NEFROLOGÍA. Vol. XXV. Número 2. 2005 Vascular damage in chronic renal failure. The increase of vascular nitrotyrosine and cytokines accumulation is accompanied by an increase in endothelial nitric oxide synthase (eNOS) expression P. Gómez-Fernández, J. Pérez-Requena*, V. Sánchez-Margalet**, J. Esteban***, M. Murillo-Carretero**** and M. Almaraz-Jiménez Nephrology Department. Hospital of Jerez. *Pathology Department. Puerta del Mar Hospital. **Clinical Biochemistry Department. Virgen de la Macarena University Hospital. Seville. ***Central Department of Health Research Area. University of Cadiz. ****Physiology Area. Faculty of Medicine. University of Cadiz. SUMMARY Patients with chronic renal failure (CRF) are at a greatly increased risk of cardiovascular mortality. This fact could be due to the presence of conventional risk factor and specific uremic as increase of oxidative stress, hyperhomocystaenemia, deranged calcium-phosphate metabolism and chronic inflammatory state. In order to analyce the vascular effects of CRF, we studied the histomorphometric characteristics (intima-media tickness and monocyte chemoattractant protein (MCP-1) accumulation (inmunohistochemical) on radial artery from 13 patients with CRF. We determined by Western blot analysis, the vascular nitrotyrosin abundance (footprint of nitric oxide (NO) inactivation by reactive oxygen species (ROS), and the endothelial nitric oxide synthase (eNOS) expression. The NOS activity was, also, determined. The results were compared with those obtained in pudenda artery from a healthy control group (n: 16). The CRF group showed a significant increase in intima and media tickness 108 ± 16 vs 14 ± 2,5 µ, p < 0,001 and 291 ± 19 vs 153 ± 15 µ, p < 0,001, respectively). The CRF group exhibited a marked elevation of MCP-1 vascular expression (2 ± 0,15 vs 0,6 ± 0,12 u, p < 0,001). A significant positive correlation was found between MCP-1 vascular expression and its inmunohistochemical deposits (r: 0,98, p < 0,0001). Nitrotyrosin abundance (western blot) was significantly increased in artery of CRF patients (2,1 ± 0,1 vs 0,42 ± 0,1 u, p < 0,0001). No significant differences was found in NOS activity between CRF and control groups. However, eNOS expression was greatly increased in the CRF patients (1,73 ± 0,1 vs 0,67 ± 0,1 u, p < 0,001). A significant positive correlation was found between nitrotyrosin and eNOS expression and systolic arterial pressure. However, the differences between CRF and control groups persisted after statistically fitting to arterial pressure. Correspondence: Dr. Pablo Gómez-Fernández Servicio de Nefrología. Hospital del SAS Ctra. Circunvalación, s/n 11407 Jerez (Cádiz) 155 P. GÓMEZ-FERNÁNDEZ y cols. The present study demonstrate that in CRF there are arterial preatherosclerotic changes and an increase of vascular nitrotyrosin accumulation, wich is the footprint of NO inactivation by ROS. The secondary NO inactivation can, in turn, contribute to eNOS vascular upregulation. Key words: Atherosclerosis. Oxidative stress. Nitrotyrosin. Chronic renal failure. Nitric oxide. DAÑO VASCULAR EN LA INSUFICIENCIA RENAL CRÓNICA. EL AUMENTO DE DEPÓSITOS VASCULARES DE NITROTIROSINA Y CITOCINAS SE ACOMPAÑA DE UNA ELEVACIÓN DE LA EXPRESIÓN VASCULAR DE LA ÓXIDO NÍTRICO SINTASA ENDOTELIAL (ENOS) RESUMEN La insuficiencia renal crónica (IRC) se acompaña de un aumento de la morbimortalidad cardiovascular debido a la concurrencia de factores de riesgo cardiovascular tradicionales y otros factores inherentes a la uremia como estrés oxidativo, hiperhomocisteinemia, anomalías del metabolismo fosfocálcico, anemia y fenómenos inflamatorios entre otros. Para analizar la repercusión vascular de la IRC, en este trabajo se hace un estudio histomorfométrico (grosor íntima-media) y de los depósitos vasculares (inmuno-histoquímica) de la proteína quimiotáctica de monocitos (MPC-1) de la arteria radial en 13 sujetos con IRC, Se determinan, también, la expresión vascular( western blot) de nitrotirosina (marcador del efecto de especies reactivas de oxígeno (ROS) sobre el óxido nítrico (ON), de la MCP-1 (citocina con efecto aterogénico) y