Combining lisinopril and L -arginine slows disease progression and reduces endothelin-1 in passive Heymann nephritis

Kidney International, Vol. 64 (2003), pp. 857–863

Combining lisinopril and l-arginine slows disease progression and reduces endothelin-1 in passive Heymann nephritis CARLA ZOJA, ARIELA BENIGNI, DAVIDE CAMOZZI, DANIELA CORNA, LORENA LONGARETTI, MARTA TODESCHINI, and GIUSEPPE REMUZZI Mario Negri Institute for Pharmacological Research, Bergamo, Italy; and Unit of Nephrology and Dialysis, Azienda Ospedaliera, Ospedali Riuniti di Bergamo, Bergamo, Italy

phropathy not completely responsive to ACE inhibition. Restoring the nitric oxide/ET-1 balance could be of benefit in halting renal disease progression.

Combining lisinopril and L-arginine slows disease progression and reduces endothelin-1 in passive Heymann nephritis. Background. Despite angiotensin-converting enzyme (ACE) inhibition is a very powerful therapy, it may not be uniformly renoprotective in patients with proteinuric nephropathies who might refer late in the course of the disease. In accelerated passive Heymann nephritis (PHN), a severe rat model of human membranous nephropathy, with proteinuria and increased urinary excretion of endothelin-1 (ET-1), early treatment with an ACE inhibition limited proteinuria as well as the exuberant formation of renal ET-1, while late treatment reduced urinary proteins not to a significant extent. Since biologic effects and production of ET-1 within the kidney are counteracted by nitric oxide, we studied the effect of combining lisinopril and l-arginine, the natural precursor of nitric oxide, starting late in the disease. Methods. Uninephrectomized PHN rats were divided in four groups (N ⫽ 10) and daily given orally: vehicle; 1.25 g/L l-arginine; 40 mg/L lisinopril; and l-arginine ⫹ lisinopril. Treatments started at 2 months, when rats had massive proteinuria, until 9 months. Six normal rats served as control. Results. Increase in systolic blood pressure was significantly limited by l-arginine. Lisinopril alone and the combination were more effective. Renal function impairment was not affected by l-arginine, partially ameliorated by ACE inhibitor and normalized by the combined therapy. In rats given l-arginine, proteinuria levels were similar to vehicle. ACE inhibitor kept proteinuria at values comparable to pretreatment and numerically lower than vehicle. Addition of l-arginine to lisinopril was more effective, with values significantly lower than vehicle. Glomerular and tubular changes were limited by the ACE inhibitor and further ameliorated by the combined therapy. Exaggerated urinary ET-1 of PHN was reduced by 23% and 40% after l-arginine and lisinopril, respectively, and by 62% with the combination. Defective urinary excretion of cyclic guanosine monophosphate (cGMP) was partially restored by lisinopril, while normalized by the combined therapy. Conclusion. Combining l-arginine with ACE inhibitors would represent a novel strategy for patients with severe ne-

Pharmacologic inhibition of renin-angiotensin system (RAS) with angiotensin-converting enzyme (ACE) inhibitors has been shown in landmark experimental and clinical studies to limit proteinuria and attenuate decline in renal function inexorably associated with chronic renal diseases [1, 2]. These effects have been attributed to the control of systemic and intraglomerular hypertension along with the ability of this class of drugs to limit excess protein ultrafiltration and its deleterious consequences [3]. When given soon after disease induction, ACE inhibition consistently limits hypertension, proteinuria, and renal injury in virtually all animal models of renal disease [4–7]. By contrast, when treatment starts late in the course of the disease at the stage of overt nephropathy, drugs that antagonize RAS are not uniformly effective [8–10]. Most patients with proteinuric nondiabetic, or diabetic renal disease are actually referred late or very late to the nephrologist, and in such circumstances treatment with ACE inhibitors is of little value, if anything, to control urinary proteins and influence disease progression. To model the human condition of advanced nephropathy, we set up accelerated passive Heymann nephritis (PHN), mimicking membranous nephropathy, in which the renal damage is worsened by unilateral nephrectomy, and ACE inhibitors are not consistently effective [11]. This setting was instrumental to verify whether combined treatments may still retain therapeutic effectiveness when ACE inhibition is no longer enough. A role for nitric oxide in limiting progressive kidney disease has recently emerged. Specifically, inhibition of nitric oxide synthesis by L-NAME worsened the course of the disease [12–15], while maneuvers that enhance nitric oxide were renoprotective [16–18]. A direct approach to increasing nitric oxide synthesis is to provide

