NEFROLOGIA. Vol. XV. Número 5. 1995 ORIGINALES Role of Endogenous Nitric Oxide. Evidence for a Nitric Oxide (NO)-Sensitive Regulation of Tubule Na Transport. Contribution of Atrial Natriuretic Peptide 99-126 1 J. C. Rodríguez Pérez, J. L. Troy, J. R. Neuringer y B. M. Brenner Renal Division and Department of Medicine, Brighan and Women's Hospital, The Harvard Center for Harvard Medical School, Boston, MA. SUMMARY Preliminary studies have suggested that nitric oxide (NO) control blood pressure in the basal state and plays a role in the water and sodium handling by the kidneys. Inhibition of NO synthesis with competitive L-arginine analogues leads to increased renal vascular resistance and raised systemic and glomerular blood pressure. We questioned whether the effects of NO synthase inhibition, such as NG-nitro Larginine methyl-ester (L-NAME) interferes with the disposal of an acute NaCl load in chronically NO-blocked (N = 8) anesthetized Munich-Wistar rats compared to controls (N = 6). Significant systemic hypertension and a marked renal vasoconstriction was accompanied with a decline in renal plasma flow, without changes in glomerular filtration rate, with filtration fraction thus being increased in the NOblocked rats. In addition, we observed a marked absolute and fractional excretion of sodium without influences in potassium excretion. These observations could suggest a pressure-natriuresis mechanism plus a reduction in tubular reabsorption of sodium, somewhere in the distal nephron through an NO-sensitive mechanism for regulating tubule Na transport. In an orally L-NAME pretreated rats (N = 6), the effects of ANP 99-126 administration resulted in a transient decrease of RPF (4.22 ± 0.54 mL/min at 60 min) compared with control (N = 5: 6.37 ± 0.77 mL/min at 60 min), with the consequent increase in filtration fraction in the former group. MAP and RVR were maintained without significative changes in each group during the experiment, though L-NAME pretreated rats showed a significant elevation as compared with control. The infusion of ANP 99-126 (0.01 µg/Kg/min) resulted in an 46fold increase in urinary sodium excretion in the L-NAME pretreated rats, as compar e d with a 26-fold increase in control rats. However, fractional and absolute potassium excretion was significantly higher in the control rats. K e y words: N i t r i c oxide. Natriuresis. Atrial natriuretic peptide. L-NAME (Nitro-L-arginine methyl ester). 1 Portion of this study were presented at the 25th Annual Meeting of the American Society of Nephrology, Baltimore, MD, in November 1992 and has been published in abstract form (J Am Soc Nephrol 1992; 3:818). Correspondencia: Dr. José Carlos Rodríguez-Pérez. Servicio de Nefrología-Unidad de Investigación. Hospital Ntra. Sra. del Pino. 35005 Las Palmas de Gran Canaria España. 445 J. C. RODRIGUEZ PEREZ y cols. PAPEL DEL OXIDO NITRICO ENDOGENO. EVIDENCIA DE UN MECANISMO REGULADOR (NO)-SENSIBLE. CONTRIBUCION DEL PEPTIDO NATRIURETICO ATRIAL 99-126 RESUMEN Estudios previos han sugerido que el óxido nítrico (NO) controla la presión arterial en situacion basal, al igual que interviene en el manejo del agua y del sodio por parte del riñón. La inhibición de la síntesis del óxido nítrico con análogos de la L-arginina, en este caso con L-NAME (Nitro-L-arginina metiléster), provoca un incremento de la presión arterial sistémica y glomerular junto a una importante elevación de las resistencias vasculares renales. Para analizar si los efectos de la inhibición crónica de la óxido nítrico sintetasa interfiere con el manejo en la excreción de una sobrecarga de sodio, hemos utilizado ratas Munich-Wistar a las que se les ha administrado L-NAME (100 mg/L) en el agua de bebida diariamente. El experimento fue llevado a cabo en ratas anestesiadas. El grupo tratado con L-NAME (n = 8) mostró frente al grupo de ratas controles (n = 6) una marcada vasoconstricción renal, acompañada de hipertensión arterial sistémica y disminución del flujo plasmático renal, sin cambios en el filtrado glomerular y elevada fracción de filtración. La excreción absoluta y fraccional de sodio se encontró aumentada sin modificaciones en la eliminación de potasio, lo que podría sugerir la existencia de no sólo un mecanismo presión-natriuresis, sino también de una reducción en la reabsorción tubular de sodio en algún lugar de la nefrona distal. Este fenómeno sugeriría un mecanismo óxido nítrico sensible que regulase el transporte tubular de sodio. En una segunda parte del experimento, un nuevo grupo de ratas pretratadas crónicamente con L-NAME oral (n = 6) fueron expuestas a la administración de factor natriurético auricular de rata (ANP 99-126), frente a un grupo control