Renoprotective effects of VPI versus ACEI in normotensive nephrotic rats on different sodium intakes

Kidney International, Vol. 63 (2003), pp. 64–71

CELL BIOLOGY – IMMUNOLOGY – PATHOLOGY

Renoprotective effects of VPI versus ACEI in normotensive nephrotic rats on different sodium intakes GOZEWIJN D. LAVERMAN, HARRY VAN GOOR, ROBERT H. HENNING, PAUL E. DICK DE ZEEUW, and GERJAN NAVIS

DE

JONG,

Division of Nephrology, Department of Medicine, Department of Clinical Pharmacology, and Department of Pathology and Laboratory Science, Groningen University Institute of Drug Exploration (GUIDE), University of Groningen, The Netherlands

Renoprotective effects of VPI versus ACEI in normotensive nephrotic rats on different sodium intakes. Background. Control of blood pressure (BP) and optimal reduction of proteinuria (Uprot) are necessary for long-term renoprotection. Unfortunately, angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II (Ang II) antagonists are not effective during sodium repletion. Vasopeptidase inhibitors (VPI) cause dual inhibition of ACE and neutral endopeptidase, the latter resulting in decreased atrial natriuretic peptide (ANP) breakdown and thus enhanced natriuresis. Therefore, in contrast with ACEI, VPI may be effective during high sodium intake. Methods. To test this hypothesis, the renoprotective actions of the new VPI gemopatrilat (GEM) were studied during low (0.05% NaCl) and high (3.0% NaCl) sodium diets in normotensive Wistar rats with established adriamycin nephrosis. The ACEI lisinopril (LIS) was used as control. Rats received either GEM (0.3 mg/g chow), an equihypotensive dose of LIS (75 mg/L drinking water), or vehicle (VEH) from week 6 (that is, established Uprot) until sacrifice. The effect of therapy was monitored by measuring systolic BP and Uprot (weekly) and structural renal damage at the end of study (week 16). Results. During low sodium, GEM effectively reduced Uprot (⫺48 ⫾ 4%), but LIS was more effective (⫺80 ⫾ 2%), while Uprot slightly increased in VEH (⫹23 ⫾ 2%). The focal glomerulosclerosis (FGS) score after GEM (38 ⫾ 14) was lower than in the VEH group (79 ⫾ 27), although this was not significant. LIS (18 ⫾ 6) reduced FGS significantly. Remarkably, on high sodium, GEM was completely ineffective in reducing BP, Uprot and structural renal injury, just like LIS. Conclusions. The renoprotective actions of VPI depend on dietary sodium intake in normotensive nephrotic rats: therapeutic efficacy is fully blunted by a high sodium diet. During a low sodium diet, gemopatrilat was renoprotective, but less effective than lisinopril. Whether higher doses of the VPI could improve its renoprotective efficacy remains to be elucidated.

Proteinuria and hypertension are main determinants of progressive renal function loss [1], and reduction of blood pressure and/or urinary protein loss is the cornerstone of renoprotective intervention [2, 3]. Agents interfering in the renin-angiotensin system (RAS) have proven particularly useful: Angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II (Ang II) antagonists lower both blood pressure and proteinuria effectively [4]. The effect on both proteinuria and blood pressure has to be optimal, since it has been shown that more renoprotection is afforded with increasing therapy effects on these parameters. Unfortunately, ACEI and Ang II antagonists are ineffective during sodium repletion; dietary sodium restriction or diuretic treatment is necessary in order to obtain full therapeutic efficacy [5, 6]. The new class of vasopeptidase inhibitors (VPI) provides dual inhibition of ACE and neutral endopeptidase (NEP) [7]. The latter results in decreased breakdown of atrial natriuretic peptide (ANP), thus promoting natriuresis, diuresis, and vasodilation [8, 9]. Therefore, vasopeptidase inhibitors can be considered as ACE inhibitors with an intrinsic diuretic action, which may not be dependent on sodium restriction for therapeutic efficacy. This assumption is supported by its strong antihypertensive action in high- as well as low-renin conditions [10]. The concept of vasopeptidase inhibition has been investigated extensively in various cardiovascular diseases and has proven remarkably effective in treating hypertension. In contrast, experience in renal disease is scarce, and limited to hypertensive renal disorders [11–13]. In these models this mode of treatment appears to be renoprotective. The renoprotective actions of VPI have not been tested, however, in normotensive models of proteinuriainduced renal damage. In the present study we therefore investigated whether the VPI gemopatrilat is renoprotective in adriamycin nephrosis, a normotensive rat model of proteinuria-induced renal damage. Assuming that VPI is effective, irrespec-