Key words: ACE inhibitor, l-arginine, passive Heymann nephritis. Received for publication November 27, 2002 and in revised form April 3, 2003 Accepted for publication May 12, 2003

 2003 by the International Society of Nephrology

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additional substrate to the endothelium-specific isoform of nitric oxide synthase (eNOS), a constitutive enzyme responsible for the basal production of nitric oxide. The semiessential amino acid l-arginine serves as the principal substrate for the enzyme, in that NOS catalyzes the 5-electron oxidation of l-arginine to l-citrulline and produces stoichiometric amounts of nitric oxide in this process [19]. Beneficial effects of dietary intervention with l-arginine have been reported in experimental renal disease [20]. l-arginine supplementation has been shown to improve renal hemodynamics [17] and protect renal tissue from inflammatory damage by decreasing macrophage infiltration [21]. Studies have documented that l-arginine may affect cell proliferation [16] and deposition of collagen [22]. The beneficial effect of nitric oxide provision to the kidney has been also ascribed to a reduction of vascular and renal endothelin-1 (ET-1) synthesis [16, 23]. This latter mechanism is particularly intriguing for PHN, in that in this model, urinary excretion of ET-1 increased over time in parallel with proteinuria [24], and renoprotection afforded by the combination of ACE inhibitors and angiotensin II receptor blocker was associated with remarkable suppression of exuberant renal ET-1 [25]. Here, we studied whether l-arginine, which provides eNOS with substrate to enhance nitric oxide synthesis, potentiated the effect of an ACE inhibitor of limiting renal injury when given to PHN rats with overt nephropathy. We also sought to evaluate whether a possible renoprotective effect of the combination therapy was associated with changes in renal ET-1, a possible trigger of progressive renal injury in this model. METHODS Experimental design Male Sprague-Dawley rats (Charles River Italia s.p.a., Calco, Italy) with initial body weights of 275 to 330 g were used in this study. Animal care and treatment were conducted in accordance with the institutional guidelines that are in compliance with national (Decreto Legislativo n.116, Gazzetta Ufficiale suppl 40, 18 febbraio 1992, Circolare n.8, Gazzetta Ufficiale 14 luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJL358-1, December 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 1996). All animals were housed in a room in which the temperature was kept constant on a 12-hour dark/12-hour light cycle and allowed free access to standard diet containing 20% protein by weight and tap water. PHN was induced in nonanesthetized rats by a single intravenous injection of 0.4 mL/100 g body weight of rabbit antiFx1A antibody. Unilateral nephrectomy at day 7, when animals are proteinuric, was performed to accelerate the onset of renal histologic damage [11]. Two months later, rats were divided into four groups (N ⫽ 10 each) and

daily treated up to 9 months as follows: vehicle (group 1); l-arginine (Farmaceutici Damor, Napoli, Italy), 1.25 g/L in the drinking water (group 2); lisinopril (AstraZeneca, Basiglio, Milan, Italy), 40 mg/L (group 3); l-arginine, 1.25 g/L, plus lisinopril, 40 mg/L (group 4). An additional group of normal rats served as control (group 5, N ⫽ 6). The dose of l-arginine was selected on the basis of previous reports documenting renal protection in other models of renal disease [18, 23]. The dose of 40 mg/L of lisinopril was chosen because of partial antiproteinuric effect when administered to PHN rats at an advanced phase of the disease [26], mimicking the clinical condition of patients who do not completely respond to ACE inhibitors. Systolic blood pressure, urinary protein excretion, and serum creatinine were measured at 0, 2 (before treatment), 3, 5, 7 and 9 months. Urinary excretion ET-1 was assessed at the end of the study. To obtain an indirect in vivo index of renal nitric oxide synthesis, 24-hour urinary excretion of cyclic guanosine monophosphate (cGMP) was measured in all groups at the end of the study. At 9 months, rats were anesthetized and kidneys were removed for morphologic analysis. Systolic blood pressure was recorded by tail plethysmography in conscious rats. Twenty-four–hour urine samples were collected using metabolic cages and proteinuria was determined by modified Coomassie blue G dye binding assay for proteins with bovine serum albumin (BSA) as standard [27]. Blood was collected from the tail vein of anesthetized animals. Serum was obtained after whole blood clotting and kept frozen at ⫺20⬚C until assayed. Serum creatinine was measured by alkaline picrate method [28]. Urinary ET-1 measurement Urinary ET-1 levels was evaluated by radioimmunoassay (RIA) after extraction with Sep-pak C18 (Waters, Milford, MA, USA) as previously described [29]. Crossreactivity of the ET-1 antibody (Peninsula Laboratories, Inc., San Carlos, CA, USA) was as follows: ET-2, 46.9%; ET-3, 17%; and big-ET-1, 9.4%. Results corrected for the recovery were expressed as picograms per day. Urinary cGMP excretion Urinary cGMP levels were determined by commercial EIA kit (range, 0.5 to 128 pmol/L) (Amersham Pharmacia Biotech, Buckinghamshire, UK). Data are expressed as nanomoles per day. Renal histology The removed kidneys were fixed overnight in DubosqBrazil, dehydrated in alcohol, and embedded in paraffin. Kidney samples were sectioned at 3 ␮m intervals and the sections were stained with Masson’s trichrome, hematoxylin and eosin, and periodic-acid Schiff (PAS) re-

