Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Calpains, cytosolic Ca²⁺-activated neutral cysteine proteases, are involved in a variety of cellular functions including the regulation of cytoskeletal structures, cell cycle progression, and cell spreading, adhesion, and migration. There is extensive evidence that calpains play a critical role in cell/tissue/organ injury and death, including renal proximal tubule (RPT) cell injury/death (Bronk and Gores, 1993; Schnellmann et al., 1994; Bednarski et al., 1995; Choi, 1995; Edelstein et al., 1995, 1996, 1997a; Trump and Berezesky, 1995; Markgraf et al., 1997; Waters et al., 1997; Schumacher et al., 2000; Yoshida, 2000; Chatterjee et al., 2001; McDonald et al., 2001). For example, increased calpain activity was observed in rat RPTs subjected to hypoxia and in rabbit RPTs exposed to antimycin A (Edelstein et al., 1995, 1996, 1997a; Harriman et al., 2002). Calpain inhibitors have been shown to be cytoprotective in primary RPT cell culture after HgCl₂ or cyclosporin A exposure, and in rat RPTs subjected to hypoxia (Elliget et al., 1991; Wilson and Hartz, 1991; Edelstein et al., 1995, 1996). Calpain inhibitors also have exhibited cytoprotective effects in rabbit RPTs exposed to hypoxia/reoxygenation or a diverse group of toxicants (Waters et al., 1997). Furthermore, in vivo animal experiments by Chatterjee et al. (2001) and McDonald et al. (2001) showed that calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal) protected against multiple organ failure produced by hemorrhagic shock or renal injury produced by ischemia/reperfusion. These results strongly support the hypothesis that calpains play a critical role in oncotic renal cell death and indicate potential benefits of calpain inhibitors in renal failure and multiple organ failure treatment. Although the exact calpain isozyme and the critical substrates of calpains during cell injury/death remain unidentified, calpains appear to act downstream of ATP depletion, endoplasmic reticulum Ca²⁺ release, Na⁺ influx, K⁺ efflux, and loss of membrane potential (Waters et al., 1997; Harriman et al., 2002). Previous data also suggest that calpains act before the influx of extracellular Ca²⁺ and Cl, and the increased plasma membrane permeability to large proteins [e.g., lactate dehydrogenase (LDH)] (Waters et al., 1997).

Two ubiquitous isoforms, µ- and m-calpain, are present in all animal tissues studied to date (Lane et al., 1992; Sorimachi et al., 1997). As their names imply, µ- and m-calpain require µ- and m-molar free Ca²⁺ ([Ca²⁺]_f) concentrations, respectively, for in vitro activation and/or autolysis (Sorimachi et al., 1997). Both µ- and m-calpains are heterodimers, consisting of distinct 80-kDa catalytic subunits and a common 30-kDa regulatory subunit. The 80-kDa large subunit contains four functional domains with the catalytic site residing in domain II and the Ca²⁺-binding sites located on domain IV (Sorimachi et al., 1997; Strobl et al., 2000). A number of synthetic small compounds including transition-state inhibitors, irreversible inhibitors, calmodulin antagonists, and polyamines have been designed to inhibit calpain activity. Currently, available calpain inhibitors are classified according to chemical structure (peptide versus nonpeptide) and mechanism of inhibition (catalytic site-directed versus Ca²⁺-binding site-directed). Most catalytic site-directed calpain inhibitors are peptides. For example, calpain inhibitor 1 (N-acetyl-Leu-Leu-norleucinal) is a tripeptidyl aldehyde. A series of peptide -keto amide inhibitors of calpains have been reported (Li et al., 1993, 1996), and the properties of these inhibitors were tested using intact RPTs and a synthetic calpain substrate SLLVY-7-amino-4-methylcoumarin (Harriman et al., 2000). No clear correlation was obtained between the in vitro inhibitory constants of µ- or m-calpain and cytoprotection, therefore leaving the role of each calpain isozyme in acute renal cell injury undetermined (Harriman et al., 2000). In addition, many of the catalytic site-directed peptidyl calpain inhibitors lack intracellular specificity, plasma membrane permeability, and/or potency (Li et al., 1993, 1996; Wang et al., 1996; Mellgren, 1997; Sorimachi et al., 1997; Harriman et al., 2000). In contrast, the compound 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) represents a nonpeptide Ca²⁺-binding site-directed calpain inhibitor and has been shown to block RPT cell death following exposure to diverse insults (Edelstein et al., 1996; Waters et al., 1997; Liu et al., 2001). In cellular systems, PD150606 is a more potent calpain inhibitor than most of the catalytic site-directed calpain inhibitors (Waters et al., 1997). However, PD150606 also can have nonspecific effects (Wang et al., 1996).