no pretratado con L-NAME (n = 5). Se encontró una disminución significativa del flujo plasmático renal y elevación de la fracción de filtración en el grupo pretratado con L-NAME. La presión arterial sistémica y las resistencias vasculares renales no se modificaron a lo largo del estudio en cada grupo de animales, aunque estaban significativamente más elevadas en el grupo que recibió L-NAME respecto al grupo control (p < 0,001 y p < 0,05, respectivamente, a los 180 minutos del experimento). La administración de ANP 99-126 a las dosis utilizadas provocó una excreción absoluta de sodio 46 veces superior a la basal en el grupo tratado previamente con L-NAME. Por el contrario, la excreción absoluta y fraccional de potasio fue significativamente más elevada (p < 0,05) en el grupo no tratado previamente con LNAME. Estos resultados sugieren una atenuación del efecto kaliurético del ANP en aquellos animales previamente tratados con L-NAME, datos que apoyarían la existencia de mecanismo NO-sensible a nivel tubular. Palabras clave: Oxido nítrico. Natriuresis. Factor natriurético auricular. L-NAME (Nitro-L-arginina metiléster). Acethylcholine (ACh)-induced relaxation of arteries is endothelium-dependent and the relaxation is mediated through the release of endothelium-derived relaxing factor(s) (EDRF) 1, 2. Subsequent studies suggested that at least one EDRF is nitric oxide (NO) 3, 4. Particularly, the association of ACh-induced vasodilatation and the release of nitric oxide has been demonstrated in the isolated perfused rabbit heart 5. The 446 synthesis of NO from L-Arginine has been proposed to represent a widely expressed process 6. NO, much like oxygen is actually a gas with an ultrashort half-life (less than 5 seconds in biological tissues) that is sparingly soluble in aqueous medium and functions biologically as a molecule in solution. Nitric oxide which is generated from L-Arginine by the constitutive type I nitric oxide synthase (NOS), a NO Y REGULACION DEL TRANSPORTE DE Na Ca2+ /calmodulin-dependent enzyme 7, stimulates increases in cGMP within the isolated aorta and in vascular smooth muscle cells and platelets 8-10. The inc r e a s e occurs via stimulation of the soluble, or citosolic guanylate cyclase in an autocrine or paracrine type of action 11. In light of the evidence indicating that intrarenal infusions or either ACh or bradykinin, endotheliumdependent vasodilators that increase NO synthesis and release, elicit an increase in sodium excretion as well as renal vasodilation 12, it was expected that the inhibition of NO synthesis would result in a decrease in sodium excretion as well as renal vasoconstriction. To try to examine some of the systemic and intrarenal effects of NO in normal animals, several investigators have given the animals different L-Arginine analogues as specific substrate competitors 12-20. Systemic blockade of EDRF/NO synthesis with these L-arginine analogue increases both arterial pressure (AP) and renal vascular resistances in anesthetized and conscious a n i m a l s 21-24. Baylis et al 24, have demonstrated in conscious rats that the decrease in renal plasma flow (RPF) caused by inhibition of EDRF/NO synthesis was accompanied by a minor decrease in glomerular filtration rate (GFR), resulting in an increase in filtration fraction (FF). The same authors based on these observations describe a new model of systemic hypertens i o n with glomerular capillary hypertension 2 5 . Variable effects of NO synthesis inhibition on sodium excretion have been reported. Several authors have reported that NO synthase inhibitors in vivo induces natriuresis and diuresis 15, 19, 24, 26, 27. Although some investigators have suggested that the increased excretion of sodium and water during NO inhibition is due to «pressure natriuresis» 16-18, 28, others have hypothesized a proximal direct tubular action 27, or a distal tub u l a r action of the NO synthase inhibition 15, 19, 24. Navarro et al 29, used increasing concentrations of LN A M E in the drinking water for five weeks, in Sprague-Dawley rats, and only found blood pressure elevation without changes in sodium excretion or diuresis. In a preliminary study, data from our laboratory 19 in normal and DOCA-salt hypertensive rats L-NAME induced natriuresis with a minimal kaliuretic response, suggesting a terminal nephron site of action of this nitric oxide synthesis inhibitor. There is a constant controversy about the difficulty in separating direct renal effects of NO synthesis inhibition from systemic hemodynamic effects in the various experimental models. In view of this apparent discrepancy, the purpose of the present experiment was twofold. Firstly, to investigate