Zoja et al: ACEi plus l-arginine in progressive nephropathy

agent. Tubular (atrophy, casts, and dilatation) and interstitial changes (fibrosis and inflammation) were graded from 0 to 4⫹ (0, no changes; 1⫹, changes affecting less than 25% of the sample; 2⫹, changes affecting 25% to 50% of the sample; 3⫹, changes affecting 50% to 75% of the sample; and 4⫹, changes affecting 75% to 100% of the sample). At least 100 glomeruli were examined for each animal and the extent of glomerular damage was expressed as the percentage of glomeruli presenting sclerotic lesions. All renal biopsies were analyzed by the same pathologist who was unaware of the nature of the experimental groups. Statistical analysis Data are expressed as mean ⫾ SE. Data were analyzed using the nonparametric Kruskal-Wallis test for multiple comparisons. The statistical significance level was defined as P ⬍ 0.05. RESULTS Systemic parameters Animals exhibited a normal behavior throughout the study period. Only one rat with PHN died in the vehicle group, whereas all rats assigned to the other groups were alive at 9 months. Food intake was comparable in control and PHN rats. The average food intake at month 9 was as follows: vehicle, 28 ⫾ 1 g/day; l-arginine, 29 ⫾ 2 g/day; lisinopril, 31 ⫾ 1 g/day; l-arginine ⫹ lisinopril, 30 ⫾ 1 g/day; control, 23 ⫾ 1 g/day. All PHN rats gained weight in a similar manner and at the end of the study body weight values were comparable (vehicle, 663 ⫾ 19 g; l-arginine, 663 ⫾ 25 g; lisinopril, 647 ⫾ 26 g; l-arginine ⫹ lisinopril, 692 ⫾ 17 g). Body weights of control rats were numerically higher than those of PHN rats, although a statistic significance was not reached (744 ⫾ 27 g). Systolic blood pressure Figure 1 shows the time course of systolic blood pressure. PHN animals were normotensive at 2 months, before starting treatment (systolic blood pressure, 128 ⫾ 2 mm Hg vs. control 123 ⫾ 1 mm Hg). Rats given vehicle exhibited a significant increase in systolic blood pressure within 5 months [145 ⫾ 3 mm Hg (P ⬍ 0.01) vs. control 122 ⫾ 4 mm Hg]. At the end of the study, at 9 months, systolic blood pressure averaged 153 ⫾ 4 mm Hg (P ⬍ 0.01) vs. control 121 ⫾ 2 mm Hg. Treatment with l-arginine alone maintained systolic blood pressure at values significantly lower than those of vehicle group, but higher than controls (132 ⫾ 4 mm Hg) during the entire observation period. In PHN rats treated with lisinopril alone or in combination with l-arginine, systolic blood pressure was reduced to values that were even lower (P ⬍ 0.01)

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Fig. 1. Time course of systolic blood pressure in passive Heymann nephritis (PHN) rats given vehicle (N ⫽ 9), L-arginine (N ⫽ 10), angiotensin-converting enzyme (ACE) inhibitor lisinopril (N ⫽ 10), L-arginine plus ACE inhibitor (N ⫽ 10), and in control rats (N ⫽ 6). Data are mean ⫾ SE. 䊉P ⬍ 0.01 vs. control; *P ⬍ 0.01 vs. vehicle, l-arginine, and control; ⫹P ⬍ 0.05; ⫹⫹P ⬍ 0.01 vs. vehicle.