Key words: adriamycin nephrosis, gemopatrilat, lisinopril, proteinuria, renoprotection, dietary sodium, vasopeptide inhibition. Received for publication December 4, 2001 and in revised form May 1, 2002 Accepted for publication August 12, 2002

 2003 by the International Society of Nephrology

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Fig. 1. Study design. At t ⫽ 6 weeks the rats were stratified according to proteinuria into the following treatment groups: gemopatrilat; lisinopril; vehicle.

tive of dietary sodium content, the main hypothesis was that—in contrast to ACEI—VPI would still exert therapeutic benefit during high sodium. The second hypothesis was that VPI would be at least as effective as ACEI during low sodium. To test this hypothesis, we studied the effects of gemopatrilat on proteinuria and development of structural renal injury in nephrotic rats on low and high sodium intake. The effects of an equihypotensive dose of the ACEI lisinopril were studied as a control, thus precluding differences in blood pressure control as a cause of different short-term (proteinuria) and longterm (glomerulosclerosis) therapeutic efficacy. METHODS Study protocol In our normotensive model of established adriamycin nephrosis, proteinuria develops gradually after a single injection of adriamycin (2.0 mg/kg in the tail vein, isoflurane-anesthesia) and interventions start six weeks afterwards, that is, at established proteinuria [14]. Efficacy of treatment is monitored by weekly measurement of proteinuria and blood pressure and, at the end of study, the degree of glomerular and interstitial injury. The experiment was approved by the local Committee for Animal Experiments. Our study protocol is shown in Figure 1. Immediately after arrival, male Wistar rats (Hsd.Cpd.Wu; Harlan Inc., Zeist, The Netherlands) were randomized to receive a low sodium (LS, 0.05% NaCl) or a high sodium (HS, 3.0% NaCl) diet (both diets contained 20% protein; Hope Farms Inc., Woerden, The Netherlands). Adriamycin was injected two weeks after arrival (t ⫽ 0, body weight 346 ⫾ 2.0 g). At six weeks, the rats of each of the sodium groups were stratified into three groups according to the amount of proteinuria:

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treatment with ACEI (lisinopril), with VPI (gemopatrilat) or vehicle until termination in week 16. Thus, there were 6 groups: group 1, LS, VPI (N ⫽ 15); group 2, LS, ACEI (N ⫽ 15); group 3, LS, vehicle (N ⫽ 6); group 4, HS, VPI (N ⫽ 15); group 5, HS, ACEI (N ⫽ 15); and group 6, HS, vehicle (N ⫽ 6). Group sizes were based on the expected differences in proteinuria and FGS between the active treatment groups. Throughout the experiment groups of rats were housed in cages with free access to food and drinking water in a room maintained at 20⬚C and 60% humidity with a 12 hour light-dark cycle. Body weight and intake of food and water (24-hour stay in metabolic cages) were measured weekly. Urine was sampled for measurement of 24-hour proteinuria and creatinine. Reference values for creatinine clearance and blood pressure were obtained from healthy Wistar rats. Dosing of drugs Gemopatrilat is a vasopeptidase inhibitor with a higher potency to inhibit ACE than NEP. It has previously been shown in rats that 24 hours after a single dose of 10 mg/kg, inhibition of renal ACE was approximately 50% and inhibition of renal NEP was approximately 30% [15]. Gemopatrilat was mixed with chow at a concentration of 0.3 mg/g (Hope Farms Inc.) resulting in a dose of 14 mg/kg⫺1/ day⫺1. This concentration was derived from preliminary experiments in adriamycin-nephrotic rats in our laboratory where we established that this dose proved equipotent to the used dose of lisinopril as to reduction of blood pressure. Lisinopril was provided at 75 mg/L in the drinking water, corresponding with a mean daily dose of 9 mg/ kg⫺1/day⫺1. Previous experiments in adriamycin nephrotic rats demonstrated that this dose results in maximal reduction of proteinuria [16]. Measurements Systolic blood pressure measurements were performed weekly after two weeks of daily training prior to induction of nephrosis. An automated multichannel system was used with tail cuffs and photoelectric sensors to detect the tail pulse (Apollo 179; IITC Life Science, Woodland Hills, CA, USA). The rats were placed in a test chamber in restrainers while temperature was maintained at 27 to 29⬚C. For each rat, the value was calculated from the mean of two consecutive measurements. The urinary concentration of protein was determined with the biuret method (Bioquant娃; Merck, Darmstadt, Germany). Creatinine concentrations were determined by a multi-analyzer (SMAC威; Technicon, Tarrytown, NY, USA). All rats were sacrificed in week 16 under isoflurane anesthesia and blood was collected by puncture of the abdominal aorta. Kidneys were perfused with saline and harvested for histological examination. Tissue was fixed