the effects on renal hemodynamics and excretory function in anesthetized rats, of chronic NO synthesis inhibition on the response to an acute NaCl load. Since NG-nitro-L-arginine is not readily soluble in water, we used NG-nitro-L-arginine methyl ester (LNAME) an easily dissolved and orally active NO inhibitor 25, 30. Secondly, we explore the role of ANP 991 2 6 (atrial natriuretic peptide 99-126) in blood pressure alteration, natriuresis and kaliuresis in L-NAME pretreated rats. Some of the ANP described actions include an elevation of GFR and renal sodium excretion, relaxation of contracted vasculature in vitro, and reduction of systemic arterial blood pressure in vivo. Therefore, the present study was undertaken to evaluate the effects of ANP on the systemic and renal circulation under NO syntesis inhibition. Since NO is a very labile substance, direct measurement of NO has proven to be difficult, especially in vivo experiments. We must limited as in other experiences to interpretation of the responses to NO synthase inhibitors. Materials and methods Studies were performed on 25 male Munich-Wistar rats (220-300 g body weight) from the Charles River, Wilmington, Mass. All experimental procedures were designed in accordance with the recommendations f r o m the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals approved b y the Council of the American Physiological S o c i e t y . Animals were maintained on a 12-hour light/dark cycle and provided normal rat chow and tap water ad libitum. In the first experiment protocol ( S t u d y I) eight rats (Group Ia) were placed on oral L-NAME (100 mg/L in drinking water, changed daily) f o r a continuous 10 to 15 day period. After 5 to 7 days of habituation, systolic blood pressure was recorded every two days in all rats by the awake tail c u f f method 31, until hypertensive state was confirmed. A control group of six rats (Group Ib) aged over a similar time period differed only in not receiving LNAME (figure 1). The day of the experiment, rats were anesthetized w i t h intraperitoneal thiobarbiturate, Inactin (BYK G u l d e n , Konstans Fed Rep. Germany) (100 mg/kg body wt.) and placed on a temperature-regulated micropuncture table. Rectal temperature was maintained at 37 ± 1.0 °C. An indwelling polyethylene catheter (PE-50) was placed in the left femoral artery for c o n t i n u o u s monitoring of mean arterial pressure (MAP) as well as for collecting blood samples. MAP was measured utilizing a pressure transducer connected to a direct recorder (Gould Inc. Cleveland, OH. with a thermal writing recorder 8000-S, model 8188-2202). After a baseline blood sample was collected and tracheostomy, both yugular veins were catheterized with PE-50 polyethylene tubing, one for 447 J. C. RODRIGUEZ PEREZ y cols. Surgical Baseline U3 U4 U5 U6 U7 U8 I/ / / / / / / / / / I- - - - - - - - I - - - - - - - - - I - - - - - - - - -I- - - - - - - -I procedure B1 U1 B2 U2 B3 B4 B5 B6 B7 B8 ­30 min 0 min 60 min 120 min 180 STUDY I GROUP Ia: L-NAME NaCl 0.9 % - - - - - - - - - - - - - - - - - - I - - - - - - - - - -I - - - - - - - - - - - - - - - -I 0 5 % BW 60 minutes GROUP Ib: CONTROL NaCl 0.9 % - - - - - - - - - - - - - - - - - - I - - - - - - - - - -I - - - - - - - - - - - - - - - -I 0 5 % BW 60 minutes STUDY II GROUP IIa: L-NAME + ANP 0,01 µg/kg/min ANP in NaCl 0.9 % - - - - - - - - - - - - - - - - - - I - - - - - - - - - -I - - - - - - - - - - - - - - - -I 0 60 minutes GROUP IIb: CONTROL + ANP 0,01 µg/kg/min ANP in NaCl 0.9 % - - - - - - - - - - - - - - - - - - I - - - - - - - - - -I - - - - - - - - - - - - - - - -I 0 60 minutes fied synthetic rat atrial natriuretic peptide (ANP 99126), from Peninsula Laboratories (Belmont CA), during a 60-min period (figure 1). The dose utilized was previously determined to yield effective natriuretic and diuretic effects without a deleterious alteration of r a t GFR and minimal changes in blood pressure. Each rat received only one peptide infusion. A control group of five rats (Group IIb) differed only in not receiving L-NAME. Analytical procedures Hematocrit was determined by the microcapillary tube method. Urinary and plasma sodium and pota ssium concentration were measured by standard flame photometry. Protein concentration in femoral arterial blood plasma was determined using the fluorom e t r i c method 32. Inulin concentrations in plasma a n d urine were measured using a macro-anthrone method 33, and PAH concentrations were measured by the method of Smith et al 34. Filtration fraction was calculated as FF= GFR/RPF. Renal vascular resistance was estimated by the expression RVR= MAP (1HCT)/RPF, where HCT is arterial hematocrit. Efferent arteriolar protein concentration was estimated by the expression CE=CA/(1-FF), where CA and CE are plasma protein concentrations in afferent (femoral artery determination) and efferent arterioles, respectively. Results are presented as mean ± 1 SE. Analysis of comparisons between groups was performed by oneway analysis of variance. We considered a P value of less than 0.05 to be statistically significant. Results Fig. 1.