Activation of Nitric Oxide—Cyclic Guanosine Monophosphate Signaling in Kidney by Extracorporeal Shock Wave Therapy

0022-5347/03/1706-2459/0 THE JOURNAL OF UROLOGY® Copyright © 2003 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 170, 2459 –2462, December 2003 Printed in U.S.A.

DOI: 10.1097/01.ju.0000094186.19728.c2

ACTIVATION OF NITRIC OXIDE–CYCLIC GUANOSINE MONOPHOSPHATE SIGNALING IN KIDNEY BY EXTRACORPOREAL SHOCK WAVE THERAPY JONG KWAN PARK,* YONG CUI, HYUNG JIN KIM, HEUI KYOUNG OH, GOU YOUNG KOH AND KYUNG WOO CHO From the Departments of Urology (JKP, YC, HJK, HKO) and Physiology (KWC), Chonbuk National University Medical School and Chonbuk National University Institute for Medical Sciences and Research Institute of Clinical Medicine of Chonbuk National University Hospital, Chonju and Pohang University of Science and Technology (GYK), Pohang, South Korea

ABSTRACT

Purpose: We defined whether extracorporeal shock wave therapy (ESWT) to the kidney activates the nitric oxide (NO)-cyclic 3⬘,5⬘-guanosine monophosphate (cGMP) pathway. Materials and Methods: A total of 90 male rabbits were randomly divided into group 1—pretreated with normal saline, group 2—pretreated intravenously (IV) with N␻ nitro-L-arginine-methyl ester (NAME) (100 mg/kg), group 3—pretreated IV with NAME and L-arginine (300 mg/kg) with ESWT to 1 kidney, group 4 —pretreated IV with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) (20 ␮g/kg) and group 5—pretreated with normal saline with ESWT to the bladder. Plasma nitrite, NO metabolite and cGMP were analyzed in peripheral blood samples before, immediately after, and 30 and 60 minutes after ESWT. Results: ESWT to the kidney but not to the bladder caused an increase before, immediately after, and 30 and 60 minutes after ESWT in plasma nitrite in group 1 (186.1 ⫾ 20.6, 217.5 ⫾ 21.6, 241.9 ⫾ 28.4 and 230.5 ⫾ 25.3 nM) and group 5 (149.0 ⫾ 14.7, 155.6 ⫾ 18.4, 131.8 ⫾ 13.6 and 140.0 ⫾ 15.7 nM), and in cGMP in group 1 (24.2 ⫾ 1.9, 33.8 ⫾ 3.2, 32.9 ⫾ 2.2 and 29.4 ⫾ 1.9 pmol/ml) and group 5 (25.5 ⫾ 2.1, 27.5 ⫾ 2.5, 28.7 ⫾ 3.1 and 25.5 ⫾ 2.6 pmol/ml, respectively). In group 2 NAME significantly inhibited the production of nitrite (113.4 ⫾ 18.6, 118.2 ⫾ 19.9, 114.6 ⫾ 18.3 and 112.5 ⫾ 17.6 nM) and cGMP (19.4 ⫾ 2.6, 20.6 ⫾ 2.8, 19.3 ⫾ 2.7 and 18.6 ⫾ 2.6 pmol, respectively). In group 3 inhibited nitrite and cGMP production caused by NAME was recovered with L-arginine. In group 4 ODQ significantly inhibited cGMP production. Conclusions: The results show that ESWT increases the level of NO and cGMP released by the kidney in an animal model. KEY WORDS: kidney, rabbits, 5⬘-guanylic acid, lithotripsy, nitric oxide