than those of control rats (90 ⫾ 4 mm Hg; 80 ⫾ 4 mm Hg, respectively). Urinary protein excretion The time course of urinary protein excretion is shown in Table 1. In rats with PHN proteinuria progressively increased with time, with mean values exceeding 350 mg/ day in all groups before treatment (month 2). In rats given l-arginine proteinuria levels were comparable to those measured in PHN rats given vehicle. Administration of ACE inhibitor maintained urinary protein similar to pretreatment along the whole study period, with values being reduced on average by 50% in respect to animals receiving vehicle. By contrast, combining l-arginine with lisinopril had a powerful antiproteinuric effect, such that proteinuria values were significantly lower than vehicle (P ⬍ 0.01). Remarkably, in the combined therapy group, urinary protein excretion was reversed in respect to pretreatment, to levels that were not different from those of age-matched controls. Renal function Renal function in PHN rats given vehicle progressively worsened, as indicated by serum creatinine levels significantly higher than controls (Table 2). PHN rats given l-arginine exhibited high levels of serum creatinine that were comparable to those of untreated PHN rats. Renal function impairment was partially ameliorated by lisinopril. Actually, mean values of serum creatinine were numerically lower than vehicle, although a statistic significance was not achieved. Conversely, in rats on l-arginine plus lisinopril, serum creatinine levels were comparable to those measured in the same animals before treatment and significantly lower than those of vehicle rats. Renal histology The results of renal histology are reported in Figure 2. The percentage of sclerotic glomeruli and the scores of

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Zoja et al: ACEi plus l-arginine in progressive nephropathy Table 1. Time course of proteinuria in passive Heymann nephritis (PHN) rats Proteinura mg/day

Groups

0

2 (before treatment)

PHN ⫹ PHN ⫹ PHN ⫹ PHN ⫹ Control

20 ⫾ 2 19 ⫾ 1 21 ⫾ 1 19 ⫾ 2 23 ⫾ 2

365 ⫾ 67 353 ⫾ 59 381 ⫾ 58 350 ⫾ 55 31 ⫾ 6

vehicle l-arginine lisinopril l-arginine ⫹ lisinopril

3

5

599 ⫾ 107 544 ⫾ 64a 315 ⫾ 84a 117 ⫾ 19a,b 36 ⫾ 6

a

7

627 ⫾ 83 634 ⫾ 49a 354 ⫾ 103a 95 ⫾ 16c,e 43 ⫾ 8 a

9 months

643 ⫾ 80 753 ⫾ 47a 342 ⫾ 111a,b 156 ⫾ 41c 43 ⫾ 7 a

709 ⫾ 62a 743 ⫾ 31a 355 ⫾ 120d 143 ⫾ 42c 80 ⫾ 16

Values are expressed as mean ⫾ SE. a P ⬍ 0.01 vs. control; bP ⬍ 0.05; cP ⬍ 0.01 vs. vehicle and l-arginine; dP ⬍ 0.05 vs. l-arginine; eP ⬍ 0.01 vs. lisinopril

Table 2. Time course of serum creatinine in passive Heymann nephritis (PHN) rats Serum creatinine mg/dL Groups

0

2 (before treatment)

PHN ⫹ PHN ⫹ PHN ⫹ PHN ⫹ Control

0.62 ⫾ 0.03 0.55 ⫾ 0.02 0.56 ⫾ 0.02 0.63 ⫾ 0.01 0.58 ⫾ 0.02

0.82 ⫾ 0.02 0.88 ⫾ 0.03b 0.86 ⫾ 0.03b 0.84 ⫾ 0.03b 0.62 ⫾ 0.02

vehicle l-arginine lisinopril l-arginine ⫹ lisinopril

b

3

5

1.08 ⫾ 0.04 0.92 ⫾ 0.03b 0.85 ⫾ 0.02b,c 0.82 ⫾ 0.03b,c 0.59 ⫾ 0.02 b

7

1.07 ⫾ 0.04 0.98 ⫾ 0.04b 0.88 ⫾ 0.02b,e 0.78 ⫾ 0.02b,d 0.61 ⫾ 0.02 b

9 months

1.08 ⫾ 0.12 1.00 ⫾ 0.06b 0.89 ⫾ 0.04b 0.77 ⫾ 0.04a,e 0.63 ⫾ 0.04 b

1.96 ⫾ 0.59b 1.80 ⫾ 0.45b 0.98 ⫾ 0.05a 0.85 ⫾ 0.05f 0.81 ⫾ 0.04

Values are expressed as mean ⫾ SE. a P ⬍ 0.05; bP ⬍ 0.01 vs. control; cP ⬍ 0.01 vs. vehicle; dP ⬍ 0.01 vs. vehicle, l-arginine alone, and lisinopril alone; eP ⬍ 0.05; fP ⬍ 0.01 vs. vehicle and l-arginine