­Schematic representation of experimental protocols. continuous iv infusion throughout the experiment of i s o n c o t i c plasma obtained from normal adult Munich-Wistar rats to maintain an euvolemic condition and for a sustained infusion of p-aminohippurate (4 00 µl) and inulin (10 ml) solution for the measurement of renal plasma flow (RPF) and glomerular filt r a t i o n rate (GFR). The left kidney was exposed through a ventral midline incision and its surface was m o i s t e n e d with saline throughout the experiment. The left ureter was cannulated with PE-10 tubing and t h e bladder vented by a curved 19-gauge needle. Urine was collected in a preweighed plastic vial for gravimetric determination of urine flow rate. After a 60-min equilibration time, when plasma inulin and PAH concentrations had plateaud, a control observation period was begun in which two 15 min urine collections were made and arterial blood samples were taken at the midpoint of each urine. After completion of control measurements, in Study I the second yugular vein was used with an isotonic infusion of NaCl (5 % BW in 60 min). Six more 30-min urine collections with midpoints bloods were taken. In Study II, six rats (Group IIa) previously placed on oral L-NAME (100 mg/L in drinking water during a continuous period of 10-15 days) followed a similar surgical protocol to those of Study I with the differenc e that, during the experimental period 0,01 µg/Kg/min was infused in saline (1.5 µg/mL), of puri448 Study I: Rats subjected to a NaCl load The mean body weight of the rats used in this study was 289 ± 4.6 g and 260.1 ± 11.6 g (NS) for groups Ia and Ib, before placing the former on oral L-NAME. The effect of NaCl load on mean arterial pressure, renal vascular resistance and urinary sodium and potassium excretion in rats of groups Ia and Ib is shown in figure 2 After 10-15 days rats placed on oral L-NAME were hypertensive. Mean arterial pressure averaged 130.5 ± 2.3 in the L-NAME group, and each value was significantly higher (P < 0.001) than the arterial pressure of 97.7 ± 3.9 mmHg for the vehicle-treated control rats (fig. 2A). A marked and significant elevation in systemic MAP was maintained throughout the experiment. The renal vascular resistance (RVR) increased in parallel with MAP and maintained for more than 180 minutes (27.1 ± 1.9 and 22.0 ± 1.1 vs 13.07 ± 1.0 and 12.9 ± 0.7 mmHg/mL/min in groups Ia and NO Y REGULACION DEL TRANSPORTE DE Na Fig. 2.­Effect of NaCl load in rats of study I in the baseline and during the experimental protocol. Mean arterial pressure (A), renal vascular resistance (B), urinary excretion rates of sodium (C), and potassium (D). * P < 0.05, ** P < 0.01, *** P < 0.001 vs baseline. P < 0.05, P < 0.01, P < 0.001 group Ia vs group Ib. Ib respectively). A slight decrease in RVR was observed in groups Ia and group Ib during NaCl infusion although it was more pronounced in the L-NAME treated rats (fig. 2B). Since the glomerular filtration rate was unchanged in L-NAME and in the control group of rats by NaCl infusion, RPF was significantly reduced (P < 0.01) in group Ia. These hemodynamic changes yielded at 120 and at 180 min with a significant increase in FF. No significant differences in urine output was seen between L-NAME and control group. The magnitude of the slight decrease in arterial hematocrit was similar in groups Ia and Ib. Associated with the pressor responses elicited by L-NAME were a striking increase in urinary sodium excretion during NaCl load (fig. 2C) also evident at 180 min (4.67 ± 0.4 µeq/min) and significantly higher (P < 0.05) than the 180 min sodium excretion rate measured in the vehicle-treated animals (3.5 ± 0.23 µeq/min). Both groups of rats excreted with minor differences (NS) t h e same sodium load throughout the experiment. On the other hand, urinary potassium excretion did not differ in Ia and Ib groups respectively (fig. 2D). In group Ia mean arterial pressure and RVR had decreased somewhat by the end of the protocol (basal vs final 130.5 ± 2.36 vs 114.2 ± 5.2 mmHg, P < 0.05, and 2 7 . 1 ± 1 . 9 vs 21.9 ± 1 . 