Introduction

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Ischemic cell injury in the kidney occurs during cardiovascular surgery, shock, and transplantation, which may lead to acute renal failure. It is difficult to determine the pathological roles of many factors involved in cellular injury and resultant organ damage, since ischemic acute renal failure (ARF) is induced not only by the ischemia itself but also by the following reperfusion. Furthermore, the kidney is a complex tissue comprising vascular and tubular networks. One of the major contributors to ischemic cell injury is an increase in intracellular Ca²⁺, which occurs in cases of ARF (Schrier et al., 1987). Although physiological and pathophysiological mechanisms of Ca²⁺ overload in ischemic kidney have not been fully elucidated, there is evidence indicating that increased cytosolic Ca²⁺ may be an important mediator of epithelial cell necrosis, which is characteristic of ischemic ARF (Schrier et al., 1984; Wilson et al., 1984; Wong and Chase, 1986). In addition, Ca²⁺ channel blockers exert a protective effect against ischemic ARF (Goldfarb et al., 1983; Shimizu et al., 1990).

The Na⁺/Ca²⁺ exchanger (NCX), which is expressed in a variety of tissues, including the kidney, is involved in the regulation of intracellular Ca²⁺ concentration. The role of this exchanger protein is best understood in studies of cardiac muscle, where NCX is encoded by a multigene family comprising three NCX isoforms, NCX 1, NCX 2, and NCX 3 (Quednau et al., 1997), and which functions as a bi-directional plasma membrane antiporter transporting three Na⁺ for one Ca²⁺. Therefore, the activity of this antiporter is influenced by electrochemical gradients for both Na⁺ and Ca²⁺. Since there is normally a large inwardly directed electrochemical gradients for Na⁺, exchange activity results in the electrogenic movements of Na⁺ into and Ca²⁺ out of the cells, which is referred to as the forward-mode operation of the exchanger. However, in ischemic cardiac cells where intracellular pH decreases, the intracellular Na⁺ concentration may rise through the Na⁺/H⁺ exchanger system, which in turn increases the intracellular Ca²⁺ concentration through the reverse mode of the NCX system (Allen et al., 1993; Scholz et al., 1993; Ver Donck et al., 1993). This process leads to Ca²⁺ overload, which induces various pathological conditions in the heart. On the other hand, little is known of the possible involvement of Ca²⁺ overload via the reverse mode of NCX system, with ischemic ARF.

KB-R7943 (2-[2-[4-nitorobenzyloxy]phenyl]ethyl)isothioureamethanesulfonate) (Fig. 1) has been reported to inhibit the Ca²⁺ influx mode of NCX in guinea pig cardiac ventricular cells (Watano et al., 1996). This compound efficiently improved the ischemia/reperfusion-induced injury in the isolated rat perfused heart (Nakamura et al., 1998). We also found that pre-ischemic treatment with KB-R7943 shows protective effects on ischemia/reperfusion-induced renal dysfunction (Kuro et al., 1999), thereby suggesting that activation of the reverse mode of NCX plays an important role in the pathogenesis of ischemic ARF. However, it is more important to examine effects of drugs administered after reperfusion, since many clinical cases of ischemic ARF cannot be predicted.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Chemical structure of KB-R7943.

Endothelin (ET-1), a 21-amino acid peptide, is a potent vasoconstrictor peptide isolated from the culture supernatant of porcine aortic endothelial cells (Yanagisawa et al., 1988). This peptide has been considered to play an important role in the pathophysiology of cardiac, vascular, and renal diseases associated with regional and systemic vasoconstriction. The kidney synthesizes ET-1 and has both ET_A and ET_B receptors (Nambi et al., 1992a,b). Recent experimental and clinical studies indicated a close relationship between the renal ET-1 system and the pathogenesis of ischemic ARF, based on findings that renal ET-1 mRNA expression is increased in the ischemic kidney (Firth and Ratcliffe, 1992; Wilhelm et al., 1999) and that ET_A-selective or nonselective ET_A/ET_B-receptor antagonists attenuate the ischemia/reperfusion-induced impairment of renal function (Mino et al., 1992; Gellai et al., 1995; Birck et al., 1998; Kuro et al., 2000). However, there is no available evidence regarding the involvement of Ca²⁺ overload for ET-1 overproduction in the kidney subjected to the ischemia/reperfusion.

