J Am Soc Nephrol 14: 653–660, 2003 Hyperhomocysteinemia Induces Renal Hemodynamic Dysfunction: Is Nitric Oxide Involved?

PATRICIA A. FISCHER,* GRACIELA N. DOMINGUEZ,* LUIS A. CUNIBERTI,† VERO´ NICA MARTINEZ,† JOSE´ P. WERBA,† AGUSTI´N J. RAMIREZ,* and LUCAS D. MASNATTA* *Section of Pharmacology, Department of Physiology, and †Lipid and Atherosclerosis Research Laboratory, Department of Pathology, Favaloro University, Buenos Aires, Argentina.

Abstract. Hyperhomocysteinemia is associated with endothe- centration (7.6 Ϯ 1.7 versus 4.9 Ϯ 1.0 ␮mol/L; P Ͻ 0.001), Ϫ. Ϯ Ϯ lial dysfunction, although the underlying mechanism is un- (O2 ; production (157.92 74.46 versus 91.17 29.03 cpm · known. Previous studies have shown that nitric oxide (NO) 103/mg protein), and nitriteϩnitrate levels (33.4 Ϯ 5.1 versus plays an important role in the regulation of systemic and renal 11.7 Ϯ 4.3 ␮mol/mg protein; P Ͻ 0.001) than the control hemodynamics. This study investigated whether hyperhomo- group. Western blot analyses showed a nitrotyrosine mass 46% cysteinemia induces renal oxidative stress and promotes renal higher in the HYC group than in the controls. Furthermore, the dysfunction involving disturbances of the NO-pathway in HYC group showed lower GFR, renal plasma flow (RPF), and Wistar rats. During 8 wk, control (C) and hyperhomocysteine- higher renal vascular resistance (RVR) than the controls. After mic (HYC) groups had free access to tap water and homocys- L-Arg administration, the responses of GFR, RPF, and RVR were teine-thiolactone (HTL, 50 mg/kg per d), respectively. At 8 wk, attenuated by 36%, 40%, and 50%, respectively; after L-NAME, plasma homocysteine concentration, renal superoxide anion the responses of RPF and RVR were exaggerated by 79% and Ϫ. ϩ (O2 ), nitrotyrosine, and nitrite nitrate levels, and renal func- 112%, respectively. This suggests a reduced NO bioavailability to tion were measured. To assess NO involvement, the responses produce vasodilation and an enhanced sensitivity to NO inhibi- G to L-Arginine (L-Arg, 300 mg/kg) and N -nitro-L-arginine- tion. In conclusion, hyperhomocysteinemia induces oxidative methyl-ester (L-NAME, 20 ␮g/kg per min for 60 min) were stress, NO inactivation, and renal dysfunction involving distur- analyzed. The HYC group showed higher homocysteine con- bances on the NO-pathway.

The endothelium plays an important role in the regulation of Homocysteine is a sulfur-containing amino acid that is an vascular tone involving vasorelaxing and vasoconstrictor fac- intermediary product in methionine metabolism. Hyperhomo- tors (1,2). Nitric oxide (NO), one of the most important va- cysteinemia is an independent risk factor of arteriosclerosis, sorelaxing factors, is produced from the amino acid L-Arginine involving coronary, cerebral, and peripheral arteries, and it is by nitric oxide synthase (eNOS) in the endothelium (3,4). It associated with an increased relative risk of cardiovascular stimulates cytosolic guanylate cyclase and increases guanosine events, comparable to hypercholesterolemia or smoking (10). 3', 5'-cyclic monophosphate (cGMP) content in vascular Depending on the definition of hyperhomocysteinemia, 10 to smooth muscle cells, thus inducing vascular relaxation (5,6). 20% of the general population, more than 85% of patients with In the normal kidney, NO plays a key role in the homeostatic end-stage kidney disease (ESRD) (11–13), and up to 32% of regulation of vascular, glomerular, and tubular function main- the individuals having premature peripheral arterial disease taining normal renal perfusion, GFR and renal vascular resis- (14) have increased homocysteine levels. On the other hand, tance, RVR (7,8). hyperhomocysteinemia is a frequent finding in heart, liver, and Ϫ. Increased superoxide anion radical ((O2 ) production inacti- renal transplant recipients and is associated with renal dysfunc- vates the released NO, reducing NO bioavailability by per- tion, even though the etiologic factors have not been clearly Ϫ oxynitrite (OONO ) formation. Thus, a greater NO inactiva- identified (15–17). In patients with ESRD, plasma homocys- tion may reduce medullary blood flow, contributing to the teine levels may become 3 to 5 times higher than normal; even development of renal failure (9). dialysis therapy or kidney transplantation fail to restore plasma homocysteine levels to a normal level (13). Several experimental and clinical studies have reported that Received July 15, 2002. Accepted November 22, 2002. hyperhomocysteinemia induces vascular oxidative stress and Correspondence to Lucas Daniel Masnatta, Section of Pharmacology, Department of Physiology, Favaloro University, Solı´s 453, Buenos Aires (C1078AAI), Ar- impaired vascular response to NO, denoting endothelial dys- gentina. Phone/Fax: 5411-4-383-9744; E-Mail: [email protected] function (18). Moreover, Fu et al. (19) showed that homocys- 1046-6673/1403-0653 teine reacts with NO, inhibiting not only the endothelial- Journal of the American Society of Nephrology derived NO but also the bioactivity of exogenously supplied Copyright © 2003 by the American Society of Nephrology NO. The precise mechanism underlying the link between mod- DOI: 10.1097/01.ASN.0000053419.27133.23 erate hyperhomocysteinemia and endothelial dysfunction re- 654 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 653–660, 2003