Extracorporeal shock wave therapy (ESWT) is widely used for ureterorenal stones. Although the main force of shock wave energy is focused on to the stone, ESWT is not completely free of trauma.1 The mechanisms underlying shock wave induced renal injury are not completely understood. Shear stress, thermal and cavitation effects, and free radical formation have been discussed.1 To prevent potential renal injury caused by impaired renal hemodynamics substances have been used, including calcium antagonists to restore renal perfusion decreased by vasoconstriction in states of enhanced vascular resistance by ESWT.1 We have also suggested that ESWT for renal stones increases the production of nitric oxide (NO) and cyclic 3⬘,5⬘-guanosine monophosphate (cGMP), and activation of the NO-cGMP pathway by ESWT may attenuate renovascular contraction by trauma in humans.2 However, little is known about the effect of ESWT on the regulation of NO-cGMP signaling. In kidney endothelial cells, including vessels, glomeruli, mesangial cells and renal tubular epithelial cells, release vasoactive substances in response to trauma.3, 4 Endothelial NO synthase (NOS) is present in renal epithe-

lium and in vivo NO may be derived from constitutive NOS activity of the adjacent glomerular capillary endothelium or it may diffuse from the nearby macula densa.5 NO has been highly effective in increasing cGMP accumulation. cGMP elevation activates cGMP dependent protein kinase, which in turn decreases intracellular calcium, resulting in smooth muscle cell relaxation.6 Activation of the NO-cGMP signaling pathway may counteract the contraction caused by vasoactive hormones locally. NO production may be changed in response to a wide variety of stimuli to renal cells.7 Shear stress and cyclic strain have been reported to be effective stimuli for the release of endogenous endothelial vasodilators, such as NO.8, 9 ESWT to the kidney can also produce vasoactive substances from renal cells as a stimulus. Previously we have suggested that only ESWT for renal stones induced changes in renal hemodynamic by activating NO-cGMP in humans.2 However, we suggested these possible mechanisms because we did not use inhibitors to dissect the pathway, such as NOS or guanylate cyclase, needed for the regulation of NO-cGMP in humans. Therefore, in the current study we clarified the origin of NO and the actual relationship between NO and cGMP. To this end we used NOS and guanylate cyclase modulators in rabbits.

Accepted for publication June 13, 2003. Supported by Grant 02-PJ1-PG10 –21401– 0003 from the Korea Health 21 R & D Project, Ministry of Health and Welfare, Republic of Korea. * Corresponding author: Department of Urology, Chonbuk NaMATERIALS AND METHODS tional University, Medical School, 2–20, Keum-Am-Dong-San, Animal. A total of 72 adult New Zealand White male rabChonju, 561–712, South Korea (telephone: 82– 63-250 –1510; FAX: bits weighing approximately 3 kg (range 2.5 to 3.5) were 82– 63-250 –1564; e-mail: [email protected]). 2459