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Fig. 2. Systolic blood pressure in the low sodium (A ) and high sodium (B ) groups over time in rats treated with gemopatrilat (䉱), lisinopril (䊉), or vehicle (⫻). *P ⬍ 0.05 vs. vehicle group. Each value represents mean ⫾ SEM of two consecutive weeks.

in 4% paraformaldehyde and embedded in paraffin. Four micrometer sections were cut and stained with periodic acid-Schiff (PAS) for determination of focal glomerulosclerosis (FGS) and interstitial fibrosis (IF). The degree of FGS was assessed in 50 glomeruli by scoring semiquantitatively on a scale of 0 to 4 as originally described by Raij, Azar and Keane [17]. FGS was scored positive when mesangial matrix expansion and adhesion of the glomerular visceral epithelium to Bowman’s capsule were present in the same segment. If 25% of the glomerulus was affected, a score of 1⫹ was adjudged, 50% was scored as 2⫹, 75% as 3⫹ and 100% as 4⫹. The ultimate score is then obtained by multiplying the degree of change by the percentage of glomeruli with the same degree of injury and adding these scores, thus rendering a theoretical range of 0 to 400. Interstitial fibrosis was scored semiquantitatively for each individual on a scale of 0 to 3⫹, where 0 indicates absence of interstitial fibrosis and 1⫹, 2⫹ and 3⫹ reflect fibrotic changes in 0 to 25%, 25 to 50% or over 50%, respectively, of the total interstitial area. Premature expiration Animals that were found to be severely ill, judged by general appearance and loss of body weight, were sacrificed. Whereas no animals were lost in the low sodium groups, a number of high sodium fed rats expired before the end of study (Table 2). These rats were excluded from the group data. Most of these rats (10 of 13) expired late in the study, that is, between weeks 12 and 15. To test whether excluding these rats from the analysis influenced the interpretation of the over-all results, individual data on proteinuria are shown also for the response at week 8, that is, at a time when all animals were still alive; thus, no selection by mortality could have occurred.

Data analysis Data are expressed as mean ⫾ SEM unless stated otherwise. Baseline values for proteinuria are the mean of the values in weeks 5 and 6. As the blood pressure (BP) remained stable during development of proteinuria, the baseline values for BP are the mean of week 1 through week 6. Repetitively determined variables with a parametric distribution were tested by two-way ANOVA for repeated measurements with a post-test according to Bonferroni. In case of non-parametric distribution of data, analysis of variance (ANOVA) on ranks for repeated measurements was used in combination with Dunn’s method as the post-test. The appropriate vehicle treated group was used as control group. Differences between pre- and post-treatment values within a group were tested using a paired t test. To test whether the incidence of capillary thrombosis was different between the high sodium groups, ␹2 test was used. Statistical significance was assumed at the 5% level. RESULTS Blood pressure The systolic blood pressure in healthy rats was 149 ⫾ 5 mm Hg. Blood pressure remained stable in all groups after induction of nephrosis until start of treatment, confirming that this is a non-hypertensive model (Fig. 2). The percentage changes of blood pressure are depicted in Table 1. In the low sodium groups, both the rats treated with gemopatrilat and those treated with lisinopril showed a clear-cut and similar drop in blood pressure that remained consistently equivalent throughout follow-up. The low sodium, vehicle group showed no change in blood pressure throughout the study. In the high sodium groups, no significant change in blood pressure was observed (Table 1). Thus, neither drug lowered blood pressure during a high sodium diet.