1 mmHg/mL/min, P < 0.05 respectively), however RPF (basal vs final 2.67 ± 0.22 vs 3.1 ± 0.11 mL/min, P = NS) were unchanged over the course of the experiment. In both groups Ia and Ib, a significant increase (P < 0.001) in urine flow rate and UNa.V was observed over the 180 minutes experimental period. Study II: Rats subjected to atrial natriuretic peptide (ANP 99-126) infusion A r t e r i a l hcts were similar between experimental (IIa) and control (IIb) groups. There were no signifi449 J. C. RODRIGUEZ PEREZ y cols. cant differences in mean body weight for both group o f rats 280 ± 2 . 2 g (group IIb) and 280.8 ± 7 . 5 g (group IIa). Figure 3 shows the data for mean arterial pressure, glomerular filtration rate and urinary sodium and potassium excretion measured before and 60, 120 and 1 8 0 minutes after infusion of ANP in L-NAME and vehicle-control groups. MAP slightly decreased (Fig. 3A) after ANP 99-126 infusion in L-NAME group, but s t i l l remained significantly higher than the vehicle group (116.2 ± 2.39 vs 93.5 ± 2.2 mmHg respectively at 180 min after ANP). In control rats and in those pretreated with L-NAME coincident with the ANP infusion, glomerular filtration rate increased but, ret u r n to basal levels straightaway (Fig. 3B), without any difference between IIa and IIb groups. The filtrat i o n fraction was significantly higher (P < 0.01 and P < 0.05 at 120 and 180 min respectively) in the group IIa associated with a decreased RPF at baseline in this group of rats (p < 0.05). The peptide caused a significant increase in urine flow in group IIa (P < 0.05) and IIb (P < 0.01) at 120 minutes respect to baseline as well as an increase in urine sodium excretion (P < 0.05) and (P < 0.01) for both groups respectively. The infusion of ANP 99-126 in group IIa animals resulted in a 46-fold increase in urinary sodium excretion averaged 13.84 ± 2.1 and 9.67 ± 1.0 at 60 (P < 0.01) and 120 (P < 0.05) min vs 5.32 ± 1.36 and 5.56 ± 0.93 µeq/min in group IIb (a 26-fold increase in UNa.V), and each value was significantly higher than the basal excretion rate in both g r o u p s 0.3 ± 0 . 0 5 (group IIa) (P < 0.05) and 0.2 ± 0.09 (group IIb) µeq/min (P < 0.01) at 120 min (Fig. 3C). Fractional excretion of potassium as well as absolute urinary potassium excretion (Fig. 3D) were sign i f i c a n t l y higher in group IIb as compared with LN A M E - A N P rats (IIa) at 120 min (P < 0.05) and at 180 min however, at that latest period this difference was not statistically significant for the fractional excretion rate (P < 0.1). Thus, the «potassium sparing» Fig. 3.­Effect of ANP (99-126) administration in control and L-NAME rats (Study II) in baseline and over the course of the experiment. Mean arterial pressure (A), gomerular filtration rate (B), urinary excretion rates of sodium (C), and potassium (D). * P < 0.05, ** P < 0.01, *** P < 0.001 vs baseline. P < 0.05, P < 0.01,P < 0.001 group IIa vs group IIb. 450 NO Y REGULACION DEL TRANSPORTE DE Na Table I. Systemic and renal variables in anesthetized male Munich-Wistar rats studied in the basal conditions and after chronic EDRF-Blockade with L-Name. Groups Ia (A) and Ib (B) were loaded with NaCl 5 % BW, and groups IIa (C) and IIb (D) with ANP 99-126 (0.01 µg/kg/min) during 60 min after basal measuresa Basal L-NAME group (la) (A) HCT (%) MAP (mmHg) GFR (mL/min) RPF (mL/min) FF RVR (mmHg/mL/min) UV (mL/min) UNa.V (µEq/min) FE Na (%) UK.V (µEq/min) FE K (%) CA (g/dl) CE (g/dl) Control group (Ib) (B) HCT (%) MAP (mmHg) GFR (mL./min) RPF (mL/min) FF RVR (mmHg/mL/min) UV (mL/min) UNa.V (µEq/min) FE Na (%) UK.V (µEq/min) FE K (%) CA (g/dl) CE (g/dl) L-NAME-ANP group (lla) (C) HCT (%) MAP (mmHR) GFR (mL/min) RPF (mL/min) FF RVR (mmHg/mL/min) UV (mL/min UNa.V (µEq/min) FE Na (%) UK.V (µEq/min) FE K (%) CA (g/dl) CE (g/dl) Control-ANP group (llb) (D) HCT (%) MAP (mmHg) GFR (mL/min) RPF (mL/min) FF RVR (mmHg /mL/min) UV (mL/min) UNa.V (µEq/min) FE Na (%) UK.V (µEq/min) FE K (%) CA (g/dl) CF: (g/dl) a 60' 40.9 ± 0.65**, 123.3 ± 3.58 1.28 ± 0.09** 4.9 ± 0.48** 0.26 ± 0.01** 16.0 ± 1.8**, 0.039 ± 0.005*** 7.5 ± 1.09*** 4.4 ± 0.78*** 2.27 ± 0.15**_ 41.8 ± 2.3* 4.0 ± 0.08** 5.6 ± 0.2** 43 ± 0.5* 101 ± 4.0 1.58 ± 0.19 5.91 ± 0.49* 0.27 ± 0.04 10.0 ± 1.0 0.04.3 ± 0.005** 8.4 ± 0.9*** 4.2 ± 0.9* 1.90 ± 0.23 37.3 ± 8.7 4.3 ± 0.07** 6.0 ± 0.34 42.6 ± 1.79 117.5 ± 4.63 1.22 ± 0.03 *** 4.22 ± 0.54 0.31 ± 0.04 17.0 ± 2.0 0.065 ± 0.0092***, 13.84 ± 2.16***, 8.07 ± 1.19***, 2.0 ± 0.2**44.8 ± 4.9* 4.56 ± 0.16*, 6.87 ± 0.63 40.7 ± 0.46**91 ± 3.06 1.22 ± 0.04* 6.37 ± 0.77* 0.20 ± 0.01 8.95 ± 1.09 0.027 ± 0.0054** 5.32 ± 1.36* 2.95 ± 0.77* 1.96 ± 0.18*** 40.1 ± 3.89* 4.08 ± 0.037** 5.1 ± 0.09*** 120' 40.6 ± 0.89**, 116.8 ± 4.23*, 1.02 ± 0.04 3.12 ± 0.15 0.33 ± 0.01 22.5 ± 1.3 0.033 ± 0.001*** 6.55 ± 0.25***, 4.55 ± 0.24***, 1.88 ± 0 06* 43.9 ± 2.3**4.3 ± 0.1** 6.5 ± 0.28* 43.5 ± 0.81* 98.8 ± 4.17 1.10 ± 0.09 4.18 ± 0.2 0.26 ± 0.01 13.6 ± 1.0 0.032 ± 0.002*** 5.0 ± 0.6*** 3.4 ± 0.7* 1.54 ± 0.14 40.8 ± 7.2 4.5 ± 0.14* 6.2 ± 0.21 41.3 ± 0.59**117.0 ± 3.48*, 0.91 ± 0.14 3.52 ± 0.57 0.26 ± 0.006*, 20.6 ± 2.6 0.026 ± 0.0046* 9.6 ± 1*, 6.59 ± 0.62**, 1.2 ± 0.20 34.3 ± 2.7*, 4.62 ± 0.094*, 6.24 ± 0.12**, 41.2 ± 0.47** 93.7 ± 3.72 1.01 ± 0.04 5.03 ± 0.34 0.20 ± 0.01 11.03 ± 0.55 0.029 ± 0.0041** 5.56 ± 0.9** 3.81 ± 0.70** 1.90 ± 0.22*** 49.7 ± 4.71** 4.3 ± 0.07** 5.4 ± 0.07** 180' 41 ± 0.85**, 114.2 ± 5.2*. 