J Am Soc Nephrol 13: 2207–2212, 2002 Renal Interstitial Fluid I and Angiotensin II Concentrations during Local Angiotensin-Converting Enzyme Inhibition

AKIRA NISHIYAMA, DALE M. SETH, and L. GABRIEL NAVAR Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana.

Abstract. It was recently demonstrated that angiotensin II (An- perfusate; n ϭ 5 and 8, respectively) significantly increased gII) concentrations in the renal interstitial fluid (RIF) of anes- RIF AngI concentrations but did not significantly alter AngII thetized rats were in the nanomolar range and were not reduced concentrations. However, perindoprilat (10 mM in the perfus- by intra-arterial infusion of an angiotensin-converting enzyme ate, n ϭ 7) significantly decreased RIF AngII concentrations (ACE) inhibitor (enalaprilat). This study was performed to by 22 Ϯ 4% and increased RIF AngI concentrations. Interstitial determine changes in RIF AngI and AngII concentrations infusion of AngI (100 nM in the perfusate, n ϭ 7) significantly during interstitial administration of ACE inhibitors (enalaprilat increased the RIF AngII concentration to 8.26 Ϯ 0.75 nM, and perindoprilat). Studies were also performed to determine whereas plasma AngI and AngII levels were not affected (0.15 the effects of enalaprilat on the de novo formation of RIF AngII Ϯ 0.02 and 0.14 Ϯ 0.02 nM, respectively). Addition of ena- elicited by interstitial infusion of AngI. Microdialysis probes laprilat to the perfusate (10 mM) prevented the conversion of (cut-off point, 30,000 D) were implanted in the renal cortex of exogenously added AngI. These results indicate that addition anesthetized rats and were perfused at 2 ␮l/min. The effluent of AngI in the interstitial compartment leads to low but sig- dialysate concentrations of AngI and AngII were measured by nificant conversion to AngII via ACE activity (blocked by RIA, and reported values were corrected for the equilibrium enalaprilat). However, the addition of ACE inhibitors directly rates at this perfusion rate. Basal RIF AngI (0.74 Ϯ 0.05 nM) into the renal interstitium, via the microdialysis probe, either and AngII (3.30 Ϯ 0.17 nM) concentrations were much higher did not reduce RIF AngII levels or reduced levels by a small than plasma AngI and AngII concentrations (0.15 Ϯ 0.01 and fraction of the total basal level, suggesting that much of the 0.14 Ϯ 0.01 nM, respectively; n ϭ 27). Interstitial infusion of RIF AngII is formed at sites not readily accessible to ACE enalaprilat through the microdialysis probe (1 or 10 mM in the inhibition or is formed via non-ACE-dependent pathways.

There is now substantial evidence indicating that angiotensin II interstitial changes (3,4,20–22). Several studies have indicated (AngII) is formed locally in the kidney and is critical in the that AngII is present at high concentrations in renal interstitial paracrine regulation of renal function and in the pathophysio- fluid (RIF) (9–13). Earlier studies demonstrated that AngII logic processes of hypertension (1–4). Intrarenal AngII forma- concentrations in the renal lymph were substantially greater tion is particularly complex, because all of the components than those in arterial or renal venous plasma (9,10). Studies needed for AngII formation are formed locally but substantial using microdialysis probes implanted in the renal cortex also intrarenal metabolism and degradation of AngI and AngII also indicated that RIF AngII concentrations were much greater occur (5). Furthermore, intrarenal AngII is not distributed in a than plasma concentrations (11–13). A recent study demon- homogeneous manner but is compartmentalized in the tubular strated that RIF AngI and AngII concentrations in anesthetized fluid (5–8), in interstitial fluid compartments (9–13), and also rats were in the nanomolar range and were substantially greater intracellularly (14–16). than the corresponding plasma concentrations and kidney con- Interstitial AngII plays an important role in the regulation of tents (13). These results support the concept that AngII is the renal microcirculation (17–19) and tubular