Endothelium-Dependent Relaxation of Small Resistance Vessels Is Impaired in Patients with Autosomal Dominant Polycystic Kidney Disease

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1 ARTICLES J Am Soc Nephrol 11: , 2000 Endothelium-Dependent Relaxation of Small Resistance Vessels Is Impaired in Patients with Autosomal Dominant Polycystic Kidney Disease DAN WANG,* JENS IVERSEN, and SVEND STRANDGAARD* Departments of *Nephrology and Medicine, and Clinical Physiology, Herlev Hospital, Herlev, Denmark. Abstract. Impaired endothelium-dependent relaxation has been demonstrated previously in resistance vessels of Han:SPRD polycystic kidney disease rats. The aim of the present study was to investigate whether endothelium-dependent relaxation is reduced also in patients with autosomal dominant polycystic kidney disease (ADPKD) and whether this is influenced by the nitric oxide (NO) system. Small subcutaneous resistance vessels from normotensive ADPKD patients with normal or nearnormal renal function (n 9) and from healthy control subjects (n 10) were mounted in a Mulvany Halpern myograph. The morphology of the vessels and acetylcholine (ACh)-induced endothelium-dependent relaxation, as well as 3-morpholino-sydnonimine (SIN-1, NO donor)-induced endotheliumindependent relaxation were investigated. The results showed that: (1) there were no significant differences in morphologic parameters of resistance vessels between the two groups; (2) the maximal ACh-induced relaxation rate was decreased in ADPKD patients compared with control subjects ( versus %, P 0.01); (3) in the presence of L- arginine (a substrate of NO synthase), a left shift of the ACh dose response curves was found in control subjects, but not in ADPKD patients; (4) in the presence of the N G -nitro-l-arginine methyl ester (an inhibitor of NO synthase), a right shift of the ACh dose response curve was found in control subjects, but not in ADPKD patients; and (5) endothelium-independent relaxation rate induced with SIN-1 was similar in patients and control subjects. In conclusion, endothelium-dependent relaxation was impaired in resistance vessels from patients with ADPKD. The reduced response of the vessels to both the substrate and inhibitor of NO synthase in ADPKD suggests that an impairment of NO synthase may be involved in the mechanism of endothelial dysfunction in ADPKD. Received September 28, Accepted December 13, Correspondence to Dr. Dan Wang, Department of Nephrology, Herlev Hospital, University of Copenhagen, Herlev Ringvej 75, DK-2730 Herlev, Denmark. Phone: ; Fax: ; wangdan9090@hotmail.com / Journal of the American Society of Nephrology Copyright 2000 by the American Society of Nephrology Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic multisystem disorder (1,2). Cardiovascular manifestations may include hypertension, cerebral and coronary artery aneurysms, mitral valve prolapse, aortic root dilation, dissection of the thoracic aorta, and aneurysm formation in the abdominal aorta (3,4). Cysts may develop not only in the kidneys, but also in the liver, spleen, and pancreas (5). The structure and function of the microvascular bed have not previously been investigated in ADPKD. Endothelium-dependent relaxation of arteries can be induced in vitro by acetylcholine (ACh), which functions as an activator of nitric oxide synthase (NOS) by increasing intracellular calcium (6). An impaired relaxation response of resistance vessels to ACh has been demonstrated and proposed as a contributory factor to vascular disease in essential hypertension and diabetes mellitus (7 10). In Han:SPRD polycystic kidney disease rats, we have demonstrated that endothelium-dependent relaxation of resistance vessels was impaired (11). Accordingly, endothelial dysfunction of resistance vessels might be present in patients with ADPKD as well, and might contribute to the development of hypertension. To test this hypothesis, the present study was designed to elucidate whether there is an impairment of ACh-induced endothelium-dependent relaxation in patients with ADPKD and whether this could be influenced by NOS substrate and inhibitor. Materials and Methods Patients The protocol was approved by the Medical Ethics Committee, Copenhagen County, Denmark, and all individuals gave written informed consent before entering the study. Ten healthy control subjects and nine normotensive ADPKD patients ages 23 to 60 yr were recruited. All ADPKD patients were diagnosed by renal ultrasound, showing five or more renal cysts distributed in both kidneys. Most of the patients had a positive family history of ADPKD. During outpatient control, the ADPKD patients consistently had a systolic BP 140 mmhg and a diastolic BP 85 mmhg. No antihypertensive medications or other drugs were taken by the patients or control subjects. Plasma creatinine was normal in nine of the ADPKD patients (68 to 105 mol/l) and slightly elevated in one patient (154 mol/l). The patients had no disease other than ADPKD, and were in good general health. Twenty-four hour ambulatory BP was measured with the Takeda 2420 monitor. GFR was measured with the 4-h one sample plasma