mains to be determined. Various reports suggest that oxidative In renal supernatants, superoxide anion radical production was stress plays a major role (20,21). This is supported by recent determined by the increase of chemiluminescence produced in re- studies showing that oral administration of antioxidants (e.g., sponse to the addition of nicotinamide adenine dinucleotide (NADH) ␮ ␮ vitamin C and vitamin E) prevents oral methionine load-in- (100 M), 5 M of dark-adapted lucigenin (30), and in presence or duced impairment of dilatation of conduit arteries in response absence of the nonenzymatic superoxide scavenger Tiron (10 mM), according to Trolliet et al. (31). The supernatant was incubated at to acetylcholine and after release of forearm occlusion (18,22). room temperature for 5 min. Chemiluminescence was measured in a On the other hand, there is evidence which suggests that liquid scintillation counter (Packard-Tricarb 2200). A blank probe was hyperhomocysteinemia induces endothelial dysfunction by a run for each sample and subtracted before data transformation. Net mechanism involving an increased generation of reactive ox- superoxide elicited chemiluminescence was calculated from the Tiron ygen species and a decreased NO bioavailability (3,20,21). inhibitable fraction and expressed as counts per min per mg protein Although the increased homocysteine level is able to impair (cpm/mg protein). endothelial-dependent vascular relaxation in coronary, bra- chial, or femoral arteries (23,24,19) and the renovascular bed is TBARS Measurement more sensitive to changes in endothelial function than other Renal tissue was homogenized in 0.3 M Tris-HCl buffer, pH 7.4. vascular beds (25), there are, to our knowledge, no reports TBARS were measured according to Ohkawa et al. (32). Protein showing the impact of hyperhomocysteinemia on renal hemo- content was determined by the Lowry method (29). Results were dynamic function. expressed as nmol of malondialdehyde per mg protein (nmol Thus, the aim of the present work was to study whether MDA/mg protein). hyperhomocysteinemia can induce renal oxidative stress and renal hemodynamic dysfunction involving disturbances on the CD Measurement NO pathway. Kidneys were homogenized in a saccharose (0.32 mM) and EDTA (3 mM) solution. Lipids were extracted with chloroform:methanol (3:1). The lipid extract was used to determine CD according to Davis Materials and Methods et al. (33). Results were expressed as Abs/mg protein. Animals ϭ Three-month-old male Wistar rats (n 62; 350 to 400 g) were Measurement of Nitrotyrosine used. The animals were maintained on a standard rat chow (Argentine Cooperative Association, Animal Nutritional Division) and tap water. Renal tissue was homogenized in a solution containing 50 mM They were housed under a 12/12 h day/night cycle at a steady Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM ␮ temperature of 25°C. After 1 wk of acclimatization, they were ran- NaCl, 1 mM EGTA, aprotinin, leupeptin, pepstatin (1 g/ml each), 1 domly allocated to either a normal control (C, n ϭ 31) or hyperho- mM Na3VO4,and1mMNaFat0to4°C using a sonic disruptor mocysteinemia (HYC, n ϭ 31) group. Control and HYC groups had (Tekmar). Nitrotyrosine mass was analyzed by Western blot technique free access during 8 wk to either tap water or homocysteine thiolac- using an anti-nitrotyrosine monoclonal antibody (Upstate Biotechnol- tone (HTL, 50 mg/kg per d), respectively (26). ogy Inc, Lake Placid) according to Vaziri et al. (34) with some modifications. Renal homogenates (100 ␮g of protein) were size- fractioned on 4 to 20% Tris-Glycine gel (Novex Inc) at 120 V for 2 h. Plasma Homocysteine Levels In preliminary experiments, we found that the protein concentrations At baseline conditions and at the end of the study, femoral arterial to be measured were within the linear range of detection for our blood samples (200 ␮l) were withdrawn and immediately cooled in Western blot technique. After electrophoresis, proteins were trans- Eppendorff tubes containing 0.1% EDTA. Plasma was separated by ferred onto a polyvinylidine difluoride (PVDF) membrane (Amer- centrifugation at 4°C and immediately frozen at Ϫ60°C until the day sham Life