de la óxido nítrico sintasa endotelial (eNOS), y la actividad de la NOS. Los hallazgos se comparan con los observados en la arteria pudenda, arteria muscular de las mismas características que la radial, en un grupo control sano (n: 16), de edad y sexo similares a los enfermos. El grosor de la íntima y de la media fue mayor en los enfermos (íntima 108 ± 16 vs 14 ± 2,5 µ, p < 0,001; media: 291 ± 19 vs 153 ± 15 µ, p < 0,001). La expresión vascular de la MCP-1 en los enfermos fue más elevada que en los controles (2 ± 0,15 vs 0,6 ± 0,12 u, p < 0,001). La expresión de la proteína se correlacionó con los depósitos inmunohistoquímicos de la misma (r: 0,98, p < 0,0001). Las arterias de los enfermos con IRC tenían mayor expresión de nitrotirosina que las de los sujetos sanos (2,1 ± 0,1 vs 0,42 ± 0,1 u, p < 0,0001). No existieron diferencias significativas en la actividad de la NOS entre los dos grupos. La expresión de la eNOS, sin embargo, fue significativamente más elevada en los enfermos con IRC (1,73 ± 0,1 vs 0,67 ± 0,1 u, p < 0,001). La expresión de la nitrotirosina y de la eNOS se correlacionó directamente con la presión arterial sistólica. No obstante, las diferencias entre los grupos persistieron tras los ajustes a los valores de presión arterial. Estos resultados demuestran que en la IRC, a nivel de la arteria radial, existen cambios preaterosclerosos, y un aumento de los depósitos de nitrotirosina, marcador del efecto de ROS sobre el ON. Secundariamente a la disminución de la bioactividad del ON, se produce un aumento compensador de la expresión vascular de la eNOS. Palabras clave: Aterosclerosis. Insuficiencia renal crónica. Nitrotirosina. Estrés oxidativo. Óxido nítrico. 156 VASCULAR DAMAGE IN CHRONIC RENAL FAILURE INTRODUCTION Several studies demonstrate that chronic renal failure (CRF) is accompanied by an increased in cardiovascular morbidity and mortality1-3. In CRF, multiple cardiovascular risk factors (arterial hypertension, diabetes, dyslipemia among others) often coexist4. This fact, however, does not completely explain the cardiovascular morbidity and mortality excess rates3. In CRF, there are metabolic impairments that may affect vascular damage and contribute to atherosclerotic phenomena, which are the substrate of most of the cardiovascular events. Nitric oxide (NO) has multiple anti-atherogenic effects: vasodilation, platelet aggregation inhibition, smooth muscle fiber proliferation inhibition, and decrease in monocyte chemotactic protein (MCP) production, among others5-7. NO abundance essentially depends on its synthesis by the different nitric oxide synthase (NOS) isoforms and on its degradation by reactive oxygen species (ROS) such superoxide anion (O2-). O2- and NO combination yields peroxinitrite (ONOO-), a potent oxidizing agent that reacts with protein tyrosine residues, producing nitrotyrosine that is used as a marker of NO inactivation by ROS8,9. In CRF, an increase in oxidative stress and metabolic impairments capable of modifying the nitric oxide pathway has been described, and this fact might determine endothelial dysfunction and promote atherosclerosis that is associated to CRF10,11. To investigate the possible vascular repercussion of these facts, the degree of vascular expression of nitrotyrosine, MCP-1, and endothelial nitric oxide synthase (eNOS) is determined, and immunohystochemical analysis of MCP-1 and a radial artery hystomorphometric study are performed in subjects with CRF in the present study. MATERIAL AND METHODS Thirteen CRF patients (6 women and 7 men), aged 49 ± 4 years, were studied. None of them had diabetes or other systemic diseases. The cause of CRF was: chronic glomerulonephritis 7, renal polycystic disease 3, vascular nephropathy 2, and chronic pyelonephritis 1. Included in the study were patients that for the last two months were not receiving treatment with renin-angiotensin blockers, calcium-channel blockers or statins, and that were not smokers for the last two years. At the time of performing the radial-cephalic fistula prior to inclusion in hemodialysis, several samples from the radial artery were taken