In the present study, we first examined whether ischemia/reperfusion-induced renal dysfunction and tissue injury would be overcome by post-ischemic treatment with KB-R7943, as well as its pre-ischemic treatment, in comparison with verapamil, a Ca²⁺ channel antagonist. Second, we asked whether KB-R7943 could suppress the enhanced production of ET-1 in the ischemic kidney. We report here that Ca²⁺ overload via the reverse mode of Na⁺/Ca²⁺ exchange, followed by the ET-1 overproduction, does play an important role in the pathogenesis of ischemia/reperfusion-induced ARF.

	Materials and Methods

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Animals and Experimental Design. Male Sprague-Dawley rats (280-320 g, 10 weeks old, Japan SLC, Shizuoka) were housed in a light-controlled room with a 12-h light/dark cycle, and access to food and water was ad libitum. Two weeks before the study (rats 8 weeks of age), the right kidney was removed through a small flank incision made following pentobarbital anesthesia (50 mg/kg, i.p.). After a 2-week recovery period, these rats were separated into six groups: 1) uninephrectomized (UN) control; 2) vehicle-treated ARF; 3) pre-ischemic treatment with KB-R7943 (2 mg/kg, 10 mg/kg, i.v.) in ARF; 4) pre-ischemic treatment with verapamil (1 mg/kg, i.v.) in ARF; 5) post-ischemic treatment with KB-R7943 (2 mg/kg, 10 mg/kg, i.v.) in ARF; 6) post-ischemic treatment with verapamil (1 mg/kg, i.v.) in ARF. To induce ischemic ARF, the rats were anesthetized with pentobarbital (50 mg/kg, i.p.), and the left kidney was exposed through a small flank incision. The left renal artery and vein were occluded for 45-min with a nontraumatic clamp. At the end of the ischemic period, the clamp was released and blood reperfused.

Measurements of Mean Arterial Pressure (MAP) and Heart Rate (HR) in Anesthetized Rats. Male Sprague-Dawley rats (280-320 g, 10 weeks old, Japan SLC, Shizuoka) were used. Two weeks before the study (8 weeks of age), the right kidney was removed. After a 2-week recovery period, animals were anesthetized with sodium thiobutabarbital (Inactin, 100 mg/kg; i.p.) and placed on a surgical tray that maintained rectal temperature between 37 and 38°C. After a tracheotomy was done, the left femoral artery was cannulated for measurement of MAP and HR. The left femoral vein was also cannulated for drug administration. MAP and HR were continuously recorded on a polygraph (RM 6000; Nihon Kohden, Tokyo, Japan). After the stabilization period, KB-R7943 or its vehicle was injected i.v., and MAP and HR were monitored for 120 min.

Blood and Urine Measurements. Blood urea nitrogen (BUN) and creatinine levels in plasma and urine were determined using the BUN-test-Wako and Creatinine-test-Wako (Wako Pure Chemical Industries, Osaka, Japan), respectively. Urinary osmolality (UOsm) was measured by freezing point depression (Fiske, MA). Urine and plasma sodium concentrations were determined using a flame photometer (Hitachi, 205 D, Hitachinaka, Japan). Fractional excretion of sodium (FENa, %) was calculated from the formula, FENa = UNaV/(PNa × Ccr) × 100, where UNaV is urinary excretion of sodium, PNa is the plasma sodium concentration, and Ccr is creatinine clearance.

Histological Studies. Excised left kidneys were processed for light microscopic observation, according to standard procedures. The kidneys were then preserved in phosphate-buffered 10% formalin, after which the kidneys were chopped into small pieces, embedded in paraffin wax, cut at 3 µm and stained with H&E. Histopathological changes were analyzed for tubular necrosis, proteinaceous casts, and medullary congestion, as suggested by Solez et al. (1974). Tubular necrosis and proteinaceous casts were graded as follows; no damage ( or 0), mild (± or 1, unicellular, patchy isolated damage), moderate (+ or 2, damage less than 25%), severe (++ or 3, damage between 25 and 50%), and very severe (+++ or 4, more than 50% damage). Degree of medullary congestion was defined as follows: no congestion ( or 0), mild (± or 1, vascular congestion with identification of erythrocytes by 400× magnification), moderate (+ or 2, vascular congestion with identification of erythrocytes by 200× magnification), severe (++ or 3, vascular congestion with identification of erythrocytes by 100× magnification), and very severe (+++ or 4, vascular congestion with identification of erythrocytes by 40× magnification). Evaluations were made in a blind manner.