Science Inc.) at 200 mA for 120 min using the Novex of analysis. Total plasma homocysteine levels were determined using transfer system. The membrane was prehybridized in buffer A (10 the Bio-Rad HPLC kit (27). mM Tris hydrochloride, pH 7.5, 100 mM NaCl, 0.1% Tween 20, and 5% nonfat milk powder) for 1 h and then hybridized overnight at 4°C Renal Oxidative Stress and NO Bioavailability in the same buffer containing 0.5 ␮g/ml anti-nitrotyrosine monoclonal antibody (clone 1A6, Upstate Biotechnology) or anti-actin monoclo- Superoxide anion radical production, thiobarbituric acid-reactive nal antibody at the final titer of 1:500 (SC-8432, Santa Cruz Biotech- species (TBARS), and conjugated dienes (CD) were determined in nology Inc.). The membrane was then washed in buffer A without fresh renal tissue. To estimate NO bioavailability, renal nitrotyrosine nonfat milk for 30 min. This wash buffer was changed every 5 min. mass, total nitrate and nitrite (NOx), and cyclic GMP (cGMP) levels The membranes were subsequently incubated for1hinbuffer A plus were also measured. For this purpose, 10 rats from each group were horseradish peroxidase-conjugated sheep anti-mouse IgG antibody at killed at 8 wk. After that, kidneys were isolated, cleaned in saline the final titer of 1:2000 (NA 931, Amersham Biosciences Inc.). solution, and immediately frozen in liquid nitrogen, pulverized, and Experiments were carried out at room temperature. The washing homogenized (25% wt/vol) in suitable solutions (see below). procedure was repeated before the membrane was developed with a light-emitting nonradioactive method using chemiluminescence Measurement of Superoxide Anion Radical Production (ECL) reagent (Amersham Biosciences Inc.). Once the membranes Renal homogenates were prepared in 50 mM phosphate-0.01 mM were incubated in the ECL reagent, they were exposed to a film EDTA buffer (pH 7.4) using a glass-to-glass homogenizer followed (Hyperfilm ECL, Amersham Biosciences Inc.) from 20 s to 20 min to by centrifugation (1000 ϫ g)at4°C for 10 min (28). The supernatants provide a hard-copy result. The autoluminographs were digitized were maintained in ice until used. Protein content was measured using a scanner (HP ScanJet) and Adobe Photoshop software (Adobe according to the Lowry method (29). Systems Inc.). The digitized image was then analyzed for total nitro- J Am Soc Nephrol 14: 653–660, 2003 Renal Hemodynamics in Hyperhomocysteinemic Rats 655 tyrosinated proteins in each lane using QuantiScan (Microbial Sys- Blood samples were used for hematocrit, inulin, and PAH deter- tems Ltd, Biosoft Software). The area under the curve of densitomet- minations. Clearances of inulin and PAH were calculated as described ric analysis was quantified by Image J (Wayne Rasband, National previously (40,41). Institutes of Health, Bethesda, MD). To evaluate equal protein load- BP and heart rate were monitored throughout the study via an ing, the membranes were stripped and reprobed with anti-actin mono- arterial catheter connected to a Statham pressure transducer (Gould clonal antibody. In addition, the transferred gels were stained with Instrument) and attached to a polygraph recorder (model 2400S, Coomassie Blue (Bio-Rad, Richmond, CA). To confirm the specific- Gould Instrument). ity of the anti-nitrotyrosine monoclonal antibody, positive and nega- tive immunoblotting controls of nitrotyrosine (Upstate Biotechnology, Chemicals and Reagents NY, USA) were performed. Protein concentration was determined by Ether (Sigma Chemical Co) and Ketamine (G & M S.A.) were used the bicinchoninic acid protein assay kit (Pierce Biotechnology) (35). as anesthetics. Lucigenin, EDTA, NADH, Tiron, L-Arg, L-NAME, and HTL were purchased from Sigma. Sterile and non-pyrogenic Renal NOx Determination inulin (Cypros Pharmaceutical Co, West Carlsbad, CA) and PAH ampules for intravenous administration (Merck & Co. Inc) were used. NO is unstable and quickly oxidized; therefore, NOx determination is considered as a marker of NO synthesis (36). Renal tissue was homogenized in 0.3 M Tris-HCl buffer, pH 7.4. Supernatants were Statistical Analyses ultrafiltered using micropore filters (Ultrafree MC microcentrifuge All values are expressed as mean Ϯ SD. Comparisons between device, Millipore), and NOx assay was performed using a commercial HYC and C groups were done using the unpaired t test. To compare nitric oxide colorimetric assay kit (Roche Diagnostics GmbH). Protein the responses to L-Arg, L-NAME, and vehicle within one group, concentration was determined by Lowry method (29). Results were percent changes from baseline were analyzed by one-way ANOVA expressed as ␮mol/mg protein. followed by the Scheffe´ test. Differences between values were con- sidered to be statistically significant when P Ͻ 0.05.

