. This inhibits the usual vasoconstrictor signalling to the preglomerular arterioles, a typical response within the tubulo-glomerular feedback mechanism (TGF) and, ultimately, RBF and GFR are increased. However, more recent studies failed to confirm the stimulatory action of glycine on proximal reabsorption (Deng and Thomson 2009). Irrespective of the prevailing mechanism (primary vascular or tubular) of glycineinduced renal hyperaemia, we found that renal haemodynamic response was not abolished or reduced in SHR. Glycine-induced changes in the perfusion of the renal medulla were parallel and most probably secondary to substantial changes in total RBF. Surprisingly, the renal haemodynamics increased also after KYNA, an inhibitor of co-agonistic glycine site on NMDA receptor. It can be assumed that under baseline conditions there was no tonic vasodilator influence of NMDA receptor, hence their inhibition by KYNA did not result in vasoconstriction. Remarkably, modest vasoconstriction did occur when KYNA was given after infusion of glycine which must have activated NMDA receptor. Nevertheless, the reason why renal haemodynamics actually increased after KYNA (given alone) remains obscure. The reduction by KYNA of glycine-induced renal hyperaemia, as shown by Deng et al. (2002) and in this study, supports NMDA receptor dependent control of renal vascular tone. One can reason that intrarenal KYNA might buffer activation of NMDA receptors induced by plasma glycine level which might be elevated after a high-protein meal. Under physiological conditions this mechanism would moderate a possible excessive mobilisation of the “renal functional reserve” as described by Pitts (1944), and help maintain minute-to-minute body fluid balance. However, the exact nature of such a balancing mechanism is unknown. There is no clear-cut evidence on the presence of active NMDA receptor in the renal vasculature. One can speculate that inhibition of proximal epithelial NMDA receptor by KYNA altered TGF activity in the direction opposite to that described for glycine (see above), leading to a decrease GFR and RBF. Indeed, MK-801, an NMDA channel blocker, was reported to decrease proximal reabsorption (Deng and Thomson, 2009). It should be remembered, however, that the responses to KYNA and MK-801 need not be quite similar: the nature of antagonistic action of the two compounds is different and, in contrast to KYNA, MK-801 readily penetrates the blood–brain barrier and may thereby have additional central effects, also in the kidney. 4.3. Glycine and KYNA effects on renal excretion
4.2. Glycine and KYNA effects on renal haemodynamics We found systemic administration of KYNA slightly increased total RBF in S–D rats, without changing perfusion of the renal medulla. Remarkably, glycine acted in the same direction and the glycine-induced increase in renal haemodynamics was even more substantial; this action in the same direction was unexpected because KYNA is an NMDA antagonist acting on glycine site. Some antagonistic effect of the two agents was observed when KYNA
We found that KYNA invariably increased renal excretion in both normotensive Sprague–Dawley strain and in SHR. This finding is in accordance with the observation that in Wistar rats renal artery infusion of D-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist, increased renal excretion and GFR and blocked the decreases thereof induced by NMDA agonist (Yang et al. 2008). Both observations strongly suggest a receptor mediated event. On the other hand, our finding that both in
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B. Bądzyńska et al. / European Journal of Pharmacology 743 (2014) 37–41
RBF 18
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Fig. 2. Total renal blood flow (RBF) and cortical perfusion (CBF, laser-Doppler flux), and renal excretion of sodium (UNaV) and urine flow (V). Data for the left kidney of normotensive Sprague–Dawley rats and SHR, showing effects of KYNA (K), in animals receiving either control saline infusion (C, blank columns) or under background glycine infusion (G, striped columns). In S–D group n¼ 8; in SHR n¼ 9 * different from the appropriate control value at P o0.05 or less, # statistically different from G þ K, † statistically different from K.