0.96 ± 0.4 3.1 ± 0.11 0 31 ± 0 01 21.9 ± 1.1*. 0.027 ± 0.001*** 4.67 ± 0.4***. 3.4 ± 0.27***. 1 59 ± 0.1 39.3 ± 2.0* 4.4 ± 0.1* 6.4 ± 0.3* 43.6 ± 0.81* 97 ± 4.89 1.08 ± 0.06 4.26 ± 0.19 0.25 ± 0.01 12.8 ± 0.7 0.024 ± 0.002*** 3.5 ±i 0.23*** 2.3 ± 0.3** 1.27 ± 0.11 32.7 ± 4.1 4.5 ± 0.12* 6.1 ± 0.17* 42.0 ± 0.4** 116.2 ± 2.39*. 0.90 ± 0.12 3.39 ± 0.37 0.26 ± 0.007*. 20.0 ± 2.0 0.024 ± 0.0048* 4.39 ± 1.37* 3.52 ± 1.04* 1.22 ± 0.12 37.3 ± 3.72* 4.65 ± 0.095*. 6.38 ± 0.14 *. 41.1 ± 0.51** 93.5 ± 2.21 0.85 ± 0.04 3.87 ± 0.61 0.23 ± 0.02 15.27 ± 2.28 0.024 ± 0.0020** 4.44 ± 0.38** 3.76 ± 0.39*** 1.45 ± 0.11** 47.3 ± 2.01** 4.3 ± 0.07** 5.6 ± 0.2* 46.5 ± 0.63 130.5 ± 2.36 0.88 ± 0.05 2.67 ± 0.22 0.33 ± 0.01 27.1 ± 1.9 0.006 ± 0.0008 0.39 ± 0.12 0.29 ± 0.09 1.24 ± 0.16 31.1 ± 2.87 5.0 ± 0.03 7.5 ± 0.1 46.7 ± 0.64 97.7 ± 3.91 1.01 ± 0.08 4.09 ± 0.31 0.24 ± 0.01 13.0 ± 1.0 0.0082 ± 0.0016 1.17 ± 0.36 0.8 ± 0.2 1.31 ± 0.12 33.7 ± 4.0 5.0 ± 0.1 6.7 ± 0.16 46.7 ± 0.36 126.0 ± 1.81 0.91 ± 0.04 2.99 ± 0.18 0.31 ± 0.015 22.9 ± 1.82 0.0054 ± 0.00047 0.30 ± 0.05 0.22 ± 0.04 1.08 ± 0.12 27.2 ± 1.6 5.18 ± 0.06 7.51 ± 0.11 45.9 ± 0.46 85.5 ± 3.49 0.89 ± 0.09 4.19 ± 0.47 0.21 ± 0.02 11.4 ± 1.09 0.0038 ± 0.00028 0.20 ± 0.09 0.17 ± 0.07 0.72 ± 0.08 22.0 ± 4.04 5.03 ± 0.033 6.45 ± 0.18 Values are means ± SE. HCT, hematocrit; MAP, mean arterial pressure; GFR, glomerular filtration rate; RPF, renal plasma flow; FF, filtration fraction; RVR, renal vascular resistance; UV, urinary volume; UNa. V, sodium excretion rate; FENa, fractional excretion of sodium; UK.V, potassium excretion rate; FE K, fractional excretion of potassium; CA and CE, afferent and efferent arteriolar protein concentration. Significant differences between subsequent time points and baseline within each group: * p < 0.05; ** p < 0.01; *** p < 0.001. Significant differences between groups la and Ib and IIa IIb: p < 0.05; p < 0.01; p < 0.001. 451 J. C. RODRIGUEZ PEREZ y cols. e f f e c t of ANP 99-126 was only maintained in the control-vehicle group. The increased renal vascular resistances in group IIa dropped slightly as well as in group IIb after the initiation of ANP 99-126 infusion. As a consequence of the duration of the experiment RVR in group IIb exhibits a tendency to be higher than basal levels but without any statistical significance. Table I illustrates the variations in the systemic and renal parameters throughout the experiment. Discussion Control of vascular function by the endothelium is quite complex and involves a balanced synthesis and release of both vasodilator and vasoconstrictor substances 35. The important modulatory effects of nitric oxide on regional hemodynamics and renal vascular tone have r e c e n t l y been well demonstrated. To examine the s y s t e m i c and renal hemodynamic effects of NO in n o r m a l animals, most investigators have given the animals NO synthesis inhibitors acutely 12-15, 19, 24 dir e c t l y into the renal artery, by venous infusion or chronically in the drinking water 25, 26, 29, 36. Some differences were observed in conscious versus anesthetized rats 37-39. These differing responses were apparently due to different animal models or to varying circulating levels of angiotensin II 38. Meanwhile acetylcholine infusion to the animals resulted in systemic hypotension and renal vasodilation with diuretic and natriuretic effect 40, the administration of NO synthesis inhibitors to animals prom o t e s a marked increase in mean arterial pressure and renal vascular resistance, with a decrease in renal plasma flow and variable (null or minor) effects on glomerular filtration rates with the concommitant elevation in filtration fraction. Baylis and coworkers 25, using 50 mg/L of L-NAME in the drinking water for a two months period, reported a mean arterial pressure of 136 ± 4 mmHg with 30.1 ± 5.6 mmHg/mL/min of renal vascular resistance. Meanwhile, Ribeiro et al 36 using 10 fold higher dose over 4-6 weeks of continual NO blockade with L-NAME reported a significantly greater hypertension. In the study presented here, the results are compatible with those of recent reports 25, 41-43, confirming a previous suggestion, wher e there is a dose-dependency to