and imme- diately put and maintained in tubes stored in liquid nitrogen until their processing. Sixteen healthy subjects were studied as a control group, 7 women and 9 men, aged 44 ± 2 years, non-smokers, and that were submitted to a scheduled surgery for varicose veins of the lower limbs. At the time of surgery, several samples were taken from the pudendal artery, which is a muscular artery with features similar to those of the radial artery, and were processed and studied in exactly the same manner as the patients' samples. All patients and controls gave their consent after being informed of the nature of the study. For the hystomorphometric study of the intimae and media layers width and the MCP-1 immunohystochemical study, a computer-based image analysis system was used (Micro Image semiautomatic, Olympus). Arterial sections stained with the antibody were digitalized with a Bx-60 microscope connected to a color video camera JVC and to an analytical system (image software by Olympus). After image acquisition, each pixel was assigned a value range from 0 to 255. Marked areas of the intimae and media layers were delineated and, after image improving and segmentation to define a threshold value, an automatic analysis was performed. Results are expressed as immunostaining area and fractional intimae-media area. For the study of eNOS, nitrotyrosine, and MCP-1 expression, samples were homogenized with a Politron in Tris 50 mM buffer, pH 7.4, 1 mM EDTA, 1% Trixon X-100, with a cocktail of proteases inhibitors. The homogenate was spun to 15,000 rpm and supernatant was recovered. Protein concentration was determined. It was denatured with Laemli buffer for SDS-PAGE electrophoresis with an 8-16% gradient. After electrophoretic spacing, proteins were electrotransferred to nitrocellulose membrane for immunoblotting analysis. The membrane was blocked with skimmed milk and was incubated with specific monoclonal antibodies for eNOS (BD Biosciences, San Jose, CA, USA), nitrotyrosine (Cell Signaling Technology) (New England Biolabs, Beverly, MA, USA), and MCP-1 (Santa Cruz, CA, USA). After membrane washing, it was incubated with a secondary peroxidaselabeled antibody. Finally, it was incubated with a chemoluminiscent substrate and exposed to a radiographic film or photo paper. Bands were scanned and the optical densities analyzed by PCBAS software. The NOS activity determination was done by measuring [3H] L-citruline formation from [3H] L-arginine according to the previously described methodology12. After centrifugation of the homogenate, the supernatant was incubated in duplicated tubes with 157 P. GÓMEZ-FERNÁNDEZ y cols. 50 mM phosphate buffer, Cl2Ca 0.2 mM, C12Mg 1 mM, L-arginine 0.05 mM, NADPH 0.1 µM, valine 8 µµ, [3H] L-arginine 9.3 mCi/L. In two other tubes, EDTA was added to the previous solution for calcium chelation and for inhibition of constitutive NOS (cNOS) calcium-dependent activity. In two other tubes, 2 µM of L-NAME were added to all previous solutions to inhibit 100% the NOS activity, which allows ruling out any NOS-independent metabolic activation of L-arginine. Thus, through the appropriate calculations cNOS and iNOS activities may be differentiated. After incubation, 900 µl of water at 0º C and 500 µl of Dowex50 resin were added. Once the resin precipitated, scintillation liquid was added to the supernatant. [3H] L-citruline was counted with a beta counter (LK 1410). The obtained NOS activity was divided by the homogenate protein concentration and expressed as pmols/g-1/min-1. Arterial samples were code-identified. None of the investigators that performed the different studies (hystomorphometry, immunohystochemistry, western blot, NOS activity) knew the sample origin. For statistical study, Mann-Whitney test was used to compare quantitative variables between the patients group and the control group, and Chi-squared test was used for qualitative variables. For analysis of the effect of any uncontrolled variable, the covariance analysis was used. Determination of the relationship between the different variables was done by simple regression analysis. The results