Renal ET-1 Assay. ET-1 was extracted from the kidney, as described elsewhere (Fujita et al., 1995). Briefly, kidneys were weighed and homogenized for 60 s in 8 volumes of ice-cold organic solution (chloroform/methanol, 2:1, including 1 mM N-ethylmaleimide). The homogenates were left overnight at 4°C, then 0.4 volumes of distilled water was added after which the homogenates were centrifuged at 1500g for 30 min and the resultant supernatant was stored. Aliquots of the supernatant were diluted 1:10 with a 0.09% trifluoroacetic acid solution and applied to Sep-Pak C18 cartridges. The sample was eluted with 3 ml of 63.3% acetonitrile and 0.1% trifluoroacetic acid in water. Eluates were dried in a centrifugal concentrator, and the dried residue was reconstituted in assay buffer for radioimmunoassay (RIA). The clear solution was subjected to RIA. The recovery of ET-1 was approximately 80%. RIA for tissue ET-1 was done, as described elsewhere (Matsumura et al., 1990b), using ET-1 antiserum (a generous gift from Dr. Marvin R. Brown, Department of Medicine, University of California, San Diego, CA). This serum dose not cross-react with big ET-1.

Western Blotting. For a quantitative analysis of NCX protein, renal tissue (0.7-1.2 g) was freeze-clamped and ground in liquid nitrogen before suspension in homogenization buffer containing 10 mM NaHCO₃, 5 mM L-histidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine; pH 7.5. This homogenized solution was lysed in buffer [2.5% sodium dodecyl sulfate (SDS), 25 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine; pH 7.4] and centrifuged 36,000g at 4°C for 30 min. Supernatants were mixed with loading buffer (1 M Tris, 0.5 M EDTA, 800 mM dithiothreitol, 15% SDS, 42% glycerol, 0.12% bromphenol blue). Protein concentration was determined, using the bicinchoninic acid assay method. To determine the molecular weights of separated proteins, the BENCH MARK Prestained Protein Ladder (Life Technologies, Gaithersburg, MD) was used. Equal amounts of protein were fractionated using 7.5% SDS-polyacrylamide gels. After transfer to nitrocellulose membranes (Amersham Pharmacia Biotech, Arlington Heights, IL) for 30 min, the blots were blocked overnight at 4°C with 5% nonfat dry milk in phosphate-buffered saline (PBS). After blocking, blots were incubated with anti-NCX 1 polyclonal antibody (a generous gift from Dr T. Iwamoto, Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Japan) at 1:200 dilution with 5% nonfat dry milk in PBS at 37°C for 30 min. After three washes with 5% nonfat dry milk in PBS (10 min per wash), the preparations were further incubated with goat anti-rabbit IgG coupled to horseradish peroxidase (Zymed Laboratories, South San Francisco, CA) at 1:1000 dilution with 5% nonfat dry milk in PBS at 37°C for 30 min. Blots were then washed three times with 5% nonfat dry milk in PBS (10 min per wash). Subsequent detection was carried out using enhanced chemiluminescence Western blotting kits (Amersham Pharmacia Biotech) and quantified by using the NIH IMAGE for Macintosh.

Drugs. KB-R7943 (Kanebo, Ltd., Osaka, Japan) was dissolved in a mixture of 15% ethanol, 15% polyethylene glycol 400, and 70% saline (0.9%), and verapamil was dissolved in 0.9% saline just before administration. Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan).

Statistical Analysis. Values are mean ± S.E.M. For statistical analysis, we used one-way analysis of variance followed by Bonferroni's multiple comparison tests. Histological data were analyzed using the Kruskal-Wallis nonparametric test combined with the Steel-type multiple comparison test. For all comparisons, differences were considered significant at P < 0.05.

	Results

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Effects of KB-R7943 on Systemic Hemodynamics. When KB-R7943 (10 mg/kg) or its vehicle was administered i.v., no significant changes were observed in MAP or HR.

Renal Function after Ischemia/Reperfusion and Effects of Pre-Ischemic Treatment with KB-R7943 or Verapamil. Table 1 shows the effect of pre-ischemic treatment with KB-R7943 or verapamil. Renal functional parameters of rats subjected to 45-min ischemia showed a marked deterioration, as measured 24 h after reperfusion. As compared with UN control rats, vehicle-treated ARF rats showed significant increases in BUN, plasma creatinine concentration (Pcr), urine flow (UF), and FENa, and significant decreases in Ccr and UOsm. Pre-ischemic treatment with KB-R7943 (2 mg/kg, 10 mg/kg, i.v.) markedly and dose-relatedly attenuated the ARF-induced renal dysfunction. As reported (Goldfarb et al., 1983), verapamil (1 mg/kg) also attenuated significantly the decreased responses of renal function to the ischemia, to a degree similar to findings with the lower dose of KB-R7943. The administration of KB-R7943 (10 mg/kg) to UN control rats produced no significant effects in their renal functional parameters (data not shown).