the magnitude of the systemic hypertension achieved with chronic NO blockade. This increase in renal vascular resistance is specifically related to the EDRF-NO synthesis inhibit i o n . The renal vasoconstriction observed in this study is not due to an autoregulatory phenomena elicited by the concurrent rise in systemic arterial pressure (using subpressor doses of L-NAME in the drin452 king water still increases the calculated RVR; unpublished observations), but rather to a greater sensibility of the vascular renal bed to L-arginine analogues or to an angiotensin II mediated renal vasoconstriction 38. The latter would be in accordance with the reversal decrease in renal plasma flow after the administration of an angiotensin II receptor antagonist 37. In the absence of high circulating levels of angiotens i n II another possibility could be the underlying m y o g e n i c mechanism, as suggested by Ito et al 44. However this vasoconstriction was found to be confined to the afferent arteriole, in controversy with this study where vasoconstriction affect predominantly postglomerular renal vasculature, as indicated by a s i g n i f i c a n t decrease in RPF with no alterations in GFR. In concordance with a Shultz and Tolins suggestion 28, concerning the difficulty to evaluate the different results of the effects of NO synthase inhibitors on renal hemodynamic and excretory function, our findings are consistent with previous reports 15, 19, 24, 27. Meanwhile Zats and De Nucci 15, and Baylis et al 24, have suggested a possible inhibitory effect associated with NO inhibition at the level of the distal or collecting tubule, De Nicola et al 27, evidenced a possible i n h i b i t o r y effect on proximal tubular reabsorption. Our results demonstrate that chronic inhibition of basal EDRF/NO synthesis in anesthetized rats produces a substantial pressor response associated with a marked natriuresis. Thus, the reduction in tubular reabs o r p t i o n of sodium (fractional excretion of sodium clearly elevated in group Ia without significant modification in the glomerular filtration rate) without any modification in potassium secretion, must suggest the existence of an NO-sensitive mechanism for regulat i n g distal tubule Na transport, more than a mere pressure natriuresis effect. These findings are compatible with a recent report and indicate that, in the kidney-rat the distribution of NO synthase examined by t h r e e different approaches (immunocytochemistry, enzymatic activity and mRNA expression) revealed the strongest signals for NO synthase in the macula densa cells of the juxtaglomerular apparatus 45. In this regard, Neuringer et al 19, reported a significant reduction in fractional reabsorption of sodium in normal and DOCA-salt hypertensive rats after infusion of L-NAME; furthermore, Radermacher et al 46, in isolated perfused rat kidney believe that reduction in fractional reabsorption of sodium after NO inhibition is p a r t i a l l y due to a specific tubular effect of NO. Previously in a recent report Green and coworkers 47, have shown that 8-bromo-cyclic GMP can also stimulate the Na+-H+ antiporter in renal brush border membranes and thus increase sodium uptake. Our observations of the effects of NO inhibition on urine flow and urinary sodium excretion are different NO Y REGULACION DEL TRANSPORTE DE Na from the results of some experiments in rats in which systemic administration of NO synthesis inhibitors induced an antidiuretic and antinatriuretic respons e 16-18, 28. In view of this apparent discrepancy we c a n n o t provide any satisfactory explanation. However, this may be due to the influence of species and strain differences, distinct methodological approach with the use of not always the same L-Arginine analogues, or the influence of some other activated mechanism like the release of ANP during NO-inhibitors administration 48. These findings could suggest a dissociation of the sodium excretory responses from the hemodynamic changes during NO synthesis inhibition. A transient increase in GFR and renal plasma flow was observed in group IIb after ANP 99-126 administration in accordance with other studies using different ANP peptides 49, 50, a similar increase was obs e r v e d in the NO synthesis blocked group. In contrast, DePriest, Zimmermann and Baylis 51, using different doses of a 28 aminoacid rat ANP for a 60 min period to conscious unstressed rats failed to produce statistically significant changes in GFR with a slight reduction in renal plasma flow and increased renal vascular resistance although these effects were not statistically significant. These and other studies have provided significant evidence that