Recently, Inoue et al. (1999) reported the development of nonpeptide, irreversible, catalytic site-directed calpain inhibitors (see Table 1 for structures and in vitro calpain-inhibitory constants). SJA7019 [chloroacetic acid N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl]hydrazide] has similar IC₅₀ values for both purified µ- and m-calpains and is approximately 20-fold more selective for calpains than cathepsin L. The absence of a methoxy group on the phenyl ring of SJA7029 [chloroacetic acid N'-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl)hydrazide] does not change the similar IC₅₀ values for both purified µ- and m-calpain, but it does decrease the potency of SJA7029 approximately 2-fold compared with SJA7019. SJA7029 is approximately 30-fold more selective for calpains than cathepsin L. 

The goals of the present study were to determine the action of these two compounds on 1) RPT calpain activity using fluorescein isothiocyanate (FITC)-casein zymography, 2) antimycin A-induced RPT extracellular ⁴⁵Ca²⁺ influx and cell death, and 3) hypoxia/reoxygenation-induced RPT cellular dysfunction and death.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Purified µ-calpain (porcine erythrocytes) and m-calpain (porcine kidney) were purchased from Calbiochem (La Jolla, CA). FITC-casein, dimethyl sulfoxide (DMSO), antimycin A, acrylamide, and bis-acrylamide were obtained from Sigma-Aldrich (St. Louis, MO). SJA7019 and SJA7029 were gifts from Dr. Jun Inoue (Senju Pharmaceutical Company, Kobe, Japan). The sources of other chemicals were reported previously (Rodeheaver et al., 1990; Groves and Schnellmann, 1996) or were obtained from Sigma-Aldrich. All glassware was silanized and autoclaved. All media and buffers were filter sterilized prior to use.

Isolation of Rabbit RPTs. RPTs were isolated and purified according to the method described by Rodeheaver et al. (1990) and Groves and Schnellmann (1996) from New Zealand White rabbits (female, 2 kg; Myrtle's Rabbitry, Thompson Station, TN). RPTs were suspended at a concentration of 2 mg/ml in an incubation buffer containing 1 mM alanine, 5 mM dextrose, 2 mM heptanoate, 4 mM lactate, 5 mM malate, 115 mM NaCl, 15 mM NaHCO₃, 5 mM KCl, 2 mM NaH₂PO_4, 1 mM MgSO₄, 1 mM CaCl₂, and 10 mM HEPES (pH 7.4, 295 mOsm/kg). RPT suspensions were incubated under air/CO₂ (95%/5%) at 37°C in a gyrating water bath (180 rpm). All experiments utilized a 15-min preincubation period with no experimental manipulations. To determine the calpain-inhibitory effects of SJA7019 and SJA7029, RPTs were incubated with SJA7019 (10, 30, or 100 µM), SJA7029 (10, 30, or 100 µM), or diluent (DMSO, 0.1% total volume) for 30 min. At the end of incubation, aliquots of RPTs were removed and processed for FITC-casein zymography. To test the cytoprotective effects of the SJA compounds, various concentrations of SJA7019 or SJA7029 were added to RPTs 30 min before the mitochondrial inhibitor antimycin A (10 µM) or diluent (DMSO, 0.25% total volume), and the incubation continued for an additional 30 or 60 min. At the corresponding time points, aliquots of RPTs were removed for LDH release analysis. Antimycin A has been shown to produce extensive cell death over an extended period of time in this RPT cell injury/death model (Schnellmann et al., 1993; Liu et al., 2001). In some experiments, SJA7029 was added 10 min after antimycin A and the incubation continued for an additional 20 min.