function (19) formed locally within the renal interstitium or is added to the and acts as a pivotal mediator in the pathogenesis of tubulo- interstitial compartment. Interestingly, we also observed that the high RIF AngII concentrations were not responsive to acute arterial infusion of an angiotensin-converting enzyme (ACE) Received January 20, 2002. Accepted June 1, 2002. inhibitor, enalaprilat. Similarly, acute volume expansion did Correspondence to Dr. Akira Nishiyama, Department of Pharmacology, Ka- not lead to significant reductions in RIF AngII concentrations, gawa Medical University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761- although plasma AngII levels were significantly decreased 0793, Japan. Phone: 81-87-898-5111, ext. 2502; Fax: 81-87-891-2126; E-mail [email protected] (13), suggesting that the regulation of RIF AngII is indepen- 1046-6673/1309-2207 dent of plasma AngII levels. Journal of the American Society of Nephrology This study was conducted to determine whether more direct Copyright © 2002 by the American Society of Nephrology and local ACE inhibition could modify RIF AngI and AngII DOI: 10.1097/01.ASN.0000026610.48842.CB concentrations. Experiments were performed in anesthetized 2208 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2207–2212, 2002 rats, to determine changes in RIF AngI and AngII concentra- (13). The dialysate concentration of inulin was below the detectable tions in response to interstitial administration of ACE inhibi- range (Ͻ0.03 mg/ml), indicating that dialysate contamination by tors, which were infused through the microdialysis probe by tubular fluid and plasma was minimal. Previous studies also demon- inclusion of the drug in the perfusate. Studies were performed strated that dialysate concentrations of AngI and AngII were elevated with enalaprilat and a more lipophilic ACE inhibitor, perindo- immediately after implantation of the microdialysis probe but de- creased within the first 30 min and then remained stable for up to 270 prilat (23,24), to determine effects on RIF AngI and AngII min (13). Therefore, all in vivo collection experiments were initiated concentrations. Furthermore, we investigated the effects of 90 min after implantation of the microdialysis probe. local administration of enalaprilat on de novo formation of RIF AngII induced by interstitial infusion of AngI substrate. Effects of Interstitial Infusion of Enalaprilat and Perindoprilat on RIF AngI and AngII Levels Materials and Methods Ninety minutes after implantation of the microdialysis probe, the Animal Preparation experimental protocol was initiated with dialysate fluid collections for The experiments were performed in accordance with the guidelines two consecutive 30-min periods. At the end of the second control and practices established by the Tulane University Animal Care and sample collection, an arterial blood sample was obtained from a Use Committee. Sprague-Dawley rats weighing 260 to 315 g were catheter placed in the left femoral artery. Enalaprilat (1 and 10 mM; anesthetized with Inactin (100 to 120 mg/kg, intraperitoneally). The n ϭ 5 and 8, respectively) or perindoprilat (10 mM, n ϭ 7) was then surgical preparation of the animals and basic experimental techniques infused interstitially through the microdialysis probe, by addition of were identical to those described previously (13,25). During surgical the drug to the perfusate. After 30 min of infusion, four consecutive preparation, Ringer’s solution containing 6% bovine serum albumin 30-min dialysate samples were collected. At the end of the final (BSA) (pH 7.4) was infused intravenously, at a rate of 20 ␮l/min, to sample collections, arterial blood samples were obtained. The kidney replace volume losses. Ringer’s solution containing 1% BSA (pH 7.4) was then removed, and the location of the microdialysis membrane was then infused, at a rate of 20 ␮l/min, for the duration of the was confirmed by surgical exposure of the probe. stabilization and experimental periods. Effects of Interstitial Infusion of Enalaprilat on De Characteristics