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Laverman et al: Vasopeptidase inhibition and sodium intake Table 1. Group characteristics Low sodium diet

Body weight g Baseline proteinuria mg/day Change proteinuria % Baseline blood pressure mm Hg Change blood pressure %

High sodium diet

Vehicle

Gemopatrilat

Lisinopril

Vehicle

Gemopatrilat

Lisinopril

396 ⫾ 9 575 ⫾ 58 23 ⫾ 2a 154 ⫾ 3 ⫺5 ⫾ 2

408 ⫾ 7 604 ⫾ 52 ⫺48 ⫾ 4a,b,c 152 ⫾ 4 ⫺29 ⫾ 2a,b

408 ⫾ 5 624 ⫾ 60 ⫺80 ⫾ 2a,b 146 ⫾ 4 ⫺31 ⫾ 2a,b

399 ⫾ 16 723 ⫾ 95 31 ⫾ 16a 147 ⫾ 4 1⫾4

389 ⫾ 4 656 ⫾ 91 20 ⫾ 7a 146 ⫾ 4 ⫺6 ⫾ 4

385 ⫾ 6 531 ⫾ 78 12 ⫾ 6a 151 ⫾ 3 ⫺5 ⫾ 5

Data reflect the group characteristics at baseline and their percentage change (mean of week six through sixteen). a P ⬍ 0.05 vs. baseline b P ⬍ 0.05 vs. vehicle c P ⬍ 0.05 vs. Lisinopril

Fig. 3. Proteinuria in the low sodium (A ) and high sodium (B ) groups over time in rats treated with gemopatrilat (䉱), lisinopril (䊉), or vehicle (⫻). *P ⬍ 0.05 vs. vehicle group, #P ⬍ 0.05 vs. gemopatrilat group. Each value represents mean ⫾ SEM of two consecutive weeks.

Proteinuria and creatinine clearance Proteinuria developed rapidly during the four weeks following injection of adriamycin and subsequently leveled off (Fig. 3). Percentage changes of proteinuria are provided in Table 1. In the low sodium groups, vehicle treated animals showed a modest further rise in proteinuria whereas gemopatrilat induced a significant reduction of proteinuria (⫺46 ⫾ 6% at week 10). Lisinopril induced a powerful antiproteinuric response that stabilized after four weeks of treatment (⫺84 ⫾ 2% at week 10) and was stronger than the reduction obtained by gemopatrilat. These differences were significant, both by repeated measurements testing, and when tested for each week separately. In the high sodium groups, neither gemopatrilat nor lisinopril induced a lowering of proteinuria. Instead, regardless of treatment, proteinuria further increased as in the vehicle group. The creatinine clearances in the study groups are shown in Table 2. As expected, the creatinine clearance in these nephrotic groups was lower than reference values obtained from healthy control rats (0.45 mL * min⫺1 * 100 g⫺1; 95% CI 0.36, 0.60), but only the difference between refer-

ence values and the nephrotic groups on low sodium was statistically significant. No significant differences were found between the nephrotic groups (Table 2). Structural renal injury The low sodium group treated with vehicle developed considerable glomerulosclerosis during the 16 weeks of nephrosis (Table 2). Gemopatrilat reduced the FGS score considerably, although it just did not reach statistical significance. Treatment with lisinopril resulted in a significant reduction in FGS score. Representative glomeruli of each group are shown in Figure 4. Scores for interstitial fibrosis showed similar trends as the FGS scores, but failed to reach statistical significance. In all three high sodium groups, massive glomerular damage was observed. A considerable number of animals in all three groups showed extensive capillary thrombosis. The incidence of capillary thrombosis in the gemopatrilat group and lisinopril group was not statistically different from the vehicle group (␹2 test, P ⫽ 0.27 and P ⫽ 0.16, respectively). Since these lesions differ by their nature from glomerulosclerosis, no reliable FGS score can be provided for the high sodium groups. No