are expressed as mean ± standard error (X ± SE). P values < 0.05 were considered significant. RESULTS There were no significant differences in age or gender distribution between patients and controls. Obviously, serum creatinine levels in the CRF group were significantly higher than those of the control group (6.5 ± 0.3 vs 0.90 ± 0.03 md/dL, p < 0.001). Systolic (SAP) and diastolic (DAS) arterial pressure values were higher in patients (SAP: 147 ± 4 vs 121 ± 3 mmHg, p < 0.001; DAS: 82 ± 2 vs 69 ± 2 mmHg, p < 0.001). In the histological study of the arterial sample from a patient with CRF, calcification of the media layer was observed. In two radial artery samples and in two pudendal artery samples material for study was considered insufficient. Both arterial intimae and media width and their coefficient were much higher in CRF patients (intimae: 108 ± 16 vs 14 ± 2.5 µm, p < 0.001; media: 291 ± 19 vs 153 ± 15 µm, p < 0.001; intimae/media: 28 ± 4 vs 8 ± 1%. P < 0.001) (Fig. 1). At the request of one of the investigators, in 158 a delayed manner and whenever possible, the lumen/wall ratio was determined. This ratio was significantly lower in patients [0.66 ± 0.15 µm (n = 5)] than in controls [1.24 ± 0.15 µm (n = 7) (p = 0.04)]. The immunohystochemical study showed a higher MCP-1arterial deposition in patients than in controls (8.5 ± 0.8 vs 2.5 ± 0.4 µm2, p < 0.001) (Fig. 2). MCP-1 protein expression (western blot) was significantly higher in patients (2.0 ± 0.15 vs 0.60 ± 0.12, p < 0.001) (Fig. 2). Arteries from patients with CRF had much higher expression of proteins with nitrosilated tyrosine (nitrotyrosine) residues than arteries from normal subjects (2.10 ± 0.1 vs 0.42 ± 0.1, p < 0.001) (Fig. 3). cNOS activity was lesser in patients, not reaching statistical significance (46 ± 5 vs 59 ± 8 pmolg of tissue-1mim-1). However, arterial eNOS protein expression was higher in CRF patients (1.73 ± 0.1 vs 0.67 ± 0.1 µ, p < 0.001). A direct relationship was shown between MCP-1 expression (western blot) and its immunohystochemical deposition (r: 0,891, p < 0.0001), and between SAP values and nitrotyrosine expression (r: 0.750, p < 0.0005) and eNOS expression (r: 0.708, p < A B Patient x 10 Fig. 1. VASCULAR DAMAGE IN CHRONIC RENAL FAILURE A 2.5 C 2 p < 0.001 1.5 Units 1 0.5 0 Control CRF B D Fig. 2. 0.0005). After adjusting for SAP values, significant differences in nitrotyrosine and eNOS expression persisted between both groups. DISCUSSION Our results demonstrate that in the radial artery of end-stage CRF patients there exist a greater width of the intimae and media layers, and an increase in nitrotyrosine deposition, which is a marker of NO inactivation by free radicals, and of cytokine MCP1 deposition, as well. These data are accompanied by an elevated eNOS vascular expression. However, vascular cNOS activity is normal. The need for a scheduled surgery to perform the arterial-venous fistula and to operate on varicose veins offers the opportunity for taking samples from the radial and pudendal arteries in CRF patients and in healthy subjects, respectively. Since hemodynamic factors and the existence of rheologic peculiarities in each vascular territory take part in vascular structure regulation, it is possible that comparing these two arteries may entail some limitation. However, both of them are intermediate size muscular arteries and, moreover, offer the possibility to conduct the study without the ethical issues that would ensue by taking radial artery samples from healthy subjects. On the other hand, the lumen/wall ratio determination in another group of subjects, a 159 P. GÓMEZ-FERNÁNDEZ y cols. A 2.5 B 2 p < 0.001 1.5 Units 1 0.5 0 Control Fig. 3. CRF parameter that «standardizes» the vessel size, shows that the vascular wall width of renal patients is increased. Although it has been considered that the radial artery is spared from atherosclerosis, which has been the rationale for its frequent use in cardiac bypass surgery, recent studies show that the atherosclerotic histopathological findings are more frequent in the radial artery than in the internal thoracic artery13. On