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used. Rabbits were divided randomly into 5 groups. Each group of rabbits was exposed to a different treatment before ESWT. In group 1, 18 rabbits were injected with normal saline 150 minutes before ESWT to the left kidney. In group 2, 18 rabbits were injected intravenously (IV) with N␻ nitroL-arginine-methyl ester (NAME) (100 mg/kg) 150 minutes before ESWT to the left kidney. In group 3, 18 rabbits were injected IV with NAME (100 mg/kg) and L-arginine (300 mg/kg) 150 minutes before ESWT. In group 4, 18 rabbits were injected IV with ODQ (1H-1, 2, 4oxadiazolo[4,3a]quinoxalin-1-one (20 ␮g/kg) 60 minutes before ESWT. In group 5, 18 rabbits were injected with 6 ml (2 ml/kg) normal saline 150 minutes before ESWT to the bladder. The injected volume of chemical agents was 6 ml (2 ml/kg). After blood sampling the rabbits were anesthetized IV with sodium thiopental (30 mg/kg) and exsanguinated. Animal treatment. The animals were anesthetized with intramuscular injection of a mixture that contained ketamine and xylazine. It was given at a dose of 1 ml/kg body weight (45 mg/kg ketamine and 3 mg/kg xylazine). For ESWT exposure the back or lower abdomen of the anesthetized animals was shaved. The animal was placed supine or prone on the lithotriptor for the kidney or bladder, respectively. ESWT. ESWT was performed using a 9200-X (Dornier Medizintechnik GmbH, Wessling Munchen, Germany) multiple purpose lithotriptor. A computer assisted, ultrasound guided system was used for kidney or bladder localization. A silicone membrane covered, water filled reflector was brought into contact with the rabbit flank or lower abdomen to provide an air-free pathway for energy delivery. Blood sampling. Blood samples were collected from ear artery using Neoflon (Becton Dickinson Corp., Helsingborg, Sweden) before, immediately after, and 30 and 60 minutes after ESWT based on the previous experiment.2 Blood sampling from the ear artery was started 10 minutes after anesthesia. An indwelling Neoflon intravenous cannula was flushed with heparin sodium solution (Green Cross Corp., Seoul, South Korea) (5,000 IU/ml) to inhibit coagulation and obstruction after blood sampling. Blood samples were centrifuged and plasma samples were maintained frozen at – 82C until analyses. Nitrite measurement. Nitrite and nitrate are the primary oxidation products of NO. Therefore, the nitrate plus nitrite concentration was used as an indicator of changes in NO production in vivo. Nitrite was measured by a method described previously.2 Briefly, blood samples were collected from each group of animals and centrifuged at 15,000 ⫻ gravity for 3 minutes at 4C to remove cells and particles. Nitrate in the plasma was reduced to nitrite by incubating sample aliquots (10 ␮l) for 30 minutes at 37C in the presence of 10 ␮l nitrate reductase (Boehringer Mannheim, Mannheim, Germany) (10 U/ml) and 10 ␮l reduced nicotinamide adenine dinucleotide phosphate (Sigma Chemical Co., St. Louis, Missouri) (5 mM) in a final volume of 100 ␮l. Subsequently plasma was mixed with an equal volume of Griess reagent (1:1 mixture of 1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in water) and incubated at room temperature for 10 minutes. Mixture absorbance at 550 nm was determined using a Model 550 (BioRad Laboratories, Hercules, California) enzyme-linked immunosorbent assay plate reader using sodium nitrite as the standard.

Ultrasonographic findings of rabbit kidney (K)

Measurement of cGMP. As described previously,2 cGMP was measured by equilibrated radioimmunoassay. Briefly, standards or samples were incubated with diluted cGMP antiserum (Calbiochem-Novabiochem Co., San Diego, California) and iodinated cGMP (10,000 cpm/100 ␮l) in sodium acetate buffer (50 mM, pH 4.85) for 24 hours at 4C. The bound form was separated from the free form by charcoal suspension. Radioimmunoassay for cGMP was done on the day of experiments and all samples in an experiment were analyzed by a single assay. Nonspecific binding was less than 2.5%. Average results of determinations are expressed as pmol cGMP generated per ml. Statistical analyses. Data are shown as the mean ⫾ SE. Values in each group before and after ESWT were compared by the paired t test. All statistical analyses were performed using Instat. RESULTS

We have previously reported that ESWT to the kidney increases plasma levels of nitrite and cGMP in humans.2 Therefore, we used these ESWT regimens in a rabbit model to define the mechanism by which ESWT increases plasma levels of NO and cGMP. ESWT. The number of exposures was 1,400 delivered at 14 kV for the kidney and bladder. The mean time exposed was 26.3 ⫾ 2.35 minutes. The kidney was focused clearly by ultrasonography (see figure). Changes in plasma nitrite concentrations. ESWT significantly increased plasma levels of nitrite in groups 1, 3 and 4 but not in group 2 or 5 (table 1). Plasma nitrite was higher in group 1 than in the other groups. This finding might have been an individual variation. Pretreatment with NAME significantly inhibited the production of nitrite (table 1). The increase in nitrite production caused by ESWT, which was blocked by pretreatment with NAME, was recovered by combined injection of L-arginine (table 1). ODQ did not inhibit the production of nitrite by ESWT (table 1). Plasma levels of cGMP. ESWT increased plasma cGMP levels in groups 1 and 3 but not in group 2, 4 or 5 (table 2).