2 1372 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 clearance of 51 Cr-ethylenediaminetetra-acetic acid (12). These measurements were done within 1 wk of the subcutaneous fat biopsy described below. Preparation of Small Subcutaneous Vessels All subjects arrived in the laboratory between 8 and 9 a.m. and had fasted since the previous evening. From each individual, a biopsy of subcutaneous fat of cm was obtained from the gluteal region under local anesthesia with 1% lidocaine hydrochloride. Arteries were carefully dissected from the biopsy under a dissecting microscope (Olympus SN450). Two segments of the same artery (about 2 mm in length with a mean diameter of 300 m) were isolated as described previously (13). Vessels were mounted as ring preparations on two 40- m stainless steel wires in an isometric Mulvany Halpern small-vessel myograph (J.P. Trading, Science Park, Aarhus, Denmark) (Figure 1) (14). One wire was attached to a force transducer and the other was attached to a micrometer (13,14). This arrangement enabled the wall tension to be measured at a predetermined internal circumference. Both dissection and mounting of the vessels were carried out in cold (4 C) PSS solution (118 mmol/l sodium chloride, 25 mmol/l sodium bicarbonate, 4.5 mmol/l potassium chloride, 2.5 mmol/l calcium chloride, 1.0 mmol/l magnesium sulfate, and 6.0 mmol/l glucose). The two segments of resistance vessels from each individual were studied in parallel. One was treated by the experimental protocol described below; the other was used as a time control and was treated only with repeated courses of contraction with noradrenaline (NA) (10 5 mol/l). Vessel Experimental Procedure Once mounted, the resistance vessels were warmed to 37 C in PSS and allowed to equilibrate for 30 min, with the vessels internal circumference set to give a wall tension of 0.2 mn/mm. The myograph chambers were bubbled with 5% CO 2 and 95% O 2 to maintain a ph of 7.4. Morphologic measurements of wall thickness were then performed with a precalibrated filar micrometer eyepiece with a resolution of 1 m. The cross-sectional wall area of each vessel could then be calculated. These measurements were made with the vessel relaxed and minimal passive stretch (wall tension of 0.2 mn/mm), and normalized results were calculated by a computer program (Myosight, J.P. Trading, Science Park, Aarhus, Denmark). The resting tension/ internal circumference relationship for each vessel was determined and then the internal circumference was set to 0.9 L 100, where L 100 is the internal circumference the vessel would have had in vivo when relaxed and under a transmural pressure of 100 mmhg (15). After this normalization process, the vessels were incubated in PSS for 30 min before further study. During this baseline period, the PSS was replaced at 10-min intervals. Vessels were then contracted by 3 PSS containing 10 5 mol/l NA, followed by one exposure to high-potassium PSS (during which sodium chloride was replaced by potassium chloride) and one exposure to PSS containing 10 5 mol/l NA, respectively. Contractions were maintained for 3 min before rinsing with PSS back to baseline. After this stimulation procedure, the vessels were rinsed three times with fresh PSS and left to recover at baseline for 20 min. Maximal contraction of the vessels was then achieved by incubation with 10 5 mol/l NA. When a plateau of contraction had been reached, relaxation was induced by adding cumulatively increasing concentrations of ACh (10 9 to 10 5 mol/l) in the presence of an unchanged concentration of NA. Afterward, the bath was rinsed with PSS three times, and the vessels were allowed to recover for at least 15 min. Then the vessels were maximally contracted with NA (10 5 mol/l) and relaxed with cumulatively increasing concentrations of the endothelium-independent vasodilator (nitric oxide [NO] donor) 3-morpholino-sydnonimine (SIN-1, 10 9 to 10 3 mol/l), again in the presence of an unchanged concentration of NA. The vessels were then rinsed to baseline and incubated with L-arginine (substrate of NOS, 10 3 mol/l) for 30 min, and subsequently the NA contraction and ACh relaxation response were studied in the presence of L-arginine. Finally, the vessels were rinsed with PSS and incubated with the NOS inhibitor N G -nitro-l-arginine methyl ester (L-NAME, 10 4 mol/l) for 30 min, after which the NA contraction and ACh relaxation response were studied in the presence of L-NAME. All solutions were freshly prepared 1 d before the experiment. ACh, NA, SIN-1, and L-NAME were purchased from Sigma (St. Louis, MO). All reagents were prepared in distilled water and diluted to the final bath concentration with PSS. Figure 1. Diagram of segment of small subcutaneous artery mounted in wire myograph.