FITC-Casein Zymography. FITC-casein zymography was performed as described previously (Arthur and Mykles, 2000; Liu et al., 2001). RPTs were centrifuged, and the pellet was resuspended in the zymography buffer containing 50 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, 5 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 mM 2-mercaptoethanol, pH 7.6. RPTs were lysed with 1% Triton X-100 at 37°C for 10 min. The total lysate was centrifuged at 14,000g for 10 min at 4°C, and the supernatant was mixed with 2× loading buffer (100 mM Tris-HCl, pH 6.8, 10 mM EDTA, 20% glycerol, 10 mM 2-mercaptoethanol, and 0.02% bromphenol blue). Matched samples were taken for protein concentration. Protein concentrations were determined by the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL) using bovine serum albumin as the standard.

Ca²⁺ Influxes. The calpain inhibitor SJA7019 or SJA7029, or DMSO was added 30 min before antimycin A (10 µM), and the incubation continued for an additional 30 min. In some experiments, the calpain inhibitor SJA7019 or SJA7029 was added 10 min after antimycin A, and the incubation continued for an additional 20 min. Ca²⁺ uptake was determined by adding a tracer amount of ⁴⁵Ca²⁺ to RPT suspensions 15 min after adding antimycin A (Miller and Schnellmann, 1993; Waters et al., 1997). After 15 min, aliquots were removed and RPTs were separated from the surrounding buffer by rapid centrifugation through a layer of dibutylphthalate/dioctylphthalate (2:1). RPT Ca²⁺ contents were determined by resuspending the pellets in Triton X-100 solubilization buffer (100 mM Tris, 150 mM NaCl, and 0.05% Triton X-100, pH 7.5), and aliquots were taken for liquid scintillation spectrometry and protein determination. Extracellular Ca²⁺ was corrected using the extracellular water marker [¹⁴C]dextran.

Hypoxia/Reoxygenation Exposure and Oxygen Consumption (QO₂) Measurement. RPTs were subjected to hypoxia (95% N₂/5% CO₂, 1 h)/reoxygenation (95% air/5% CO₂, 1 h) as described previously (Moran and Schnellmann, 1997; Liu et al., 2001). The calpain inhibitor SJA7029 (30 µM) or diluent (DMSO, <0.1% total volume) was added at the onset of hypoxia. Immediately after the hypoxic period, aliquots of RPTs were removed for determination of LDH release. After reoxygenation, aliquots of RPTs were removed for determination of LDH release or QO₂. QO₂ was measured polarographically using a Clark-type electrode as described previously (Schnellmann, 1994). After basal QO₂ was obtained, ouabain-insensitive QO₂ was measured in the presence of 0.1 mM ouabain and the ouabain-sensitive QO₂ was calculated as a difference between basal and ouabain-insensitive QO₂. Results are expressed as percentage of controls.

Cell Death. The release of LDH into the incubation buffer was measured as a marker of cell death as described previously (Moran and Schnellmann, 1996).

Statistical Analysis. RPTs isolated from one rabbit represent one individual experiment (n = 1). Data were expressed as means ± S.E. and analyzed by one-way ANOVA, and multiple means were compared using Fisher's protected least significant difference test with a level of significance of P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