B. Bądzyńska et al. / European Journal of Pharmacology 743 (2014) 37–41
normotensive and hypertensive rats a glycine-induced increase in renal excretion was substantially augmented by subsequent administration of KYNA suggests that the baseline activity of NMDA receptor is not a crucial determinant of KYNA-induced natriuresis and diuresis. The additive effects of glycine and KYNA, both enhancing renal excretion, can be explained by involvement of two different mechanisms, primarily glomerular in the case of glycine and primarily tubular for KYNA. Our observations may have a therapeutic potential: combined systemic application of both drugs would deplete body fluids and help lower elevated arterial blood pressure. Owing to glycine the risk is eliminated of kidney ischaemia and likely deterioration of its function, which are common side-effects of standard diuretic therapy. Notably, natriuretic and diuretic effects of the combined treatment were visible also in SHR. Conflict of interest None declared Acknowledgements. The study was funded in part by the Foundation for Polish Science. References Bądzyńska, B., Sadowski, J., 2006. Renal hemodynamic responses to intrarenal infusion of acetylcholine: comparison with effects of PGE2 and NO donor. Kidney Int. 69, 1774–1779. Bergamaschi, C., Campos, R.R., Schor, N., Lopes, O.U., 1995. Role of the rostral ventrolateral medulla in maintenance of blood pressure in rats with Goldblatt hypertension. Hypertension 26, 1117–1120. Crow, B., Bishop, M., Paliakov, E., Norton, D., George, J., Bralley, J.A., 2008. Analysis of urinary aromatic acids by liquid chromatography tandem mass spectrometry. Biomed. Chromatogr. 22, 1346–1353. De Nicola, L., Blantz, R.C., Gabbai, F.B., 1991. Renal functional reserve in treated and untreated hypertensive rats. Kidney Int. 40, 406–412. Deng, A., Thomson, S.C., 2009. Renal NMDA receptors independently stimulate proximal reabsorption and glomerular filtration. Am. J. Physiol. Renal Physiol. 296, F976–F982. http://dx.doi.org/10.1152/ajprenal.90391.2008.
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Deng, A., Valdivielso, J.M., Munger, K.A., Blantz, R.C., Thomson, S.C., 2002. Vasodilatory N-methyl-D-aspartate receptors are constitutively expressed in rat kidney. J. Am. Soc. Nephrol. 13, 1381–1384. Hilmas, C., Pereira, E.F., Alkondon, M., Rassoulpour, A., Schwarcz, R., Albuquerque, E.X., 2001. The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J. Neurosci. 21, 7463–7473. Hiratsuka, C., Fukuwatari, T., Shibata, K., 2012. Fate of dietary tryptophan in young Japanese women. Int. J. Tryptophan Res. 5, 33–47. Ito, S., Komatsu, K., Tsukamoto, K., Sved, A.F., 2000. Excitatory amino acids in the rostral ventrolateral medulla support blood pressure in spontaneously hypertensive rats. Hypertension 35, 413–417. Ito, S., Komatsu, K., Tsukamoto, K., Sved, A.F., 2001. Tonic excitatory input to the rostral ventrolateral medulla in Dahl salt-sensitive rats. Hypertension 37, 687–691. Kapoor, V., Kapoor, R., Chalmers, J., 1994. Kynurenic acid, an endogenous glutamate antagonist, in SHR and WKY rats: possible role in central blood pressure regulation. Clin. Exp. Pharmacol. Physiol. 21, 891–896. Mills, E., Minson, J., Drolet, G., Chalmers, J., 1990. Effect of intrathecal amino acid receptor antagonists on basal blood pressure and pressor responses to brainstem stimulation in normotensive and hypertensive rats. J. Cardiovasc. Pharmacol. 15, 877–883. Mizutani, K., Sugimoto, K., Okuda, T., Katsuya, T., Miyata, T., Tanabe, T., Higaki, J., Ogihara, T., Yamori, Y., Tsujita, Y., Tago, N., Iwai, N., 2002. Kynureninase is a novel candidate gene for hypertension in spontaneously hypertensive rats. Hypertens. Res. 25, 135–140. Pawlak, D., Tankiewicz, A., Matys, T., Buczko, W., 2003. Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure. J. Physiol. Pharmacol. 54, 175–189. Pitts, R.F., 1944. The effect of infusing glycine and varying the dietary protein intake on renal hemodynamics in the dog. Am. J. Physiol. 142, 355–365. Stone, T.W., 1993. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45, 309–379. Turski, M.P., Turska, M., Zgrajka, W., Kuc, D., Turski, W.A., 2009. Presence of kynurenic acid in food and honeybee products. Amino Acids 36, 75–80. Wang, J., Simonavicius, N., Wu, X., Swaminath, G., Reagan, J., Tian, H., Ling, L., 2006. Kynurenic acid as a ligand for orphan G protein-coupled receptor GPR35. J. Biol. Chem. 281, 22021–22028. Wallenstein, S., Zucker, C.L., Fleiss, J.L., 1980. Some statistical methods useful in circulation research. Circ. Res. 47 (1), 1–9. Yang, C.C., Chien, C.T., Wu, M.H., Ma, M.C., Chen, C.F., 2008. NMDA receptor blocker ameliorates ischemia‐reperfusion‐induced renal dysfunction in rat kidneys. Am. J. Physiol. Renal Physiol. 294, F1433–F1440. http://dx.doi.org/10.1152/ ajprenal.00481.2007. Zhao, J., Chen, H., Ni, P., Xu, B., Luo, X., Zhan, Y., Gao, P., Zhu, D., 2011. Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879, 2720–2725.