Renal cGMP Determination Results Kidneys were homogenized in ice-cold 50 mM Na PO and 10% 3 4 Animals TCA. Levels of cGMP were measured according to Pradelles et al. No significant differences were found in body weight or (37) using a cGMP enzyme immunoassay kit (Cayman Chemical). fluid or food consumption between HYC and C groups at 8 wk.

Renal Hemodynamic Function Determination Plasma Homocysteine Levels Hemodynamic studies were performed in conscious unrestrained Basal plasma homocysteine concentration did not differ rats as described previously (38). Briefly, these animals were anes- between the two groups. After 8 wk of HTL administration, thetized to cannulate both the femoral artery and the femoral vein. total plasma homocysteine levels were 55% higher in HYC After recovery from surgery and in euvolemic conditions, a priming group compared with the C group (7.6 Ϯ 1.7 ␮mol/L versus dose of inulin (16 mg/kg) and sodium para-aminohippurate (PAH, 8 4.9 Ϯ 1.0 ␮mol/L; P Ͻ 0.001). mg/kg) was administered. Immediately thereafter, a continuous intra- venous infusion of 0.9% NaCl containing inulin (36 mg/ml) and PAH (11.6 mg/ml) was given at a rate of 0.0267 ml/min. After a 105-min Renal Oxidative Stress and NO Bioavailability equilibration period, baseline arterial blood samples were taken. Sub- Renal parameters of oxidative stress are shown in Figure 1. Ϫ. ϩ ϩ sequently, still under intravenous infusion of inulin and PAH to In the HYC group, the production of O2 ( 74%), CD ( 40%), maintain steady-state conditions throughout the study, the involve- and TBARS (ϩ75%) were significantly higher when compared ment of the NO pathway was investigated. For this purpose, vehicle with the C group. Greater concentrations of NOx (33.4 Ϯ 5.1 (saline solution), L-Arg, and L-NAME treatments were performed. versus 11.7 Ϯ 4.3 ␮mol/mg protein; P Ͻ 0.001) and cGMP To evaluate the effect of NO synthesis induction on renal hemo- (5.3 Ϯ 1.5 versus 2.8 Ϯ 1.5 pmol/mg protein; P Ͻ 0.05) were dynamics, a bolus of L-Arg (300 mg/kg, intravenously) was adminis- observed in HYC rats, showing that NO synthesis was 185% tered to both HYC (n ϭ 7) and C groups (n ϭ 7). Blood sampling was higher, whereas the cGMP production was only 88% higher performed 30 min after L-Arg administration (39). On the other hand, NO production from HYC (n ϭ 7) and C (n ϭ compared with the C group. 7) groups was inhibited by L-NAME administration. For this purpose, A representative Western blot analysis using anti-nitroty- the infusion was changed to another one containing the same concen- rosine monoclonal antibody is shown (Figure 2A). Higher tration of inulin and PAH plus L-NAME (0.075 mg/ml per 100 g body levels of nitrotyrosinated proteins were observed in HYC rats. wt) at the same infusion rate (20 ␮g/kg per min for 60 min, intrave- By quantitative densitometric analysis, HYC group showed a nous infusion). Previous studies have shown that this dose induces a 46% higher mass of nitrotyrosinated proteins as compared with significant increase (more than 20%) in RVR in normal rats without controls (Figure 2B). Positive and negative immunoblotting changing mean arterial pressure, MAP (39). Blood samples were controls of nitrotyrosine were shown in Figure 3, denoting the taken 60 min after the change. high specificity of anti-nitrotyrosine monoclonal antibody. Sham rats from HYC (n ϭ 7) and C (n ϭ 7) groups were subjected to the same surgical instrumentation and received identical infusion of inulin and PAH at the same rate for 105 min. Immediately thereafter, Renal Hemodynamic Function Determination a bolus or infusion of vehicle was given and blood samples were Under baseline conditions, MAP and RVR were signifi- taken. cantly higher in the HYC group than in controls (Figure 4, A 656 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 653–660, 2003

Figure 2. Effect of hyperhomocysteinemia on nitrotyrosine-contain- ing proteins in kidney. (A) Representative Western blot analysis indicating all nitrotyrosinated proteins in renal homogenates from control (C, open column) and hyperhomocysteinemic (HYC, dark column) groups. The position of nitrotyrosine signals was distributed in a broad range of renal nitrated proteins. The same membranes with Figure 1. Renal effects of hyperhomocysteinemia on superoxide (up- nitrotyrosine signals were restripped and reprobed with actin antibody per panel), conjugated dienes (middle panel), and TBARS production as internal protein loading control. The actin signals were indicated. (bottom panel). C, control (open columns); HYC, hyperhomocysteine- (B) Quantitative densitometric analysis of total nitrotyrosine-contain- mic (dark columns) groups; TBARS, thiobarbituric acid-reactive spe- ing proteins corrected for actin. aP Ͻ 0.05 compared with controls. cies; MDA, malondialdehyde. aP Ͻ 0.05 compared with controls.