Fig. 2. Renal morphologic parameters at 9 months in passive Heymann nephritis (PHN) rats given vehicle (N ⫽ 9), L-arginine (N ⫽ 10), angiotensin-converting enzyme (ACE) inhibitors (N ⫽ 10), L-arginine plus ACE inhibitor (N ⫽ 10), and in control rats (N ⫽ 6). Data are mean⫾ SE. ⬚P ⬍ 0.05; •P ⬍ 0.01 vs. control; ⫹P ⬍ 0.01 vs. vehicle and l-arginine; *P ⬍ 0.01 vs. vehicle.

tubular damage and interstitial inflammation were significantly increased in untreated PHN rats at 9 months in respect to age-matched controls. Treatment with l-arginine did not affect the development of glomerulosclerosis and tubulointerstitial lesions that were instead significantly reduced by lisinopril. Combination of lisinopril and l-arginine further ameliorated glomerulosclerosis and tubular damage.

Urinary excretion of ET-1 Urinary excretion of ET-1, taken as an index of renal synthesis of the peptide [29, 30], was fivefold increased in PHN rats over age-matched controls at 9 months (Fig. 3), confirming our previous data in the two-kidney PHN model at 12 months. l-arginine alone reduced urinary ET-1 by 23%. By contrast, excessive urinary ET-1 was remarkably reduced by lisinopril alone [40% (P ⬍ 0.05)

Zoja et al: ACEi plus l-arginine in progressive nephropathy

Fig. 3. Urinary excretion of endothelin-1 (ET-1) at 9 months in passive Heymann nephritis (PHN) rats given vehicle (N ⫽ 9), L-arginine (N ⫽ 10), angiotensin-converting enzyme (ACE) inhibitors (N ⫽ 10), L-arginine plus ACE inhibitor (N ⫽ 10), and in control rats (N ⫽ 6). Data are mean ⫾ SE. ⬚P ⬍ 0.05; •P ⬍ 0.01 vs. control; *P ⬍ 0.05; **P ⬍ 0.01 vs. vehicle; #P ⬍ 0.05 vs. l-arginine.

vs. vehicle] and even to a greater extent by the combined treatment [62% (P ⬍ 0.01) vs. vehicle]. Urinary excretion of cGMP Urinary excretion of cGMP, used as an indirect measure of nitric oxide, was significantly decreased in PHN rats compared to controls (Fig. 4). l-arginine slightly increased urinary cGMP levels that were instead enhanced by lisinopril and almost normalized by l-arginine plus lisinopril treatment. DISCUSSION Results from this study demonstrate that in a severe rat model of PHN, which mimics an advanced phase of renal disease in humans, adding l-arginine to an ACE inhibitor had a potent antiproteinuric effect and remarkably retarded the progression of renal disease, while ACE inhibition alone was less effective. Angiotensin II and bradykinin levels within the vascular wall are controlled by ACE, which generates angiotensin II and catalyzes the hydrolysis of bradykinin into biologically inactive products [31]. Bradykinin through stimulation of the B2-receptor subtype induces release of vasodilating substances, including nitric oxide, from vessel endothelium. Data are indeed available that ACE inhibitors, by preventing bradykinin degradation, enhance the release of nitric oxide from vascular endothelium and this would translate into defense mechanisms against hypertension and vascular dysfunction [32–34].

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Fig. 4. Urinary excretion of cyclic guanosine monophosphate (cGMP) at 9 months in passive Heymann nephritis (PHN) rats given vehicle (N ⫽ 9), L-arginine (N ⫽ 10), angiotensin-converting enzyme (ACE) inhibitors (N ⫽ 10), L-arginine plus ACE inhibitor (N ⫽ 10), and in control rats (N ⫽ 6). Data are mean ⫾ SE. •P ⬍ 0.01 vs. control; *P ⬍ 0.05 vs. vehicle.