GFR-enhancing effect of ANP may result from the use of the surg i c a l l y anesthetized preparation. Although in the present study we do not conduct any micropuncture studies, the increased GFR could result from the suggested afferent arteriolar vasodilatation and concurrent efferent arteriolar vasoconstriction 50. In the pres e n t study, this was in accordance with a stable f i l t r a t i o n fraction and renal vascular resistance in both (IIa and IIb) groups without significant modific a t i o n s in MAP, probably in relation with the low doses or particular properties of the peptide reached. T h e high renal vascular resistance obtained in the group IIa after pretreatment with L-NAME, declined slightly coinciding with an increase in renal plasma flow after infusion of ANP 99-126 but without statist i c a l significance, in contrast with previous works where ANP infusion can vasodilate renal blood vessels under conditions of high vasoconstrictor tone or where renal vasculature is precontracted with norepinephrine and angiotensin II 50, 52, 53. This apparent discrepancy could be associated with the different ANP molecule or doses used, as well as duration of the experience. Some investigators have proposed that the increase in GFR alone can account for the natriuresis and diuresis induced by ANP 53,54, whereas others supported that ANP also directly alters tubule Na+ and water rea b s o r p t i o n 55-57. As in our study, ANP 99-126 has b e e n capable of stimulate natriuresis and diuresis without producing detectable and maintained alterations of GFR 56, 58, suggesting that this may be due to the lower doses used. At higher dosis, an increase in GFR is marked 56, 59. Maack 60, has argued that even during brisk natriuresis, undetectable changes in GFR could contribute to the proper natriuresis observed with ANP administration. Nevertheless, several observations like in toadfish 61, a species that lacks glomeruli, ANP induces an accentuated natriuresis, indicating that changes in GFR alone do not account for the important natriuresis and diuresis observed in response to ANP infusion. As was indicated in Table I, potassium excretion was much more variable and much less pronounced than sodium excretion rate. The absolute and fractional potassium excretion increased in groups Ia and I b , but without statistical significance between the vehicle-control group and L-NAME treated group. In contrast, the L-NAME prevented the increased rate of potassium excretion reached with the use of ANP 99126 alone in the study II. Such findings suggest that L-NAME blunted the action of ANP 99-126 at distal portions of the nephron, however, there is also little evidence of an action of ANP in the distal or collecting duct. Presently, the in vivo studies do not assess whether the hormone acts directly on the epithelial cells or alters the transepithelial driving forces 62, 63, and whether these segments are devoid or not of receptors for ANP 64. The current study therefore suggests, by the use of t h e NO synthase inhibitor L-NAME, that NO is an important modulatory agent in both the renal vascular and tubular function, and probably independent of changes in the renal perfusion pressure. In conclusion, the present investigation indicates that EDRF/NO exerts a substantive role in maintaining the normally low renal vascular tone, the renal vasoconstriction observed in this study is not due to an autoregulatory phenomena elicited by the concurrent rise in systemic arterial pressure. The use of LNAME an orally active NO-synthase inhibitor was fol l o w e d in our model with a marked natriuretic response without any modification in the potassium e x c r e t i o n rate, suggesting an NO-sensitive mechanism for regulating distal tubule Na-transport. With t h e use of the peptide ANP 99-126 a potent and m a i n t a i n e d diuresis and natriuresis was observed even in the L-NAME pretreated group with minimal and excretion response the L-NAME treated transient elevations in GFR. The potassium reached with this p e p t i d e was attenuated in animals. Further studies are needed to clarify the exact mechanism responsible for NO induced changes in tubular function, and the physiological role of this particular ANP 99-126 peptide in the regulation of salt, water and potassium homeostasis. 453 J. C. RODRIGUEZ PEREZ y cols. Acknowledgments These studies were supported by a grant 91/5469 f r o m the Fondo de Investigación Sanitaria (FISss), from the National Institute of Health of Spain, to Dr. J. C. Rodríguez-Pérez. The authors thank Miguel A. Zayas and Myriam Lee for expert technical assistance. Bibliografía 1. 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