smaller. The RPF decrease in response to L-NAME was 79% and D). Conversely, GFR (Figure 4B) and RPF (Figure 4C) higher in the HYC compared with the C group. Finally, L-Arg were significantly lower in the HYC than in the C group. decreased RVR in both groups, although the reduction was In sham rats from both HYC and C groups, vehicle treatment 50% smaller in the HYC group, and the RVR increase induced failed to induce any significant alterations of systemic and by L-NAME was 112% higher in HYC than in C rats (Figure renal hemodynamics (data not shown). To evaluate whether 5C). NO was involved in the renal hemodynamic effects induced by hyperhomocysteinemia, percent changes from baseline in Discussion GFR, RPF, and RVR after L-Arg and L-NAME treatments were The balance between vasodilating and vasoconstricting analyzed. L-Arg induced a percent increase in GFR in both agents produced by the endothelium plays a key physiologic groups, but this was 36% lower in the HYC group (Figure 5A). role in the control of renal hemodynamics (2,42). Although On the other hand, the GFR reductions induced by L-NAME hyperhomocysteinemia induces vascular endothelial dysfunc- were not different between the HYC and C groups. In addition, tion and the renal vasculature is very sensitive to changes in after L-Arg treatment, the RPF increase (Figure 5B) was 40% endothelial function (25), no studies reporting the effects of J Am Soc Nephrol 14: 653–660, 2003 Renal Hemodynamics in Hyperhomocysteinemic Rats 657

Figure 3. Specificity of anti-nitrotyrosine monoclonal antibody. A combination of three nitrotyrosine-containing proteins: nitrated bo- vine superoxide dismutase (SOD, 56 ␮g/ml, approximately 16 kD), nitrated bovine serum albumin (BSA, 39 ␮g/ml, approximately 66 kD), and nitrated rabbit muscle myosin (19 ␮g/ml, approximately 215 kD), as positive nitrotyrosine immunoblotting control (lane A). Neg- ative immunoblotting control of nitrotyrosine: a combination of the same non-nitrated proteins in matched concentration (lane B). Cali- brated molecular weights of Kaleidoscope Standards (lane C).

hyperhomocysteinemia on renal function are available. Thus, the present work is the first to show that hyperhomocysteine- mia induces renal oxidative stress and promotes renal vasoconstriction. Our results show that hyperhomocysteinemia induces higher Ϫ. O2 production and greater levels of TBARS and CD denoting an increased renal oxidative stress and lipid peroxidation. This is supported by strong evidence of increased production of superoxide anion radical and markers of lipoperoxidation being involved in reduced NO bioavailability (43,44). In addition, it Figure 4. Systemic and renal effects induced by hyperhomocysteine- is known that the association of higher NO synthesis and a mia. C, control (open columns); HYC, hyperhomocysteinemic (dark Ϫ. Ϫ columns) groups; MAP, mean arterial pressure; RPF, renal plasma greater O2 production induces rapid formation of OONO a b (45), which is responsible for nitrating the tyrosine residues of flow; RVR, renal vascular resistance. P Ͻ 0.001 and P Ͻ 0.05 proteins. This is consistent with the increased renal nitrotyrosi- compared with controls. nated proteins observed in HYC rats. Reduced NO synthesis or increased NO inactivation decreases RPF and GFR and in- creases RVR (46); therefore, our results suggest that hyperho- thesis increased to compensate the NO inactivation induced by mocysteinemia is able to alter the L-Arg-NO pathway. reactive oxygen species (ROS), with the aim of maintaining a Higher NO synthesis and cGMP production suggest that the normal cGMP production. endothelial dysfunction induced by hyperhomocysteinemia is With the exception of microalbuminuria, neither clinical nor not related to reduced NO synthesis or production of the laboratory variables can be reliably used to differentiate pa- second messenger responsible for NO effects. However, the tients with progressive renal injury from those who follow a magnitudes of increase of NO synthesis (185%) and cGMP more benign evolution (47). Using low doses of L-Arg and production (88%) were dissimilar. This suggests that NO syn- L-NAME, several investigators have been able to avoid 658 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 653–660, 2003