of the Microdialysis Probe Novo Formation of RIF AngII during Interstitial An in vivo microdialysis method (11–13,26) was used for the Infusion of AngI determination of RIF concentrations of AngI and AngII. The dialysis Experiments were performed to determine the effects of local membrane was made from polyethersulfone fiber (Toyobo Co., Otsu, administration of enalaprilat on de novo formation of RIF AngII from Japan) measuring 10 mm in length, with a 30,000-D transmembrane AngI infused into the interstitium through the probe (n ϭ 7). During diffusion cut-off. Thin steel needles were inserted into both ends of the control period, two interstitial fluid samples and an arterial blood the polyethersulfone fiber. The inflow of each probe was connected to sample were obtained. Then, AngI (100 nM) was infused interstitially polyethylene tubing (PE-10) and a CMA/100 microinfusion pump through the microdialysis probe. After 15 min of AngI infusion, two (Carnegie Medicine, Stockholm, Sweden). The probes were perfused consecutive 30-min dialysate samples were collected. A continuous with Ringer’s solution containing 1% BSA (pH 7.4), at a rate of 2 interstitial infusion of enalaprilat (10 mM) was then added to the AngI ␮l/min. Before the initiation of each experiment, the probe was infusion. After 15 min of enalaprilat infusion, two additional consec- perfused for 60 min with Ringer’s solution containing 1% BSA and utive 30-min sample collections were performed. At the end of each was then soaked in 40% for 20 min. The dialysate was experiment, an arterial blood sample was obtained. collected directly from the outflow steel tubing, for analysis of AngI and AngII concentrations. The dead space of the dialysis membrane Analytical Procedures and outflow steel tubing was Ͻ1.0 ␮l. The tip of the outflow steel Plasma AngI and AngII levels were measured by RIA, as described tubing was placed into a small tube containing a solution of inhibitors previously (7,8,14). The dialysate samples in 100% methanol were (30 ␮l of 500 mM ethylenediaminetetraacetate, 15 ␮lof1mM evaporated to dryness in a vacuum centrifuge, reconstituted in assay enalaprilat, and 30 ␮l of 125 mM o-phenanthrolene and 0.2 mM buffer, and assayed by RIA (13). in 95% ethanol), so that dialysate effluent would be imme- diately mixed with the inhibitors. We recently demonstrated that use of the inhibitor mixture effectively blocked in vitro generation and Statistical Analyses degradation of AngII (13). After 30 min of sample collection, the The values are presented as means Ϯ SEM. Statistical comparisons samples were quickly vortex-mixed. Immediately thereafter, 10 ␮lof of the differences were performed by using one-way or two-way mixed solution for AngII measurements and 50 ␮l of mixed solution ANOVA for repeated measures, combined with the Newman-Keuls for AngI measurements were transferred to two glass tubes containing post hoc test. P Ͻ 0.05 was considered statistically significant. 1 ml of chilled 100% methanol. These samples were stored at Ϫ20°C until the day before analysis. Results The efficiency of the microdialysis probe was determined in vitro Under resting conditions, the RIF AngI and AngII concentra- (11–13,26). At a perfusion rate of 2 ␮l/min, the relative equilibrium Ϯ Ϯ tions calculated by using the in vitro relative equilibrium rates rates for AngI and AngII were 47 1 and 51 1%, respectively, Ϯ Ϯ ϭ which did not deteriorate with time (13). Because the microdialysis were 0.74 0.05 and 3.30 0.17 nM, respectively (n 27). probe was implanted in the renal cortex, it was possible that AngII These values were, in all cases, much higher than the respective derived from tubular fluid contributed to the AngII levels in the plasma levels (AngI, 146 Ϯ 12 pM; AngII, 138 Ϯ 10 pM; P Ͻ dialysate. Previously, we administered inulin intravenously, to assess 0.01). Furthermore, RIF AngII concentrations were substantially possible contamination of the dialysate by plasma and tubular fluid higher than RIF AngI levels (P Ͻ 0.01). J Am Soc Nephrol 13: 2207–2212, 2002 RIF AngII Levels and ACE Inhibitors 2209