the other hand, by means of non-invasive methods, it has been demonstrated that, as compared with the healthy group, individuals with coronary artery atherosclerosis have greater radial artery structural changes14. These facts allow us to reinforce that the study of the radial artery may serve as a marker of the biological and structural changes that accompany CRF. Differences in blood pressure values between patients and controls, and the direct correlation between blood pressure values and vascular nitrotyrosine and MCP-1 abundance suggest the existence of a relationship between arterial hypertension and the structural and biochemical vascular findings observed in our study. Arterial hypertension may be associated with an increase in oxidative stress that, in turn, induces an increase in nitrotyrosine, cytokines and a higher width intimae-media15,16. From another perspective, the decrement in nitric oxide bioactivity, as shown by nitrotyrosine increase, might contribute to blood pressure increase in renal failure, as has been demonstrated at an experimental level17. Persistence of significant differences in nitrotyrosine and MCP160 1 abundance between patients and controls after adjusting for arterial pressure values suggests that other factors related to azotemia exist, which are responsible for these facts. In chronic renal failure there is an increase in oxidative stress11. The greater amount of vascular nitrotyrosine in patients studied by us reflects this fact. The increase in oxidative stress, in turn, may contribute to cytokines production, such as MCP-1, and to early atherosclerosis in CRF18,19. These phenomena are enhanced by nitric oxide inactivation by ROS, a fact also reflected by increased nitrotyrosine deposition, which promotes NO functional deficit and endothelial dysfunction20. We cannot define the cause for the increase in eNOS expression in the arteries of renal patients. Shearing forces that accompany the hemodynamic changes secondary to arterial hypertension and anemia may contribute to this issue21,22. High blood pressure does not seem to be the only argument since a higher eNOS expression persists after statistical adjustment for arterial pressure values. On the other hand, in a previous study we verified that subjects with primary arterial hypertension have a decrease in eNOS expression at the arteries23. In cellular cultures it has been demonstrated that NO exerts a negative feedback regulation on eNOS expression through a c-GMP-mediated pathway24. Thus, it is possible that NO functional deficit, secondary to inactivation by ROS or to a functional defect in uncoupled eNOS by oxidative strees25, may induce a compensatory elevation in eNOS expression. VASCULAR DAMAGE IN CHRONIC RENAL FAILURE Contrary to an elevated eNOS expression, although lower the cNOS activity (formed by eNOS and nNOS) did not reach significant differences as compared to the control group. Although our assay does not allow us to discriminate eNOS and nNOS activities, it may be speculated that a normal activity together with an increased expression reflects a relative functional deficit. In CRF, false NOS substrates accumulate, such as asymmetrical methyl-Larginine (ADMA), which decreases NOS activity12,26, and furthermore, there may exist a deficit of the cofactors needed for NOS activity such as tetrahydrobiopterin (TH4)27.We believe that none of these facts justify a NOS functional defect since, in our assay, as described in Methods, there are insufficient amounts of L-arginine and TH4. It may be possible that, in CRF, there exist other eNOS phosphorylation impairments that may reduce its activ i t y 28. In summary, in CRF there exist, at the radial artery level, pre-atherosclerotic histological changes and an increase in nitrotyrosine deposition, which reflects the action of ROS on NO. In the studied group of CRF patients, it can also be observed an increase in eNOS vascular expression which mechanism is not fully understood, although it might represent a compensatory increase to the decrement in NO bioactivity. 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