conditions, tonically produced NO plays a pronounced role in the maintenance of normal renal function, suggesting that synthesis and/or release of NO is enhanced to compensate the increased NO inactivation (48). This is consistent with some reports (49,50) in which both acute and chronic NO inhibition in rats causes exaggerated renal vasoconstriction. Thus, the functional response to NO inhibition allows evaluation of the degree of NO involvement in maintaining renal function. Ac- cordingly, our results showed an exaggerated vasoconstrictor response to L-NAME, suggesting enhanced sensitivity to NO inhibition. This is consistent with an increased renal vascular dependence on endogenous NO, reflecting a physiologic ad- aptation to increased NO inactivation. The altered responses to L-Arg and L-NAME suggest that NO plays a critical role in the maintenance of renal hemodynamic function and that the adap- tive NO increase might mask the real magnitude of renal hemodynamic dysfunction. On the other hand, it is also pos- sible that the alteration of NO bioavailability induced by oxi- dative stress could not be the only mechanism involved in homocysteine-induced renal hemodynamic dysfunction. Thus, amplification and/or activation of other vasoconstrictor sys- tems might also be involved and must be further investigated (3). In conclusion, the present study provides the first evidence that hyperhomocysteinemia produces renal oxidative stress and renal hemodynamic dysfunction involving altered L-Arg-NO pathway. In addition, our results suggest that follow-up of renal function should be taken into account in all subjects with high homocysteine levels, because renal failure may aggravate the vascular disease. Finally, oxidative stress plays a major role on homocysteine-induced endothelial dysfunction, antioxidant therapy may be an alternative clinical approach to prevent and/or treat the vascular damage.

Figure 5. Percent changes from baseline on GFR (upper panel), RPF (middle panel), and renal vascular resistance (RVR, bottom panel) Acknowledgments G receiving either vehicle, L-Arginine (L-Arg) or N -nitro-L-arginine We kindly acknowledge Dr. Alberto Crottogini and Dr. Peter methyl ester (L-NAME). C, control (closed columns); HYC, hyper- Willshaw for their collaborative assistance in the correction of this homocysteinemic (open columns) groups. aP Ͻ 0.001 compared with manuscript. This study was supported by Favaloro University, Id # vehicle within the corresponding group. bP Ͻ 0.05 and cP Ͻ 0.001 008-01 and 009-01. compared with the corresponding controls. References 1. Furchgott RF, Zawadzki JV: The obligatory role of endothelial changes of arterial BP, and thus could dissociate the direct cells in the relaxation of arterial smooth muscle by acetylcholine. (renal) effects of NO from the indirect effects (46) resulting Nature 288: 373–376, 1980 from changes in systemic BP. L-Arg has been shown to induce 2. Luscher TF: Imbalance of endothelium-derived relaxing and renal vasodilation by promoting NO formation in the kidney. contracting factors. Am J Hypertens 3: 317–330, 1990 Despite the sparsity of evidence about the real importance of 3. Bagi Z, Ungvari Z, Koller A: Xanthine oxidase-derived reactive the altered renal vasodilatory response to L-Arg, it could be oxygen species convert flow-induced arteriolar dilation to con- proposed that the L-Arg study is sensitive enough to detect striction in Hyperhomocysteinemia. Arterioscler Thromb Vasc subtle endothelium-related renal hemodynamic changes (47). Biol 22: 28–33, 2002 4. Higashi Y, Oshima T, Ozono R, Watanabe M, Matsuura H, Thus, the attenuated response to L-Arg observed in HYC rats Kajiyama G: Effects of L-Arginine infusion on renal hemody- suggests that a higher superoxide anion radical production namics in patients with mild essential hypertension. Hyperten- induced by hyperhomocysteinemia favors a greater NO inac- sion 25: 898–902, 1995 tivation by sequestration, which, in turn, reduces the ability of 5. Arnold WP, Mittal CK, Katuki S, Murad F: Nitric oxide citrates NO to produce renal vasodilation. Therefore, higher NO syn- guanylate cyclase and increases guanosine 3'.5'-cyclic mono- thesis does not suffice to preserve normal renal vasodilation. phosphate levels in various tissue preparations. Proc Natl Acad On the other hand, it is known that under oxidative stress Sci USA 74: 3203–3206, 1997 J Am Soc Nephrol 14: 653–660, 2003 Renal Hemodynamics in Hyperhomocysteinemic Rats 659