Table 1 summarizes the effects of interstitial infusion of enalaprilat and perindoprilat on mean arterial pressure (MAP) and plasma AngI and AngII concentrations. Interstitial infusion of enalaprilat at a dose of 1 mM did not significantly reduce MAP (n ϭ 5); at 10 mM, however, sufficient enalaprilat entered the systemic circulation to significantly decrease MAP (n ϭ 8). Interstitial infusion of perindoprilat (10 mM) also significantly reduced MAP (n ϭ 7). These reductions in arte- rial pressure indicated that the ACE inhibitors administered via the microdialysis probe infiltrated the renal interstitium and entered the intravascular volume. The average plasma AngI concentrations increased slightly with a dose of 1 mM enala- prilat, but these changes were not statistically significant. Plasma AngII concentrations were not altered by administra- tion of 1 mM enalaprilat. The higher dose of enalaprilat (10 mM) and perindoprilat significantly increased plasma AngI concentrations and decreased plasma AngII concentrations (Table 1). At a dose of 1 mM, enalaprilat infusion for 150 min significantly increased RIF AngI concentrations from 0.67 Ϯ 0.05 to 0.85 Ϯ 0.05 nM; however, the RIF AngII concentra- tions were not significantly altered (from 3.20 Ϯ 0.50 to 3.28 Ϯ 0.50 nM). Similarly, enalaprilat infusion at a dose of 10 mM failed to significantly reduce RIF AngII concentrations, al- though RIF AngI concentrations were significantly increased from 0.75 Ϯ 0.07 to 1.21 Ϯ 0.11 nM (Figure 1, A and B). As indicated in Figure 1C, enalaprilat significantly decreased the AngII/AngI ratio in the RIF from 4.7 Ϯ 0.8 to 2.8 Ϯ 0.2. In contrast, interstitial infusion of perindoprilat significantly de- creased RIF AngII concentrations from 3.36 Ϯ 0.16 to 2.62 Ϯ 0.18 nM, in association with increases in RIF AngI concentra- tions from 0.77 Ϯ 0.02 to 1.35 Ϯ 0.10 nM (Figure 2, A and B). The AngII/AngI ratio in the RIF was significantly decreased by perindoprilat, from 4.4 Ϯ 0.2 to 2.0 Ϯ 0.1 (Figure 2C). Figure 1. (A) Effects of interstitial infusion of enalaprilat (10 mM in Interstitial infusion of AngI (100 nM) alone for 75 min did ϭ the perfusate) on renal interstitial fluid (RIF) concentrations of angio- not alter MAP and plasma AngI and AngII levels (n 7) tensin I (AngI). (B) Effects of interstitial infusion of enalaprilat (10 (Table 1). During AngI infusion, enalaprilat significantly in- mM in the perfusate) on RIF concentrations of AngII. (C) Effects of creased plasma AngI concentrations and decreased AngII con- interstitial infusion of enalaprilat (10 mM in the perfusate) on the centrations (Table 1). AngI infusion for 75 min significantly AngII/AngI ratio in the RIF. C, control. *P Ͻ 0.05 versus control 2; increased RIF AngII concentrations from 2.85 Ϯ 0.24 to 8.26 n ϭ 8.

Table 1. Effects of interstitial infusion of enalaprilat, perindoprilat, and AngI on mean arterial pressure and plasma AngI and AngII concentrations in anesthetized ratsa

Mean Arterial Plasma AngI Plasma AngII AngII/AngI Pressure (mmHg) Level (pM) Level (pM) Ratio

Control (n ϭ 5) 116 Ϯ 2 119 Ϯ 18 115 Ϯ 15 1.11 Ϯ 0.12 Enalaprilat (1 mM in perfusate, 150 min) 114 Ϯ 3 153 Ϯ 13 100 Ϯ 23 0.67 Ϯ 0.15b Control (n ϭ 8) 115 Ϯ 4 202 Ϯ 24 172 Ϯ 20 0.86 Ϯ 0.06 Enalaprilat (10 mM in perfusate, 150 min) 106 Ϯ 4b 1246 Ϯ 251b 122 Ϯ 20b 0.11 Ϯ 0.02b Control (n ϭ 7) 108 Ϯ 595Ϯ 7 106 Ϯ 18 1.11 Ϯ 0.15 Perindoprilat (10 mM in perfusate, 150 min) 99 Ϯ 5b 690 Ϯ 143b 74 Ϯ 11b 0.12 Ϯ 0.02b Control (n ϭ 7) 110 Ϯ 4 147 Ϯ 24 138 Ϯ 21 1.12 Ϯ 0.25 AngI (100 nM in perfusate, 75 min) 110 Ϯ 4 152 Ϯ 19 134 Ϯ 15 0.99 Ϯ 0.17 AngI plus enalaprilat (10 mM in perfusate, 75 min) 100 Ϯ 3b 1603 Ϯ 246b 94 Ϯ 12b 0.06 Ϯ 0.01b

a Values are means Ϯ SEM. AngI, angiotensin I. b P Ͻ 0.05 versus control. 2210 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2207–2212, 2002

Figure 3. Effects of interstitial infusion of AngI (100 nM in the perfusate) and AngI plus enalaprilat (10 mM in the perfusate) on RIF concentrations of AngII. C, control; A, AngI; E, enalaprilat. *P Ͻ 0.05 versus control 2; n ϭ 7.