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Laverman et al: Vasopeptidase inhibition and sodium intake Table 2. Parameters at the end of the study Low sodium diet

Body weight g Survival rate CCr mL * min⫺1 * 100 g⫺1 Focal glomerulosclerosis Capillary thrombosis Interstitial fibrosis

High sodium diet

Vehicle N⫽6

Gemopatrilat N ⫽ 15

Lisinopril N ⫽ 15

Vehicle N⫽6

Gemopatrilat N ⫽ 15

Lisinopril N ⫽ 15

454 ⫾ 11 6/6 0.29 ⫾ 0.04 (N ⫽ 5) 79 ⫾ 27 N⫽0 1.7 ⫾ 0.5

446 ⫾ 4 15/15 0.36 ⫾ 0.02 (N ⫽ 13) 38 ⫾ 14 N⫽0 1.4 ⫾ 0.2

428 ⫾ 8 15/15 0.31 ⫾ 0.02 (N ⫽ 14) 18 ⫾ 6a N⫽0 1.0 ⫾ 0.1

427 ⫾ 30 5/6 0.32 ⫾ 0.10 (N ⫽ 4) ND N⫽2 1.6 ⫾ 0.4

434 ⫾ 9 12/15 0.35 ⫾ 0.04 (N ⫽ 7) ND N⫽9 2.1 ⫾ 0.4

427 ⫾ 10 9/15 0.42 ⫾ 0.04 (N ⫽ 8) ND N ⫽ 10 2.2 ⫾ 0.3

Parameters at end of study are shown. Focal glomerulosclerosis (scale 0 to 400) and interstitial fibrosis (scale 0 to 3) are in arbitrary units. ND is not done. a P ⬍ 0.05 vs vehicle b P ⬍ 0.05 vs Lisinopril

Fig. 4. Renal morphology of adriamycin rats on low sodium diet (A-C ) and high sodium diet (D ). (A) A 2⫹ focal glomerulosclerosis (FGS) lesion (arrows) from a vehicle-treated rat. (B) An unaffected glomerulus from a lisinopril-treated rat. (C) A 1⫹ lesion (arrows) from a gemopatrilattreated rat. (D) Lesions that were found in a subset of adriamycin rats on a high sodium diet, consisting of endocapillary thrombosis and hyalinosis (arrows).

significant changes in interstitial fibrosis score were found between the high sodium groups. Thus, in high sodium groups, consistent with the absence of a decline in proteinuria and blood pressure, neither drug resulted in less histological damage than vehicle-treatment.

Rats that expired prematurely in the high sodium groups (Table 2) were excluded from the group analysis. To explore the possible impact of this selection on the overall results, the effects of active treatment on proteinuria during high sodium were plotted for individual rats

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Fig. 5. Proteinuria during high sodium in individual rats treated with gemopatrilat (A ) or lisinopril (B ). All individual animals that expired prematurely (⫻) featured thrombotic lesions. Of the individuals surviving until the end of the study (circles), some featured capillary thrombosis (䊉), whereas others did not (䊊).

(Fig. 5). It shows, first, that the individuals that expired before end of study were those with highest proteinuria, that is, the most severe nephrosis. Second, it shows that gemopatrilat and lisinopril were equally ineffective, both in the deceased and surviving animals. Finally, this individual analysis also allowed an assessment of the impact of glomerular capillary thrombosis on the renal therapy response. In these high sodium groups, the antiproteinuric response was invariably absent as well as in the animals without capillary thrombosis and in the animals in which proteinuria was less severe. An individual analysis of the blood pressure response yielded a similar picture (data not shown). DISCUSSION To our knowledge, this is the first study providing information on the renoprotective actions of vasopeptidase inhibition in a normotensive rat model with nephrotic range proteinuria. Most remarkably, during a high sodium diet, the VPI gemopatrilat was completely ineffective in lowering both blood pressure and proteinuria as well as in preventing histological renal damage. This is exactly like the actions previously found with ACE inhibition. In contrast, during a low sodium diet, gemopatrilat rendered a clear-cut and consistent fall in proteinuria and blood pressure. This demonstrates that gemopatrilat has renoprotective potential in this model, although the difference in FGS did not reach statistical significance compared to vehicle. Thus, in this model, the actions of gemopatrilat depend on dietary sodium intake in a similar fashion as those of an ACEI.