TABLE 1. Plasma nitrite before and after ESWT Group

Mean Before ESWT ⫾ SE (nM)

Mean Immediately After ESWT ⫾ SE (nM)

Mean 30 Mins After ESWT ⫾ SE (nM)

Mean 60 Mins After ESWT ⫾ SE (nM)

1 2 3 4 5

186.1 ⫾ 20.6 113.4 ⫾ 18.6 118.6 ⫾ 8.2 144.0 ⫾ 30.1 149.0 ⫾ 14.7

217.5 ⫾ 21.6 (p ⬍0.01) 118.2 ⫾ 19.9 144.9 ⫾ 11.8 (p ⬍0.05) 170.7 ⫾ 34.4 (p ⬍0.05) 155.6 ⫾ 18.4

241.9 ⫾ 28.4 (p ⬍0.01) 114.6 ⫾ 18.3 167.3 ⫾ 17.5 (p ⬍0.01) 163.4 ⫾ 33.3 (p ⬍0.05) 131.8 ⫾ 13.6

230.5 ⫾ 25.3 (p ⬍0.01) 112.5 ⫾ 17.6 142.3 ⫾ 10.5 (p ⬍0.01) 147.4 ⫾ 29.8 140.0 ⫾ 15.7

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TABLE 2. Plasma cGMP before and after ESWT Group

Mean Before ESWT ⫾ SE (pM)

Mean Immediately After ESWT ⫾ SE (pM)

Mean 30 Mins After ESWT ⫾ SE (pM)

Mean 60 Mins After ESWT ⫾ SE (pM)

1 2 3 4 5

24.2 ⫾ 1.9 19.4 ⫾ 2.6 18.5 ⫾ 2.1 9.6 ⫾ 1.8 25.5 ⫾ 2.1

33.8 ⫾ 3.2 (p ⬍0.001) 20.6 ⫾ 2.8 23.0 ⫾ 2.5 (p ⬍0.001) 8.9 ⫾ 1.5 27.5 ⫾ 2.5

32.9 ⫾ 2.2 (p ⬍0.001) 19.3 ⫾ 2.7 24.1 ⫾ 2.4 (p ⬍0.001) 8.4 ⫾ 1.6 28.7 ⫾ 3.1

29.4 ⫾ 1.9 (p ⬍0.001) 18.6 ⫾ 2.6 21.7 ⫾ 2.9 (p ⬍0.01) 8.7 ⫾ 1.7 25.5 ⫾ 2.6

Pretreatment with NAME or ODQ significantly inhibited the production of cGMP by ESWT (table 2). DISCUSSION