sion of AngII from the peritubular capillaries and conversion of exogenously added AngI to AngII in the renal interstitium. Consistent with our recent data (13), the studies presented here confirm that RIF AngII concentrations are in the nanomolar range and are substantially greater than the corresponding plasma concentrations. In addition, it seems clear that, when exogenous AngI is delivered via the microdialysis probe, in- terstitial ACE converts some of it to AngII. However, under basal conditions, the addition of ACE inhibitors directly into Figure 2. (A) Effects of interstitial infusion of perindoprilat (10 mM the renal interstitium via the microdialysis probe either did not in the perfusate) on RIF concentrations of AngI. (B) Effects of reduce the RIF AngII concentration or reduced it by only a interstitial infusion of perindoprilat (10 mM in the perfusate) on RIF small fraction of the total basal level. These results support the concentrations of AngII. (C) Effects of interstitial infusion of perin- previously suggested concept (13) that much of the RIF AngII doprilat (10 mM in the perfusate) on the AngII/AngI ratio in the RIF. C, control. *P Ͻ 0.05 versus control 2; n ϭ 7. is formed at sites that are not readily accessible to ACE inhibition or is formed via non-ACE-dependent pathways. These results in the kidney are consistent with those noted in Ϯ 0.75 nM, demonstrating significant conversion of the in- the heart by Dell’Italia et al. (26), who demonstrated that fused AngI (Figure 3). The addition of enalaprilat to the AngI interstitial fluid concentrations of AngII in the heart were not infusion prevented the increases in RIF AngII concentrations reduced by infusion of the ACE inhibitor . That study caused by the administration of exogenous AngI but did not also demonstrated that captopril almost completely inhibited decrease RIF AngII levels below the basal values (3.41 Ϯ 0.31 the interstitial ACE activity (26). nM). The source of the AngII collected from the renal interstitium remains unclear. Previous studies demonstrated that, when Discussion 14C-labeled AngII was administered into the renal artery, no Several studies have indicated that RIF AngII exerts biologic significant amount of 14C-labeled AngII appeared in the renal effects, including the regulation of renal function (2,17–19), lymph, suggesting that most of the AngII in renal lymph is not and may cause tubulointerstitial injury when levels are inap- derived from circulating AngII (9). Therefore, it seems that propriately elevated (3,4,20–22). Mitchell and Navar (18,19) most of the RIF AngII is formed from locally generated AngI. demonstrated that AngI and AngII added to the renal intersti- Collectively, these observations suggest that, under basal con- tium via perfusion into peritubular capillaries caused afferent ditions, high concentrations of interstitial AngII are formed via arteriolar vasoconstriction, decreases in the single-nephron ACE localized at sites that are not accessible to the adminis- GFR, and increases in the proximal fractional reabsorption tered ACE inhibitors or via non-ACE pathways. rate. These results indicate the occurrence of significant diffu- In this study, we perfused enalaprilat and perindoprilat in- J Am Soc Nephrol 13: 2207–2212, 2002 RIF AngII Levels and ACE Inhibitors 2211 terstitially, through the microdialysis probe, by adding the drugs to AngII (30,31). Substantial increases in interstitial fluid Ang- the perfusate containing 10% BSA. It is thus possible that differ- (1–7) levels during interstitial infusion of AngI in dog heart ences in the protein binding ratios of these drugs might account were recently reported (32). Furthermore, Wei et al. (33) for the observation that RIF AngII concentrations were signifi- reported that administration of the ACE inhibitor did cantly decreased by perindoprilat but not by enalaprilat. The not alter interstitial AngII concentrations but significantly in- protein binding of enalaprilat is much greater than that of perin- creased interstitial Ang-(1–7) concentrations in rat heart. Al- doprilat; therefore, a