Here, we found that in PHN, defective renal nitric oxide documented by low urinary cGMP excretion, can be partially restored by lisinopril, while normalized by adding l-arginine to the ACE inhibitor. A previous study in PHN model showed that urinary cGMP started to decline as early as 6 weeks after disease induction [13]. Captopril administered concomitantly with the immune insult enhanced cGMP excretion, indicating that dysfunctional nitric oxide system is blunted by ACE inhibitors. In the remnant kidney model characterized by impaired renal synthesis of nitric oxide, chronic administration of the nitric oxide donor, molsidomine, decreased blood pressure, partially reduced proteinuria and renal dysfunction, with an effect even higher than lisinopril at an early stage of the disease, suggesting a key role of renal nitric oxide in the early development of the lesions [35]. Beside a direct effect on determinants of renal injury via nitric oxide, molsidomine may also attenuate the damage by limiting renal ET-1, given the fact that in the remnant kidney model nitric oxide donor [35] has a well-documented capacity of modulating ET-1 pathway. In kidneys of rats with PHN, expression of ET-1 mRNA and synthesis of the corresponding peptide were dramatically increased and strongly localized in proximal tubules and in mononuclear inflammatory cells infiltrating the renal interstitium [25]. Excess synthesis of ET-1 in proximal tubules in proteinuric rats with PHN could derive from the continuous protein overreabsorption that occurs in vivo as a consequence of enhanced glomerular protein trafficking. Relevant to this interpretation are in vitro

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data showing that proximal tubular cells in culture exposed to albumin or other proteins at concentrations comparable to those in proximal tubular fluid of proteinuric animals synthetize and release an excessive amount of ET-1 [36]. Moreover, in various experimental models of progressive renal injury, enhanced renal synthesis of ET-1 correlates with the severity of renal damage, and pharmacologic manipulation of the ET pathway retards the rate at which renal function is lost, and also prolongs animal survival [37, 38]. That both ET-1 and angiotensin II conspire to promote renal damage in animals is consistent with previous findings that a combination of an ACE inhibitor and an unselective ET-1 receptor antagonist had an unequivocal antiproteinuric effect and protected against deterioration of renal function, while each drug alone was remarkably less effective [11]. These findings support the value of simultaneously interrupting disparate mediators of injury to effectively slow progression of proteinuric nephropathies, particularly in advanced disease. Along this line, in the present study, adding l-arginine to lisinopril was more effective in respect to single agents in limiting proteinuria and progression, which was paralleled by a significant reduction of ET-1. Actually, l-arginine failed to ameliorate renal dysfunction and lisinopril had only a partial protective effect. Previous studies showed that dietary supplementation with l-arginine ameliorated renal function and limited renal structural injury and interstitial infiltration of inflammatory cells in experimental models of progressive nephropathies, including subtotal nephrectomy and ureteral obstruction [16–18, 21]. The discrepancy with results presented here can be explained by the different starting time of the amino acid administration and/or the severity of the model used. A remarkable feature of the combined therapy is represented by its antihypertensive action mainly sustained by lisinopril. It is well known that control of systemic blood pressure by ACE inhibitors translates into a reduction of intraglomerular hypertension, possibly due to the property of this class of drugs of reducing efferent arteriolar resistance [39]. In this context, the association of l-arginine, which increases nitric oxide availability, to ACE inhibitors would possibly contribute to the amelioration of glomerular hemodynamics. On the other hand, l-arginine might have renal effects independent of the generation of nitric oxide. In normal animals and humans, the intravenous administration of amino acids causes significant changes in renal hemodynamics as a result of a combination of local factors, including their direct vasodilatory effect or the induction of vasoactive hormones, and the release of the growth hormone and glucagone in the systemic circulation [40]. CONCLUSION The present study showed that in a severe model of progressive nephropathy only partially responsive to

ACE inhibitor, combining l-arginine with lisinopril reversed proteinuria and protected against renal function impairment. Possible targets of the combined agents’ action are the glomerular permselective dysfunction and secondary pathways of damage triggered by nitric oxide and ET-1, which contribute to perpetuate renal injury in accelerated PHN. The proposed therapeutic approach could add to the available armamentarium for patients with advanced renal disease in which ACE inhibitors alone fail to prevent progressive renal injury. ACKNOWLEDGMENTS Part of this work was presented at the ASN/ISN World Congress of Nephrology, San Francisco, CA, October 10-17, 2001. l-arginine was kindly provided by Dr. Rodolfo Riccio (Farmaceutici DAMOR, s.p.a, Naples, Italy) and lisinopril by Astra Zeneca (Basiglio, Milan, Italy). The authors are indebted to Dr. Mauro Abbate and Dr. Marina Noris for their precious contribution. Daniela Rottoli helped preparing samples for histology. Reprint requests to Carla Zoja, Ph.D., “Mario Negri” Institute for Pharmacological Research Via Gavazzeni 11, 24125 Bergamo, Italy. E-mail: [email protected]

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