Renal Function after Ischemia/Reperfusion and Effects of Post-Ischemic Treatment with KB-R7943 or Verapamil. Figures 2 and 3 show the effect of post-ischemic treatment with KB-R7943 or verapamil. The impairment of renal function induced by ischemia/reperfusion was also markedly and dose-relatedly attenuated by KB-R7943 administered after reperfusion. At the higher dose, each renal functional parameter was significantly improved. In contrast, no significant improvements were observed with post-ischemic treatment with verapamil.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effects of KB-R7943 or verapamil administered after reperfusion on BUN (A), Pcr (B), and Ccr (C) at 24 h after ischemia/reperfusion. At 24 h after reperfusion, 5-h urine was collected. Each value represents the mean ± S.E.M. #P < 0.01, compared with UN control rats; **P < 0.01, compared with vehicle-treated ARF rats. , UN control (n = 7); , vehicle-treated ARF (n = 10); dark gray, verapamil (1 mg/kg, n = 7); light gray, KB-R7943 (2 mg/kg, n = 7); and , KB-R7943 (10 mg/kg, n = 7).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effects of KB-R7943 or verapamil administered after reperfusion on UF (A), UOsm (B), and FENa (C) at 24 h after the ischemia/reperfusion. At 24 h after reperfusion, 5-h urine was collected. Each value represents the mean ± S.E.M. #P < 0.01, compared with UN control rats; **P < 0.01, compared with vehicle-treated ARF rats. , UN control (n = 7); , vehicle-treated ARF (n = 10); dark gray, verapamil (1 mg/kg, n = 7); light gray, KB-R7943 (2 mg/kg, n = 7); and , KB-R7943 (10 mg/kg, n = 7).

Histological Renal Damage after Ischemia/Reperfusion and Effects of Post-Ischemic Treatment with KB-R7943 or Verapamil. Histopathological examination revealed severe lesions in the kidney of vehicle-treated ARF rats (1 day after the ischemia and reperfusion). These changes were characterized by tubular necrosis (Fig. 4, outer zone outer stripe of medulla), proteinaceous casts in tubuli (Fig. 5, inner zone of medulla), and medullary congestion and hemorrhage (Fig. 6, outer zone inner stripe of medulla). Post-ischemic treatment with KB-R7943 dose-relatedly attenuated the development of all these lesions. In the case of a higher dose, there were significant improvements (Table 2). On the other hand, post-ischemic treatment with verapamil exerted no protective effects against any of these lesions.

View larger version (70K): [in this window] [in a new window]   Fig. 4.   Light microscopy of the outer zone outer stripe of medulla of the kidney of ARF rats treated with vehicle (B), verapamil (C, 1 mg/kg), KB-R7943 (D, 2 mg/kg), and KB-R7943 (E, 10 mg/kg) at 24 h after ischemia/reperfusion, and UN control rats (A). Drugs were given i.v. after reperfusion. Arrows indicate tubular necrosis (hematoxylin and eosin staining).

View larger version (71K): [in this window] [in a new window]   Fig. 5.   Light microscopy of the inner zone of medulla of the kidney of ARF rats treated with vehicle (B), verapamil (C, 1 mg/kg), KB-R7943 (D, 2 mg/kg), and KB-R7943 (E, 10 mg/kg) at 24 h after ischemia/reperfusion, and UN control rats (A). Drugs were given i.v. after reperfusion. Arrows indicate proteinaceous casts in tubuli (hematoxylin and eosin staining).

View larger version (68K): [in this window] [in a new window]   Fig. 6.   Light microscopy of the outer zone inner stripe of medulla of the kidney of ARF rats treated with vehicle (B), verapamil (C, 1 mg/kg), KB-R7943 (D, 2 mg/kg), and KB-R7943 (E, 10 mg/kg) at 24 h after ischemia/reperfusion, and UN control rats (A). Drugs were given i.v. after reperfusion. Arrows indicate congestion and hemorrhage (hematoxylin and eosin staining).