greater amount of perindoprilat might have though we did not measure Ang-(1–7) levels in this study, traversed the renal interstitium. However, we infused enalaprilat those data suggest the possibility that, during local ACE inhi- and perindoprilat at very high concentrations (10 mM), and the bition, some of the increased interstitial fluid AngI is converted MAP was significantly decreased during interstitial infusion of to Ang-(1–7), rather than AngII. Nevertheless, shunting to this both enalaprilat and perindoprilat. These data suggest that both possible pathway does not explain why RIF AngII levels failed ACE inhibitors infiltrated and entered the intravascular vol- to decrease. ume. It is thus reasonable to conclude that this concentration is Although AngII formation in rodents is primarily ACE- sufficient to inhibit the activity of ACE existing in the RIF related (2), non-ACE-dependent AngII formation has also been around the microdialysis probe. Nevertheless, the differences observed in rat heart (34) and vasculature (35,36). Guo et al. in the effects of enalaprilat and perindoprilat might be the (36) demonstrated that chymase expression and chymostatin- result of differences in their binding to BSA, in their affinity inhibitable ACE activity were significantly increased in spon- for interstitial ACE, or in their ability to diffuse within the taneously hypertensive rats, suggesting a possible role for this interstitium. enzyme in AngII formation in rats. ACE-related carboxypep- Recent studies suggested that significant amounts of renal An- tidase (ACE2) is also expressed in rat kidney (37). Interest- gII are formed intracellularly (5,14–16), and it is possible that the ingly, recent studies demonstrated that ACE2 generated Ang- intracellularly produced AngII is secreted into the interstitial (1–9) from AngI but did not further convert Ang-(1–9) to space. In this study, we also investigated changes in RIF AngII AngII (38). However, the extent to which these processes concentrations in response to local administration of the lipophilic regulate RIF AngI and AngII remains unknown. ACE inhibitor perindoprilat (23,24). It is possible that some of the In conclusion, this investigation demonstrated the de novo administered perindoprilat is distributed more uniformly within formation of RIF AngII with local AngI administration, via the interstitium and partially permeates cell membranes. We ACE activity (blocked by enalaprilat). Nevertheless, the find- observed that locally administered perindoprilat significantly ing that local ACE inhibition either did not reduce RIF AngII decreased RIF AngII concentrations by 0.75 Ϯ 0.22 nM, but levels or reduced levels by a small fraction of the total basal the decrease was still only a small fraction of the basal value levels suggests intrarenal formation of AngII via ACE local- (3.36 Ϯ 0.16 nM). To the extent that ACE bound to the tubular ized at sites not easily accessed by the administered ACE basolateral membranes is responsible for the formation of inhibitors and/or via non-ACE pathways. AngII in the renal interstitium (2,5), it is possible that the convective force attributable to continuous tubular fluid reab- Acknowledgments sorption might limit the concentrations of ACE inhibitors that This work was supported by National Heart, Lung, and Blood can be achieved at or near the basolateral membranes. Institute Grant HL26371 (to Dr. Navar) and an award from the In this study, we observed that interstitial infusion of AngI American Heart Association, Southeast Affiliate (to Dr. Nishiyama). significantly increased RIF AngII concentrations. Furthermore, We are grateful to Daiichi Pharmaceutical Co. for supplying perin- these increases were prevented by coadministration of enala- doprilat. We are grateful to Drs. Hidehiko Sakurai and Kimihiro prilat. These data indicate that interstitial ACE converts exog- Mabuchi (Toyobo Co., Otsu, Japan) for supplying the dialysis mem- enously administered AngI. As already stated, however, the branes and steel tubes and for helpful advice. increases in RIF AngI levels during enalaprilat