The presented data are robust, as they were obtained during a long follow-up period of ten weeks treatment. Moreover, the main parameters, that is, proteinuria and blood pressure, were measured weekly with consistent results throughout the study. Adriamycin nephrosis is a normotensive model of proteinuria-induced renal fibrosis and glomerular sclerosis [18]. The importance of proteinuria is underlined by the fact that the efficacy of proteinuria-reduction consistently predicts long-term renal outcome during therapy in this model [16], which is in accord with proteinuric renal disease in humans [19]. High levels of proteinuria persisted in all high sodium groups. Accordingly, no improvement on histological damage was found in the high sodium groups on active treatment. Moreover, a number of animals expired prematurely in the last study weeks. Individual analysis of the antiproteinuric responses showed that gemopatrilat and lisinopril were both invariably ineffective during high sodium, irrespective whether proteinuria was high or not. Thus far, to our knowledge our study in adriamycin nephrosis is the first to document annihilation of the response to VPI by a high sodium diet. No data are available on sodium-dependency of the VPI effects in models of renal failure. However, our results are at variance with studies reporting efficacy of VPI in sodiumloaded, low-renin models of hypertension [10]. On the other hand, the present findings are in line with the results from early ANP infusion-studies in adriamycininduced [20] and aminonucleoside-induced nephrotic rats [21]. Those studies revealed that there is resistance against the acute natriuretic and diuretic actions of this peptide in the nephrotic syndrome. Thus, ANP resistance

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could explain the present findings. If so, no actions other than inhibition of ACE could be expected of gemopatrilat in the present study. It should be recollected, however, that resistance to the acute actions of ANP also was observed in dogs with heart failure [22], that is, another disorder characterized by sodium-retention, whereas longterm treatment with VPI is effective in heart failure [23, 24]. Lisinopril was used as a positive control as to the sodium dependency of the therapy response. Both drugs were completely ineffective during high sodium intake. As anticipated, during the low sodium intake, blood pressure control by both drugs was equally effective. Nevertheless, gemopatrilat was less effective in reducing proteinuria than lisinopril. What could be the mechanism behind this? Atrial natriuretic peptide has been shown to cause preglomerular vasodilation and post-glomerular vasoconstriction in experimental conditions [25, 26]. Consistent with this observation, studies in diabetic [27, 28] and non-diabetic patients [29] have shown that ANP infusion causes a rise in albuminuria, and acute administration of a NEP inhibitor in renal patients increased albuminuria [8]. In the present study, an ANP-induced increase of intraglomerular pressure partially could have offset the antiproteinuric effects of the VPI, and consequently the effects on glomerulosclerosis, but intraglomerular pressure measurements would be needed to support this assumption. It also should be recollected that NEP not only catalyzes the degradation of ANP, but also of several other vasoconstrictive and vasodilative peptides. For example, in vivo inhibition of NEP resulted in endothelin-1 mediated vasoconstriction in human forearm vessels [30]. Effects on other peptides, therefore, may have resulted in unforeseen effects in our study. We used an equihypotensive dose of the ACEI to discern possible renoprotective effects of the VPI gemopatrilat independent of its effect on blood pressure. This is relevant as both blood pressure and proteinuria are independent treatment targets in renal disease. Some previous studies in renal models have not used equipotent doses of VPI and ACEI, which hampers a comparison with our data (abstract; Wenzel UO, J Am Soc Nephrol 11:343A, 2000) [13, 31]. These studies showed a better antihypertensive and renoprotective effect of a VPI over an ACEI in two-kidney 1-clipped (2K1C) rats, 5/6 nephrectomized rats and diabetic spontaneously hypertensive rats (SHR), respectively. However, the superiority of the VPI in these studies could be blood pressure dependent, and it cannot be excluded that a higher dose of the ACEI also would have improved renal outcome. Other studies reported on the renal effects of equipotent antihypertensive doses of a VPI and an ACEI. In the 5/6 nephrectomy model, omapatrilat resulted in better prevention of structural renal injury than enalapril