View this table: [in this window] [in a new window]   TABLE 2 Histopathological changes in kidneys in UN control and ARF rats Each value represents the mean ± S.E.M. Drugs were given i.v. immediately after reperfusion. Grades: no changes ( or 0), mild (± or 1), moderate (+ or 2), severe (++ or 3), very severe (+++ or 4).

ET-1 Levels in the Kidney. To confirm the contribution of ET-1 to ischemic ARF, we measured renal ET-1 levels at the end of 45-min ischemia and at 2, 6, and 24 h after reperfusion. As shown in Fig. 7, renal ET-1 contents were not significantly increased at the end of the ischemic period. After reperfusion, there were significant increases in renal ET-1 contents; and a maximum increase was seen 6 h after reperfusion. KB-R7943 (10 mg/kg) almost completely suppressed the ischemia/reperfusion-induced increases in renal ET-1 contents. The same dose of KB-R7943 did not alter renal ET-1 contents in UN controls rats (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of KB-R7943 administered before ischemia/reperfusion on immunoreactive ET-1 content in the kidney of ARF rats at the end of 45-min ischemia and at 2, 6, and 24 h after reperfusion. Each column and bar represents the mean ± S.E.M. #P < 0.01, compared with UN control rats; **P < 0.01, compared with vehicle-treated ARF rats. , UN control (n = 10); , vehicle-treated ARF (n = 10); and , KB-R7943 (10 mg/kg, n = 10).

NCX Protein Expression at the End of 45-min Ischemia and at 6 h after Reperfusion and Effects of Pre-Ischemic Treatment with KB-R7943. We measured NCX protein expression at the end of 45-min ischemia and at 6 h after reperfusion (Fig. 8). No significant changes in NCX protein expression in renal tissue were observed with 45-min ischemia, 6-h after reperfusion, and KB-R7943 (10 mg/kg) treatment.

View larger version (29K): [in this window] [in a new window]   Fig. 8.   NCX protein expression at the end of 45-min ischemia and at 6 h after reperfusion. Each column and bar represents the mean ± S.E.M. , UN control (n = 6); , vehicle-treated ARF (n = 6); and , KB-R7943 (10 mg/kg, n = 6).

	Discussion

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In the present study, we found that KB-R7943 overcame the ischemia/reperfusion-induced renal dysfunction with post-ischemic as well as pre-ischemic treatments. In addition, the post-ischemic treatment showed a protective effect against ischemic ARF-induced histological injuries, in the same manner as seen with the pre-ischemic treatments of this compound, as noted earlier (Kuro et al., 1999). Thus, this selective inhibitor of Na⁺/Ca²⁺ exchanger may be useful in the treatment of ischemia/reperfusion-induced ARF. In contrast to the findings observed with KB-R7943, post-ischemic treatment with verapamil failed to attenuate the renal dysfunction and tissue injury induced by ischemia/reperfusion, although pre-ischemic treatment at the same dose efficiently suppressed the development of ischemic ARF. The mechanisms underlying the ischemic renal damage are complex and not fully understood, but it is known that Ca²⁺ overload to renal epithelial cells may be one of the causal factors of these diseases (Schrier et al., 1987). Taken together, Ca²⁺ influx, mainly via the voltage-dependent Ca²⁺ channel and via the reverse mode of NCX, might occur during the ischemia and after reperfusion, respectively, both of which are likely to be responsible for the development and progression of renal damage.

KB-R7943 was reported to selectively inhibit the reverse mode of NCX (Iwamoto et al., 1996) in cardiomyocytes, smooth muscle cells, and NCX-1-transfected fibroblasts. Similar inhibitory effects of KB-R7943 on the Ca²⁺ influx mode of NCX were observed in guinea pig cardiac ventricular cells (Watano et al., 1996). In ischemic cardiac cells, where the intracellular pH is decreased by anaerobic glycolysis and intracellular acidosis, intracellular Na⁺ concentration rises through increased Na⁺/H⁺ exchanger activity (Lazdunski et al., 1985). Moreover, Na⁺/K⁺-ATPase activity is inhibited during ischemia (Cross et al., 1995). These phenomena led to an increase in intracellular Ca²⁺ concentrations through the reverse mode of the NCX system (Dennis et al., 1990). Ca²⁺ overload via this system seems to contribute to the ischemia/reperfusion injury in the heart (Tani and Neely, 1989). KB-R7943 was reported to reduce the cytosolic Ca²⁺ overload in isolated rat cardiomyocytes exposed to ischemic condition and to protect against reoxygenation-induced injury in the whole heart (Ladilov et al., 1999). Nakamura et al. (1998) found that KB-R7943 significantly improved ischemia/reperfusion-induced injury in the isolated rat perfused heart, by post- as well as pre-ischemic treatment, thereby suggesting that the activation of Na⁺/Ca²⁺ exchange mainly occurs immediately after the reperfusion.