infusion were not accompanied by changes in RIF AngII concentrations. At References present, we do not have a satisfactory explanation for why 1. Arendshorst WJ, Brannstrom K, Ruan X: Actions of angiotensin enalaprilat resulted in increases in RIF AngI levels without II on the renal microvasculature. J Am Soc Nephrol 10[Suppl 11]: reductions in RIF AngII levels. However, it seems important to S149–S161, 1999 recognize that basal RIF concentrations of AngI were consis- 2. Navar LG, Inscho EW, Majid SA, Imig JD, Harrison-Bernard tently lower than those of AngII and that the increases in AngI LM, Mitchell KD: Paracrine regulation of the renal microcircu- levels after ACE inhibition were quantitatively modest, com- lation. Physiol Rev 76: 425–536, 1996 pared with RIF AngII concentrations. Therefore, it is possible 3. Hamaguchi A, Kim S, Yano M, Yamanaka S, Iwao H: Activation that blockade of the conversion of AngI to AngII in the of glomerular mitogen-activated protein kinases in angiotensin II-mediated hypertension. J Am Soc Nephrol 9: 372–380, 1998 interstitial compartment might not be reflected by significant 4. Kim S, Iwao H: Molecular and cellular mechanisms of angio- reductions in RIF concentrations of AngII. It should also be tensin II-mediated cardiovascular and renal diseases. Pharmacol noted that ACE inhibitors have been demonstrated to increase Rev 52: 11–34, 2000 both AngI and Ang-(1–7) levels in vitro (27) and in vivo (28). 5. Navar LG, Harrison-Bernard LM, Wang CT, Cervenka L, Mitch- AngI can be converted directly to Ang-(1–7) (29–31), which ell KD: Concentrations and actions of intraluminal angiotensin circulates in the blood at concentrations similar to those of II. J Am Soc Nephrol 10[Suppl 11]: S189–S195, 1999 2212 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2207–2212, 2002

6. Seikaly MG, Arant BS Jr, Seney FD Jr: Endogenous angiotensin 24. Moursi MG, Ganten D, Lang RE, Unger T: Antihypertensive concentrations in specific intrarenal fluid compartments of the action and inhibition of tissue converting enzyme (CE) by three rat. J Clin Invest 86: 1352–1357, 1990 prodrug CE inhibitors, , ramipril and , in 7. Braam B, Mitchell KD, Fox J, Navar LG: Proximal tubular stroke-prone spontaneously hypertensive rats. J Hypertens Suppl secretion of angiotensin II in rats. Am J Physiol 264: F891–F898, 4: S495–S498, 1986 1993 25. Wang CT, Chin SY, Navar LG: Impairment of pressure-natri- 8. Navar LG, Lewis L, Hymel A, Braam B, Mitchell KD: Tubular uresis and renal autoregulation in ANG II-infused hypertensive fluid concentrations and kidney contents of I and II rats. Am J Physiol 279: F319–F325, 2000 in anesthetized rats. J Am Soc Nephrol 5: 1153–1158, 1994 26. Dell’Italia LJ, Meng QC, Balcells E, Wei CC, Palmer R, Hage- 9. Bailie MD, Rector FC Jr, Seldin DW: Angiotensin II in arterial man GR, Durand J, Hankes GH, Oparil S: Compartmentalization and renal venous plasma and renal lymph in the dog. J Clin of angiotensin II generation in the dog heart: Evidence for Invest 50: 119–126, 1971 independent mechanisms in intravascular and interstitial spaces. 10. Wilcox CS, Dzau VJ: Effect of captopril on the release of the J Clin Invest 100: 253–258, 1997 components of the -angiotensin system into plasma and 27. Chappell MC, Gomez MN, Pirro NT, Ferrario CM: Release of lymph. J Am Soc Nephrol 2: 1241–1250, 1992 angiotensin-(1–7) from the rat hindlimb: Influence of angioten- 11. Siragy HM, Howell NL, Ragsdale NV, Carey RM: Renal inter- sin-converting enzyme inhibition. Hypertension 35: 348–352, stitial fluid angiotensin: Modulation by anesthesia, epinephrine, 2000 sodium depletion, and renin inhibition. Hypertension 25: 1021– 28. Luque M, Martin P, Martell N, Fernandez C, Brosnihan KB, 1024, 1995 Ferrario CM: Effects of captopril related to increased levels of