3 J Am Soc Nephrol 11: , 2000 Endothelial Dysfunction in ADPKD Patients 1373 Statistical Analyses All data are expressed as mean SD. Statistical differences were evaluated by two-tailed t test or by Newman-Keuls statistics. ACh sensitivity is expressed in terms of ped 50, which is the ( Log) concentration of the drug required to produce 50% of the maximum response. Values of relaxation response to ACh and SIN-1 were expressed as a percentage decline in the maximum contractile response. Statistical significance was defined as P Statistica 5.0 (StatSoft, Tulsa, OK) was used as software. Results Clinical Characteristics of the Subjects There were no significant differences between ADPKD patients and control subjects in age, 24-h ambulatory BP, and GFR (Table 1). Table 2. Morphologic data of vessels from study groups a Parameter Control Subjects ADPKD Patients n 10 9 Diameter ( m) Wall thickness ( m) Wall area ( m 2 ) 39, ,572 40,372 Wall to lumen ratio (%) a Results are given as mean SD. Morphology of Vessels There were no significant differences in vessel diameter, wall thickness, wall area, and wall to lumen ratio between ADPKD patients and healthy control subjects (Table 2). Endothelium-Dependent Relaxation ACh Response. The maximum relaxation rate (E max ) and ped 50 by ACh were significantly attenuated in maximumcontracted resistance vessels from patients with ADPKD (E max : 71.5% 12.1%; ped 50 : ) compared with the healthy control subjects (E max : 85.2% 8.7%; ped 50 : ) (P 0.01) (Figure 2). Effect of L-Arginine. Incubation with the substrate of NOS, L-arginine, resulted in an increased maximum relaxation to ACh of resistance vessels from healthy control subjects (from to %, P 0.05) (Figure 3A). The ACh dose response curves of the vessels from healthy control subjects were shifted to the left in the presence of L-arginine (ped 50 from to ; P 0.001) (Figure 3A). A slight, nonsignificant leftward shift of the response to ACh in the presence of L-arginine was observed in vessels from patients with ADPKD (Figure 3B). The dose response curve to ACh was significantly different in vessels from healthy control subjects and patients with ADPKD in the presence of L-arginine (E max : % versus %, control versus ADPKD, P 0.001) (Figure 4A). Incubation with L-arginine hence increased the difference in response to ACh between patients and control subjects (Figure 4A). Table 1. Characteristics of study groups a Characteristic Control Subjects ADPKD Patients Female/male 5/5 5/4 Age (yr) h average arterial BP (mmhg) Plasma clearance of 51 Cr-EDTA (ml/min) a Results are given as mean SD. Figure 2. Acetylcholine-induced relaxation curve of noradrenaline (NA)-precontracted subcutaneous small arteries from healthy control subjects (E, n 10) and patients with autosomal dominant polycystic kidney disease (ADPKD) (f, n 9). P 0.05 versus healthy control subjects. Results are given as mean SD. Effect of L-NAME. Incubation with the NOS inhibitor L-NAME resulted in a significant decrease in the maximum relaxation response to ACh of resistance vessels from healthy control subjects (from to %) (Figure 3A). The ACh dose response curves showed a significant rightward shift in the presence of L-NAME (ped 50 : from to , P 0.01) (Figure 3A). Incubation with L-NAME did not influence the response to ACh in vessels from patients with ADPKD (Figure 3B). Hence, in the presence of L-NAME, the dose response curve to ACh was not significantly different in vessels from patients and control subjects (Figure 4B). Endothelium-Independent Relaxation (Relaxation Response to SIN-1) The SIN-1 dose response was identical in resistance vessels from ADPKD patients and healthy control subjects (Figure 5). Time Control of Vessel Response The time-control studies showed no difference in vessel constriction response to NA for the duration of the experiment. The relaxation response of the time-control result to ACh in increasing dose was identical at start at baseline and after incubation in PSS for 30 min. We have found previously that the maximum response of ACh is similar between the dose