In the kidney, NCX plays an important role in transcellular Ca²⁺ reabsorption in distal and connecting nephron tubules (Yu et al., 1992; Reilly et al., 1993). One of the physiological roles of NCX in these epithelial cells is to regulate the intracellular Ca²⁺ level against its hormone-induced increment (Dai and Quamme, 1994). However, there has been little available information on the pathological role of NCX in renal ischemic conditions. In the present study, we clearly noted the pathological importance of NCX in the ischemia/reperfusion-induced renal injury.

There is growing evidence that ET-1 is closely related to the development of the ischemic ARF. It has been demonstrated that ET-1 content (Shibouta et al., 1990) and ET-1 mRNA expression (Firth and Ratcliffe, 1992; Wilhelm et al., 1999) are elevated in renal tissues after ischemia/reperfusion. We also found that daily oral administration of the ET_A-selective antagonist ABT-627, but not the ET_B-selective antagonist A-192621, had a marked effect on ischemia/reperfusion-induced renal dysfunction and on tissue injury (Kuro et al., 2000). In addition, an ET-converting enzyme inhibitor, phosphoramidon (Matsumura et al., 1990a), was found to overcome ischemia/reperfusion-induced renal injury (Vemulapalli et al., 1993; Bird et al., 1995). These findings suggest that the up-regulation of renal ET-1 production and its ET_A receptor-mediated action contribute to the pathogenesis of ischemic ARF. In the present study, we investigated the possible involvement of NCX-mediated Ca²⁺ overload for ET-1 overproduction in the kidney subjected to ischemia/reperfusion. KB-R7943 markedly suppressed the increased levels of renal ET-1 content at 2, 6, and 24 h after reperfusion. Taken together, it seems likely that KB-R7943 overcame the renal dysfunction and tissue injury induced by ischemia/reperfusion, by inhibiting the Ca²⁺ overload through the reverse mode of NCX, and consequently, ET-1 overproduction in the kidney was halted. In the present study, we did not determine the localization of ET-1 overproduction in endothelial or tubular cells, although a recent study by Wilhelm et al. (1999) reported that the expression of ET-1 peptide was increased in the endothelium of cortical peritubular capillaries of the ischemically injured kidney. Ongoing studies should determine whether or not the overproduction of ET-1 occurs in renal tubular cells.

It was reported that NCX expression and its function are up-regulated in an arrhythmogenic rabbit model of heart failure (Pogwizd et al., 1999). Overexpression of cardiac NCX increases the likelihood of ischemia/reperfusion injury (Cross et al., 1998). In our study, however, NCX protein expression in renal tissue was unchanged during the ischemia and after reperfusion, thereby suggesting that activation of the reverse mode of NCX without protein overexpression occurs during ischemia/reperfusion of the kidney. Figure 9 outlines our working hypothesis regarding the development of ischemic ARF. The accumulation of intracellular Na⁺ cannot be efficiently halted, because there is decreased Na⁺/K⁺-ATPase activity. The Na⁺/H⁺ exchanger would be activated in the presence of an intracellular acidosis, consequently, intracellular Na⁺ levels would be increased and Ca²⁺ overload would occur via the reverse mode of NCX, to be followed by ET-1 overproduction. ET-1 seems to play an important role in the pathogenesis of the ischemia/reperfusion-induced ARF.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   Schematic diagram of ischemia/reperfusion-induced renal injury. VDC, voltage-dependent Ca2+ channel; NHE, Na+/H+ exchanger; PLC, phospholipase C; ER, endoplasmic reticulum; CaM, calmodulin.

In summary, both pre- and post-ischemic treatments with KB-R7943 overcame the ischemia/reperfusion-induced renal injury, which suggests that activation of the reverse mode of NCX mainly occurs after reperfusion and plays an important role in the development of ischemic ARF. Ca²⁺ overload via the reverse mode of NCX seems to be followed by renal ET-1 overproduction, which leads to renal function impairment. Selective Na⁺/Ca²⁺ exchange inhibitors such as KB-R7943 may prove to be effective agents against the ischemic ARF in humans.