The current study clearly shows that ESWT caused an increase in NO concomitantly with cGMP, which was completely blocked by the NOS inhibitor NAME. The production of cGMP was also significantly blocked by the soluble guanylate cyclase inhibitor ODQ. Therefore, the current study provides more information on the activation of NO-cGMP signaling by the physical stimulus, corporeal shock waves. In a previous human study we did not use blocking agents and, therefore, we suggested only that ESWT activates the NO-cGMP pathway and may counteract vasoconstriction.2 However, in this animal study we used NOS inhibitor and the soluble guanylate cyclase inhibitor ODQ to evaluate the NO-cGMP pathway and we noted the activation of NO-cGMP signaling by ESWT. The response was rapid. Since the procedure was done in rabbits without kidney stones, the response should not have been related to mechanical damage from broken stone particles. ESWT is a minimally invasive, almost pain-free standard treatment modality for urolithiasis that is now used to treat about 90% of renal and ureteral stones.1, 10 The procedure is not completely free from side effects and, therefore, ESWT may induce significant changes in glomerular and tubular functions, and permanent histological damage.11 However, the mechanisms of the change in renal function induced by ESWT have not yet been fully clarified in detail. The kidney is subject to various types of stimuli, such as shear stress, cyclic strain and pressure. Unfortunately we could not clarify the main stimulus that activated the NO-cGMP pathway during ESWT in this animal model. Endothelial cells release vasoactive substances such as NO, endothelin and other endothelial derived factors.12 The endothelial lining of the whole vasculature is constantly exposed to its mechanically imposed forces, such as traction and pressure. More than 30 endothelial genes are transcriptionally regulated by shear stress.13 Renal function is modulated by various vasoactive substances in response to various humoral agents and physical stimuli, such as shear stress. ESWT significantly induced vessel vasoconstriction in each kidney and 2 explanations were suggested for the decrease in blood blow, that is renal vasoconstrictor nerves may be activated and a vasoconstrictor substance may be released by ESWT.14, 15 If the impairment in renal hemodynamics associated with ESWT is related to the vasoconstriction caused by ESWT, it is reasonable to expect that the NO-cGMP pathway may be a counteracting mechanism that can attenuate the aggravation of renal impairment caused by renal vasoconstriction due to ESWT. Renal plasma flow and the glomerular filtration rate were significantly decreased by ESWT induced vasoconstriction.14 A compensatory response to prevent potential cellular damage due to hypoxia, as threatened by an overall decrease in renal plasma flow, is needed. The responsiveness of endothelial genes to stress depends on the magnitude of stress.16 However, the possible roles of endothelial derived factors on renal hemodynamics during ESWT remain unclear.

NO synthesized from L-arginine activates guanylate cyclase, thereby, stimulating cGMP formation in smooth muscle and initiating relaxation. NO has strong vasorelaxing effect in vascular smooth muscle and it also has an important role in the regulation of renal blood flow, glomerular hemodynamics and tubular function.17 NO is released in response to a wide variety of signals, including shear stress, cyclic strain and pressure in the vascular endothelial and renal mesangial cells, and it evokes a vasodilatory effect to control systemic blood pressure and/or local blood flow.18 The endothelial NOS gene is included in the genes that are rapidly induced by the onset of stress but then slowly decrease to their static level of expression.13 In our experiment NO and cGMP rapidly increased due to ESWT, which continued up to 60 minutes. However, we do not know when the level of NO and cGMP decreased back to the static level, which should be clarified. Factors that determine the up-regulation or downregulation of endothelial NOS in macula densa cells at the transcriptional and/or protein level depend on renal perfusion, systemic volume status and distal tubular fluid flow.19 The NO synthase inhibitor NAME inhibited the increasing production of nitrite and cGMP by ESWT to the kidney and L-arginine restored the NOS function that was inhibited by NAME. Shear stress and cyclic strain increase eNOS gene expression and protein synthesis in bovine aortic endothelial cells8 and a wide variety of stimuli releases NO in renal cells.7 We suggest that NO is released from renal endothelial cells, possibly due to the cyclic strain or pressure induced by ESWT. NO appears to have many beneficial properties that may have an important role in the pathophysiology of cardiovascular disease in vivo. Little is known about the effect of activating the NO-cGMP pathway in response to ESWT, although we expect that the activated NO-cGMP pathway may prevent the constriction of renal vasculature to decrease renal damage when the kidney is exposed to ESWT. Briefly, the current study shows that extracorporeal shock waves activate NO-cGMP signaling, which may be relevant to the pathophysiological sequelae of ESWT. CONCLUSIONS

NO-cGMP signaling is stimulated by ESWT in the acute stage. Stress caused by ESWT may increase NO production, which in turn may prevent the renal damage by excessive renal vasoconstriction, although the mechanism should be clarified. ESWT could be another modality for investigating the renal hemodynamic response to shock wave stress. Instat was provided by GraphPad Software, San Diego, California. REFERENCES