4 1374 Journal of the American Society of Nephrology J Am Soc Nephrol 11: , 2000 Figure 3. Acetylcholine-induced relaxation curve of NA-precontracted subcutaneous small arteries from healthy control subjects (n 10) (A) and patients with ADPKD (n 9) (B) in the absence (E) and in the presence (f) ofl-arginine (10 3 mol/l), and in the presence of N G -nitro-l-arginine methyl ester (L-NAME) (Œ) (10 4 mol/l). a P 0.01 versus in PSS and in the presence of L-arginine. b P 0.01 versus in PSS. Results are given as mean SD. response curves performed after incubation in PSS for 30 min and after incubation in PSS for 60 min and 120 min. Moreover, in another time-control experiment with subcutaneous resistance arteries from healthy control subjects and ADPKD patients, we found that NA maximum responses and sensitivity to ACh remained unchanged within a 10-h period (data not shown). Discussion ACh-mediated relaxation of resistance vessels is predominantly mediated by the production of NO in vascular endothelial cells. NO is generated by the endothelial enzyme NOS from the substrate L-arginine. ACh may have other effects on the endothelium, such as the release of prostaglandin H 2 (16), endothelial-derived contracting factor, or endothelial-derived hyperpolarizing factor (17). Vasodilation induced by ACh via mechanisms other than NOS activity are resistant to the blocking effect of L-NAME. In the absence of endothelium, ACh constricts the small resistance vessels (18). The results of the present study demonstrated that AChinduced endothelium-dependent relaxation was impaired in Figure 4. Acetylcholine-induced relaxation curve of NA-precontracted subcutaneous small arteries from healthy control subjects (n 10) and patients with ADPKD (n 9) in the presence of L-arginine (A) and in the presence of L-NAME (10 4 mol/l) (B). *P 0.01 versus healthy control subjects. Results are given as mean SD. resistance vessels from patients with ADPKD. Hence, endothelial dysfunction was present in ADPKD, even though the subjects were still in the early normotensive phase of the disease. By contrast, endothelium-independent relaxation response to an NO donor (SIN-1) was similar in patients and control subjects (Figure 4), demonstrating that the impairment of endothelium-dependent relaxation was not due to a decreased ability of vascular smooth muscle to respond to exogenous NO. The role of NO was further assessed by the effect of incubation with the NOS substrate L-arginine and the NOS inhibitor L-NAME on ACh-induced vasodilation. In healthy control subjects, the effect of ACh was increased by L-arginine and impaired by L-NAME, whereas in ADPKD neither of these substances significantly influenced the ACh relaxation response. Hence, L-arginine increased the difference between the ACh response in healthy control subjects and ADPKD patients, while L-NAME-resistant or NO-independent vasodilation was the same in both groups (Figure 4). In ADPKD, dysfunction of endothelium-dependent vasorelaxation thus seemed to be as-

5 J Am Soc Nephrol 11: , 2000 Endothelial Dysfunction in ADPKD Patients 1375 Figure 5. 3-Morpholino-sydnonimine (SIN-1)-induced relaxation response of NA-precontracted subcutaneous small arteries from healthy control subjects (E, n 10) and patients with ADPKD (f, n 9). P 0.05 versus healthy control subjects. Results are given as mean SD. sociated with a defective NO release from the endothelium. Interestingly, in a recent study of blood vessels from endothelial NOS (enos) knockout ( / ) mice (19), isolated aorta and carotid and coronary arteries did not relax in response to ACh and endothelial-derived hyperpolarizing factor. In enos ( / ) control mice, the endothelium-dependent relaxation to ACh involved either NO or the combination of NO plus a product of cyclo-oxygenase. These findings demonstrate that enos plays an important role in endothelium-dependent