12. Siragy HM, Carey RM: Protective role of the angiotensin AT2 prostacyclin and angiotensin-(1–7) in essential hypertension. receptor in a renal wrap hypertension model. Hypertension 33: J Hypertens 14: 799–805, 1996 1237–1242, 1999 29. Tallant EA, Diz DI, Ferrario CM: Antiproliferative actions of 13. Nishiyama A, Seth DM, Navar LG: Renal interstitial fluid con- angiotensin-(1–7) in vascular smooth muscle. Hypertension 34: centrations of angiotensins I and II in anesthetized rats. Hyper- 950–957, 1999 tension 39: 129–134, 2002 30. Senanayake PD, Moriguchi A, Kumagai H, Ganten D, Ferrario 14. Imig JD, Navar GL, Zou LX, O’Reilly KC, Allen PL, Kaysen CM, Brosnihan KB: Increased expression of angiotensin peptides JH, Hammond TG, Navar LG: Renal endosomes contain angio- in the brain of transgenic hypertensive rats. Peptides 15: 919–

tensin peptides, converting enzyme, and AT1A receptors. Am J 926, 1994 Physiol 277: F303–F311, 1999 31. Lawrence AC, Evin G, Kladis A, Campbell DJ: An alternative 15. van Kats JP, van Meegen JR, Verdouw PD, Duncker DJ, Schale- strategy for the radioimmunoassay of angiotensin peptides using kamp MA, Danser AH: Subcellular localization of angiotensin II amino-terminal-directed antisera: Measurement of eight angio- in kidney and adrenal. J Hypertens 19: 583–589, 2001 tensin peptides in human plasma. J Hypertens 8: 715–724, 1990 16. Zhuo JL, Imig JD, Hammond TG, Orengo S, Benes E, Navar LG: 32. Wei CC, Ferrario CM, Brosnihan KB, Farrell DM, Bradley WE, Ang II accumulation in rat endosomes during Ang II-induced Jaffa AA, Dell’Italia LJ: Angiotensin peptides modulate brady-

hypertension: Role of AT1 receptor. Hypertension 39: 116–121, kinin levels in the interstitium of the dog heart in vivo. J Phar- 2002 macol Exp Ther 300: 324–329, 2002 17. Ito S, Amin J, Ren Y, Arima S, Abe K, Carretero OA: Hetero- 33. Wei CC, Ferrario CM, Brosnihan KB, Bradley WE, Dell’Italia geneity of angiotensin action in renal circulation. Kidney Int LJ: Acute volume overload induces a balance of Ang II and Suppl 63: S128–S131, 1997 BK/Ang-(1–7) formation in the interstitium of the rat heart in 18. Mitchell KD, Navar LG: Enhanced tubuloglomerular feedback vivo [Abstract]. FASEB J 15: A780, 2001 during peritubular infusions of angiotensins I and II. Am J 34. Muller DN, Fischli W, Clozel JP, Hilgers KF, Bohlender J, Physiol 255: F383–F390, 1988 Menard J, Busjahn A, Ganten D, Luft FC: Local angiotensin II 19. Mitchell KD, Navar LG: Superficial nephron responses to peri- generation in the rat heart: Role of renin uptake. Circ Res 82: tubular capillary infusions of angiotensins I and II. Am J Physiol 13–20, 1998 252: F818–F824, 1987 35. Leife R, Estevao R, Resende AC, Salgado MC: Role of endo- 20. Satoh M, Kashihara N, Yamasaki Y, Maruyama K, Okamoto K, thelium in angiotensin II formation by the rat aorta and mesen- Maeshima Y, Sugiyama H, Sugaya T, Murakami K, Makino H: teric arterial bed. Braz J Med Biol Res 30: 649–656, 1997 Renal interstitial fibrosis is reduced in angiotensin II type 1a 36. Guo C, Ju H, Leung D, Massaeli H, Shi M, Rabinovitch M: A receptor-deficient mice. J Am Soc Nephrol 12: 317–325, 2001 novel vascular smooth muscle chymase is upregulated in hyper- 21. Wolf G: Angiotensin II as a mediator of tubulointerstitial injury. tensive rats. J Clin Invest 107: 703–715, 2001 Nephrol Dial Transplant 15[Suppl 6]: 61–63, 2000 37. Zhang H, Wada J, Hida K, Tsuchiyama Y, Hiragushi K, Shikata 22. Largo R, Gomez-Garre D, Soto K, Marron B, Blanco J, Gazapo K, Wang H, Lin S, Kanwar YS, Makino H: Collectrin, a collect- RM, Plaza JJ, Egido J: Angiotensin-converting enzyme is up- ing duct-specific transmembrane glycoprotein, is a novel ho- regulated in the proximal tubules of rats with intense proteinuria. molog of ACE2 and is developmentally regulated in embryonic Hypertension 33: 732–739, 1999 kidneys. J Biol Chem 276: 17132–17139, 2001 23. Zhuo J, Casley D, Murone C, Mendelsohn FA: Acute and 38. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, chronic in vivo inhibition of angiotensin-converting enzyme by Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, perindopril in the endothelium and adventitia of large arteries Breitbart RE, Acton S: A novel angiotensin-converting enzyme- and organs of the rabbit. J Cardiovasc Pharmacol 29: 297–310, related carboxypeptidase (ACE2) converts angiotensin I to an- 1997 giotensin 1–9. Circ Res 87: E1–E9, 2000