6. Palmer RM, Ashton DS, Moncada S: Vascular endothelial cells 23. Willems FF, Aengevaeren WR, Boers GH, Blom HJ, Verheugt synthesize nitric oxide from L-arginine. Nature 333: 664–666, FW: Coronary endothelial function in hyperhomocysteinemia: 1988 Improvement after treatment with folic acid and cobalamin in 7. Kone BC, Baylis C: Biosynthesis and homeostatic roles of NO in patients with coronary artery disease. J Am Coll Cardiol 40: the normal kidney. Am J Physiol 272: F561–F578, 1997 766–772, 2002 8. Raij L, Baylis C: Glomerular actions of nitric oxide. Kidney Int 24. Woo KS, Chook P, Chan LL, Cheung AS, Fung WH, Qiao M, 48: 20–32, 1995 Lolin YI, Thomas GN, Sanderson JE, Metreweli C, Celermajer 9. Nath KA, Norby SM: Reactive oxygen species and acute renal DS: Long-term improvement in homocysteine levels and arterial failure. Am J Med 109: 665–678, 2000 endothelial function after 1-year folic acid supplementation. 10. Graham IM, Daly LE, Refsum HM, Robinson K, Brattstrom LE, Am J Med 112: 535–539, 2002 Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson 25. Salazar FJ, Pinilla JM, Lopes F, Romero JC, Quesada T: Renal B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman effects of prolonged synthesis inhibition of endothelium-derived J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales Luis AC, nitric oxide. Hypertension 20: 113–117, 1992 Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Andria G: 26. Frauscher G, Karnaukhova E, Muehl A, Hoeger H, Lubec B: Plasma homocysteine as a risk factor for vascular disease: The Oral administration of homocysteine leads to increase plasma European concerted action project. JAMA 277: 1775–1781, 1997 triglycerides and homocysteic acid additional mechanism in ho- 11. Chauveau P, Chadefaux B, Coude M, Aupetit J, Hannedouche T mocysteine induced endothelial damage? Life Sci 57: 813–817, Kamoun P, Jungers P: Hyperhomocysteinemia, a risk factor for 1995 atherosclerosis in chronic uremic patients. Kidney Int 4: S72– 27. Ueland PM, Refsum H, Stabler SP, Mallinow MR, Andersson A, S77, 1993 Allen RH: Total homocysteine in plasma or serum: Methods and 12. Robinson K, Gupta A, Dennis VW, Arheart K, Chaudhary D, clinical applications. Clin Chem 39: 1764–1779, 1993 Green R, Vigo P, Mayer EL, Selhub J, Kutner M, Jacobsen D: 28. Rajagopalan S, Kurz S, Mu¨nzel T, Tarpey M, Freeman BA, Hyperhomocysteinemia confers and independent increased risk Griendling KK, Harrison DG: Angiotensin II mediated hyper- of atherosclerosis in end-stage renal disease and is closely linked tension in the rat increases vascular superoxide production via to plasma folate and pyridoxine concentrations. Circulation 94: membrane NADH/NADPH oxidase activation. J Clin Invest 97: 2743–2748, 1996 1916–1923, 1996 13. Ningjun L, Ya-Fei C, Ai-Ping Z: Implications of hyperhomocys- 29. Lowry O, Rosebroughn N, Farr A, Randall L: Protein determi- teinemia in glomerular sclerosis in hypertension. Hypertension nation with the Folin phenol reagent. J Biol Chem 193: 265–275, 39: 443–448, 2002 1951 14. Jong S: Hyperhomocysteinemia in patients with peripheral arte- 30. Warnholtz A, Nickening G, Schultz E, Macharzina R, Brasen JR, rial occlusive disease. Clin Chem Lab Med 39: 714–716, 2001 Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison 15. Cook RC, Tupper JK, Parker S, Kingsbury K, Frohlich JJ, Abel DG, Bohm M, Meinertz T, Munzel T: Increase NADH-oxidase JG, Gao M, Ignaszewski AP: Effect of immunosuppressive ther- mediated superoxide production in the early stages of atheroscle- apy, serum creatinine and time after transplant on plasma total rosis. Evidence for the involvement of the renin-angiotensin homocysteine in patients following heart transplantation. J Heart system. Circulation 99: 2027–2033, 1999 Lung Transplant 18: 420–424, 1999 31. Trolliet MR, Rudd MA, Loscalzo J: Oxidative stress and renal 16. Herrero JI, Quiroga J, Sangro B, Beloqui O, Pardo F, Cienfuegos dysfunction in salt-sensitive hypertension. Kidney Blood Press JA, Prieto J: Hyperhomocysteinemia in liver transplant recipi- ents: prevalence and multivariate analysis of predisposing fac- Res 24: 116–123, 2001 tors. Liver Transpl 6: 614–618, 2000 32. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in 17. Ducloux D, Fournier V, Rebibou JM, Bresson-Vautrin C, Gibey animal tissues by thiobarbituric acid reaction. Anal Biochem 95: R, Chalopin JM: Hyperhomocyst(e)inemia in renal transplant 351–358, 1979 recipients with and without cyclosporine. Clin Nephrol 49: 232– 33. Davis M, Mehendale H: Functional and biochemical correlates of 235, 1998 chlordecone exposure and its enhancement of CCl4 hepatotoxic- 18. Nappo F, De Rosa N, Marfella R, De Lucia D, Ingrosso D, Perna ity. Toxicology 15: 91–103, 1980 AF, Farzati B, Giugliano D: Impairment of endothelial function 34. Vaziri ND, Linag K, Ding Y: Increased nitric oxide inactivation by acute hyperhomocysteinemia and reversal by antioxidant vi- by reactive oxygen species in lead induced hypertension. Kidney tamins. JAMA 281: 2113–2118, 1999 Int 56: 1492–1498, 1999 19. Fu WY, Dudman NP, Perry MA, Wang XL: Homocysteine 35. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, attenuates hemodynamic responses to nitric oxide in vivo. Ath- Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk erosclerosis 161: 169–176, 2002 DC: Measurement of protein using bicinchoninic acid. Anal 20. Bellamy MF, Mc Dowell IF: Putative mechanisms for vascular Biochem 150: 76–85, 1985 damage by homocysteine. J Inherit Metab Dis 20: 307–315, 36. Kim SW, Lee J, Paek YW, Kang DG, Choi KC: Decreased nitric 1997 oxide synthesis in rats with chronic renal failure. J Korean Med 21. Woo KS, Chook P, Lolin YI, Cheung AS, Chan LT, Sun YY, Sci 15: 425–30, 2000 Sanderson JE, Metreweli C, Celermajer DS: Hyperhomocys- 37. Pradelles P, Grassi J: Enzyme immunoassays of adenosine cyclic t(e)inemia is a risk factor for arterial endothelial dysfunction in 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate humans. Circulation 96: 2542–2544, 1997 using acetylcholinesterase. Anal Chem 61: 447, 1989 22. Raghuveer G, Sinkey CA, Chenard C, Stumbo P, Haynes WG: 38. Fischer PA, Bogoliuk CB, Ramirez AJ, Sa´nchez RA, Masnatta Effect of vitamin E on resistence vessel endothelial dysfunction LD: A new procedure for evaluation of renal function without induced by methionine. Am J Cardiol 88: 285–290, 2001 urine collection in rat. Kidney Int 58: 1336–1341, 2000 660 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 653–660, 2003

39. Demeilliers B, Mimran A, Jover B: Nitric oxide participated in the renal cortex of rats with diabetes mellitus. J Am Soc Nephrol renal vasodilatory effect of Condesartan in anesthetized rats. 12: 1630–1639, 2001 J Am Soc Nephrol 10: S208-S212, 1999 46. Baylis C, Slangen B, Hussain S, Weaver C: Relationship be- 40. Smith HW, Finkelstein N, Aliminosa L, Crawford B, Graber M: tween basal NO release and cyclooxygenase products in the The renal clearances of substituted hippuric acid derivatives and normal rat kidney. Am J Physiol 271: R1327–R1334, 1996 other aromatic acids in dogs and men. J Clin Invest 24: 388–404, 47. Bello E, Caramelo C, Martell N, Alca´zar JM, Gonza´lez J, Lo´pez 1945 MD, Ruilope LM, Gonza´lez FR, Rovira AM, GazapomR, Sol- 41. Schnurr E, Lahme W, Ku¨ppers H: Measurement of renal clear- devilla MJ, Casado S: Impairment of renal vasodilation with ance of Inulin and PAH in the steady state without urine collec- L-Arginine is related to more severe disease in untreated hyper- tion. Nephrology 13: 26–29, 1980 tensive patients. Hypertension 38: 907–912, 2001 42. Vanhoutte PM: Endothelium and control of vascular function. 48. Hill CH, Lateef AM, Engels K, Samsell L, Baylis C: Basal and Hypertension 13: 658–667, 1989 stimulated nitric oxide in control of kidney function in the aging 43. Lozcalso J: The oxidant stress of Hyperhomocyst(e)inemia. rat. Am J Physiol 272: R1747–R1753, 1997 J Clin Invest 98: 5–7, 1996 49. Reckelhoff JF, Manning RD: Role of the endothelium-derived 44. Tolins JP, Shultz PJ: Endogenous nitric oxide synthesis deter- nitric oxide in control of renal microvasculature in aging male mines sensibility to the pressor effect of salt. Kidney Interna- rats. Am J Physiol 265: R1126–R1131, 1993 tional 46: 230–236, 1994 50. Tank JE, Vora JP, Houghton DC, Anderson S: Altered renal 45. Ishii N, Patel KP, Lane PH, Taylor T, bian KA, Murad F, Pollock vascular responses in the aging rat kidney. Am J Physiol 266: JS, Carmines PK: Nitric oxide synthesis and oxidative stress in F942–F948, 1994