1. Strohmaier, W. L., Bichler, K.-H., Koch, J., Balk, N. and Wilbert, D. M.: Protective effect of verapamil on shock wave induced renal tubular dysfunction. J Urol, 150: 27, 1993 2. Park, J. K., Cui, Y., Kim, M. K., Kim, Y. G., Kim, S. H., Kim, S. Z. et al: Effects of extracorporeal shock wave lithotripsy on plasma levels of nitric oxide and cyclic nucleotides in human subjects. J Urol, 168: 38, 2002 3. Raij, L. and Shultz, P. J.: Endothelium-derived relaxing factor, nitric oxide: effects on and production by mesangial cells and the glomerulus. J Am Soc Nephrol, 3: 1435, 1993

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4. Mundel, P., Bachmann, S., Bader, M., Fischer, A., Kummer, W., Mayer, B. et al: Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int, 42: 1017, 1992 5. Bachmann, S., Bosse, H. M. and Mundel, P.: Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am J Physiol, 268: F885, 1995 6. Bachmann, S. and Mundel, P.: Nitric oxide in the kidney: synthesis, localization, and function. Am J Kidney Dis, 24: 112, 1994 7. Pfeilschifter, J., Kunz, D. and Muhl, H.: Nitric oxide: an inflammatory mediator of glomerular mesangial cells. Nephron, 64: 518, 1993 8. Awolesi, M. A., Sessa, W. C. and Sumpio, B. E.: Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells. J Clin Invest, 96: 1449, 1995 9. Ranjan, V., Xiao, Z. and Diamond, S. L.: Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress. Am J Physiol, 269: H550, 1995 10. Bataille, P., Cardon, G., Bouzernidj, M., El Esper, N., Pruna, A., Ghazali, A. et al: Renal and hypertensive complications of extracorporeal shock wave lithotripsy: who is at risk? Urol Int, 62: 195, 1999 11. Benyi, L., Weizheng, Z. and Puyun, L.: Protective effects of nifedipine and allopurinol on high energy shock wave induced acute changes of renal function. J Urol, 153: 596, 1995 12. Vane, J. R., Anggard, E. E. and Botting, R. M.: Regulatory

13.

14.

15.

16. 17.

18.

19.

functions of the vascular endothelium. N Engl J Med, 323: 27, 1990 Resnick, N., Wolfovitz, E. and Zilberstein, S.: Endothelial gene regulation by fluid shear forces. In: Mechanical Forces and the Endothelium. Edited by P. I. Lelkes. Amsterdam, The Netherlands: Harwood Academic Publishers, pp. 127–147, 1999 Willis, L. R., Evan, A. P., Connors, B. A., Blomgren, P., Fineberg, N. S. and Lingeman, J. E.: Relationship between kidney size, renal injury, and renal impairment induced by shock wave lithotripsy. J Am Soc Nephrol, 10: 1753, 1999 Mostafavi, M. R., Chavez, D. R., Cannillo, J., Saltzman, B. and Prasad, P. V.: Redistribution of renal blood flow after SWL evaluated by Gd-DTPA-enhanced magnetic resonance imaging. J Endourol, 12: 9, 1998 Malek, A. M. and Izumo, S.: Control of endothelial cells gene expression by flow. J Biomech, 28: 1515, 1995 Bosse, H. M., Bohm, R., Resch, S. and Bachmann, S.: Parallel regulation of constitutive NO synthase and renin at JGA of rat kidney under various stimuli. Am J Physiol, 269: F793, 1995 Baylis, C., Harton, P. and Engels, K.: Endothelial derived relaxing factor controls renal hemodynamics in the normal rat kidney. J Am Soc Nephrol, 1: 875, 1990 Bachman, S. and Theilig, F.: Juxtaglomerular apparatus, nitric oxide, and macula densa signaling. In: Advances in Nephrology. London: Mosby, pp. 95–107, 2000