relaxation, and that enos impairment in ADPKD endothelium may be the cause of the findings of the present study. The changes in small artery function in ADPKD patients observed in the present study seemed to be independent of structural vascular changes, because parameters of resistance arterial structure were identical in both ADPKD patients and healthy control subjects. Similar observations have been made by our group in mesenteric arteries from young polycystic kidney disease rats (11) and suggest that endothelial dysfunction and defective endothelial NO generation may be early features in ADPKD, contributing to the development of hypertension and vascular disease well before renal function starts to decline. A decreased endothelium-dependent relaxation response in isolated small arteries has also been demonstrated in patients with essential hypertension (20 22), and in patients with diabetes mellitus (9,23). In most of these studies, as in the present one, there was no change in the relaxation response to endothelium-independent vasodilators such as sodium nitroprusside. The question of whether endothelial dysfunction is an early feature of vascular disease in hypertension and diabetes mellitus, as it appears to be in ADPKD, has not been studied. The relationship between the endothelial NO system and the well-described genetic defect in the various types of ADPKD is uncertain. Polycystin 1, which is the gene product of the most frequent form of ADPKD, has been demonstrated in the wall of aneurysms from patients, and also in large vessels from patients and control subjects (4). It is uncertain whether polycystins also are present in small resistance vessels such as those used in the present study. The functional role of vascular polycystin has not been elucidated, but it would be a likely participant in the chain of events leading to vascular disease in ADPKD, possibly with NOS dysfunction also involved. An impairment of the NO system may also be operative in the kidneys in animal models of polycystic disease. Thus, in the kidneys of rats with polycystic disease, expression of NOS isoenzymes decreases as cyst development progresses (24). Taxol, which is an inducer of NOS, inhibits cyst growth and loss of renal function in mice with polycystic kidney disease (25,26). Interestingly, expression of endothelin receptors increases in the course of cyst growth (27). In conclusion, the present study demonstrated impaired endothelium-dependent relaxation in resistance vessels from patients with ADPKD with a normal BP and renal function. This impairment may be a factor that contributes to the development of hypertension and vascular disease later in life. A reduced ACh response to substrate and inhibitor of NOS was also demonstrated, suggesting that an impaired function of this enzyme may be involved in the mechanism of endothelial dysfunction in ADPKD. It may be speculated that treatment with an exogenous NO donor could reduce the cardiovascular manifestations of disease in these patients. Acknowledgments This study was supported by the Daloon Foundation, the Becket Foundation, the Danish Kidney Foundation (Nyreforeningen) and the Foundation for the Advancement of Medical Research, Denmark. The authors thank technician Pia Linne Olsen for her meticulous laboratory work during the study. References 1. Perrone RD, Grubman SA, Murray SL, Lee DW, Alper SL, Jefferson DM: Autosomal dominant polycystic kidney disease decreases anion exchanger activity. Am J Physiol 272: C1748 C1756, Gabow PA: Definition and natural history of autosomal dominant polycystic kidney disease. In: Polycystic Kidney Disease, Oxford Clinical Nephrology Series, edited by Watson ML, Torres VI, Oxford, Oxford University Press, 1996, pp Grantham JJ, Geiser JL, Evan AP: Cyst formation and growth in autosomal dominant polycystic kidney disease. Kidney Int 31: , Griffin MD, Torres VE, Grande JP, Kumar R: Vascular expression of polycystin. 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