[11] and benazepril [12]. The VPI S21402, but not the ACEI captopril, reduced albuminuria significantly in SHR [31]. Thus, these data—all obtained in hypertensive models—seem at variance with our findings. Differences in the pathophysiology of renal damage in those models and ours may be relevant in the discrepancies. In the adriamycin model, proteinuria is typically in the nephrotic range, and the development of renal structural damage is proteinuria-driven, resulting from the tubulointerstitial toxicity of leaked proteins, without a contribution of systemic or glomerular hypertension. In the above models, systemic and/or glomerular hypertension are the important driving forces, proteinuria being much less prominent, and hardly ever in the nephrotic range. Thus, nephrosis-associated ANP-resistance might be absent in those models or—when afferent vasodilation is already maximal such as in 5/6 nephrectomy—NEP inhibition may not be able to exert further (unfavorable) vasodilation of the afferent arteriole. More or less in accord with our findings, omapatrilat was less effective than captopril in preventing glomerular damage in Dahl salt-sensitive rats [32], in spite of a similar antihypertensive effect and in spite of marked improvement of endothelial function in the renal vasculature. Whereas this is still unexplained, those data together with our results indicate that it is not warranted to extrapolate data on the renoprotective potency of VPI in one experimental model to another. Finally, dosing and type of VPI and ACEI could have contributed to the observed discrepancies. We chose a dose of lisinopril known to induce optimal antihypertensive and antiproteinuric efficacy. In the discussed studies, the use of different ACEIs and/or dosing below the optimal dose for proteinuria might partially explain the favorable results for the VPI. The relative efficacy of omapatrilat and the newer compound gemopatrilat also should be considered. However, in vivo rat studies have indicated that the potency to inhibit ACE and NEP are similar for omapatrilat and gemopatrilat [15, 33]. Nevertheless, we suggest that, in order to find the optimal renoprotective dose, additional studies with omapatrilat and gemopatrilat that specifically aim for the dose-response for proteinuria need to be performed [34]. Since VPI intervene at several levels in two physiological systems, their actions are rather multiple than dual. Therefore, the clinical outcome of so-called dual blockade of ACE and NEP is intricate and depends on patient factors such as type and severity of disease in combination with environmental factors such as dietary sodium intake. We demonstrate that the renoprotective actions of vasopeptidase inhibition in normotensive nephrotic rats depend on dietary sodium intake: therapeutic efficacy is fully blunted by a high sodium diet. During a low sodium diet, the VPI gemopatrilat provided renoprotection that

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was less effective than the ACEI lisinopril, despite equivalent blood pressure control. This finding is potentially important for clinical practice, implicating that VPI might have no benefit over ACEI alone in the treatment of non-hypertensive nephrotic syndrome. Future studies are necessary to further elucidate the mechanisms behind these findings and to prove whether higher doses of gemopatrilat could improve its renoprotective efficacy in this proteinuria-driven model of renal failure. ACKNOWLEDGMENTS This study was supported by a grant from Bristol-Myers-Squibb Pharmaceuticals (Princeton, NJ, USA). GDL is appointed due to a grant of the Dutch Kidney Foundation (grant 5023). Gemopatrilat was a gift from Bristol-Myers-Squibb. Lisinopril was a gift from Merck, Sharpe and Dohme (Haarlem, The Netherlands). The authors thank C. Klein, B. Meijeringh, E. Scholtens, J. Duker and A. Kluppel for their technical assistance. Reprint requests to G.D. Laverman, M.D., Division of Nephrology, University Hospital Groningen, Hanzeplein 1, PO Box 30.001, 9700 RB Groningen, The Netherlands. E-mail: [email protected]

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