SJA7019 and SJA7029 Inhibit Basal RPT µ- and m-Calpain. FITC-casein zymography is a sensitive method of distinguishing µ- and m-calpain activities (Arthur and Mykles, 2000; Liu et al., 2001). As shown in Fig. 1A, FITC-casein zymography of purified calpains revealed one band for µ-calpain and a doublet for m-calpain, demonstrating the ability of this method to separate µ- from m-calpain activities. The presence of double bands for purified m-calpain is consistent with previous observations in the literature (Arthur and Mykles, 2000; Liu et al., 2001). The FITC-casein gel also revealed the presence of two bands in control RPTs, corresponding to those obtained with purified µ- and m-calpains (Fig. 1A). The µ- and m-calpain activities in control RPTs did not change as the incubation continued up to 30 min (data not shown). Thirty minutes of SJA7019 or SJA7029 treatment resulted in concentration-dependent inhibition of basal RPT µ- and m-calpain activities with approximate IC₅₀ at 30 µM (Fig. 1, B-E). Neither inhibitor displayed selectivity toward µ- or m-calpain. The results show that the SJA compounds inhibit RPT µ- and m-calpain with equal potency and efficacy.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   A, a FITC-casein zymogram of purified µ-, m-, and RPT calpains. Purified µ- or m-calpain (30 ng), or 20-µg RPT samples were subjected to FITC-casein zymography. B through E, the effects of SJA7019 and SJA7029 on basal µ- or m-calpain activity in RPTs. B, SJA7019 on RPT µ-calpain; C, SJA7019 on RPT m-calpain; D, SJA7029 on RPT µ-calpain; and E, SJA7029 on RPT m-calpain. RPTs were incubated with SJA7019 (10, 30, or 100 µM), SJA7029 (10, 30, or 100 µM), or diluent DMSO (CON, <0.1% total volume) for 30 min. RPT samples (20 µg) were subjected to FITC-casein zymography. The densities of the corresponding bands were determined by National Institutes of Health image software, and the results were expressed as percentage of controls. Data are means ± S.E., n = 3 to 6. Bars with different letters are significantly different from one another; P < 0.05.

SJA7019 and SJA7029 Protect RPTs against Antimycin A-Induced Cell Death. To determine the cytoprotective effects of the SJA compounds, RPTs were treated with SJA7019 or SJA7029 (10, 30, or 100 µM) for 30 min and then exposed to the mitochondrial inhibitor antimycin A. LDH release into the surrounding medium was measured as a marker of cell death at 30 and 60 min after addition of antimycin A. SJA7019 or SJA7029 reduced LDH release in RPTs exposed to antimycin A for 30 min in a concentration-dependent manner, and the cytoprotective effects of both compounds continued with 100 µM in RPTs exposed to antimycin A for 60 min (Fig. 2, A and B). The cytoprotective effects of both compounds are closely correlated with the inhibitory effects on RPT calpains, supporting the hypothesis that calpains play a critical role in the process of RPT cell injury/death.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   The effects of SJA7019 or SJA7029 on antimycin A-induced RPT cell death. RPTs were exposed to antimycin A (AA, 10 µM) or diluent DMSO (CON, <0.25% total volume) for 30 min (A) or 60 min (B). SJA7019 (10, 30, or 100 µM) or SJA7029 (10, 30, or 100 µM) was added 30 min before antimycin A. LDH release was determined 30 or 60 min, respectively, after antimycin A addition. Data are means ± S.E., n = 6 to 12. Bars with different letters are significantly different from one another; P < 0.05.

SJA7019 and SJA7029 Block Antimycin A-Induced Influx of Extracellular Ca²⁺. Loss of Ca²⁺ homeostasis is one mediator of RPT cell death. Increased influx of extracellular Ca²⁺ through a nifedipine-sensitive pathway occurs between 15 and 30 min after antimycin A exposure, and addition of nifedipine 15 min after antimycin A exposure prevented LDH release, suggesting that influx of extracellular Ca²⁺ is a late event during RPT cell injury/death (Waters et al., 1997). Since we suggested that calpains mediate influx of extracellular Ca²⁺ in RPTs exposed to antimycin A (Waters et al., 1997), the effects of SJA7019 (100 µM) and SJA7029 (100 µM) on antimycin A-induced influx of extracellular Ca²⁺ were determined. Antimycin A exposure resulted in an increase in ⁴⁵Ca²⁺ content in RPTs, and SJA7019 or SJA7029 blocked the antimycin A-induced uptake of extracellular Ca²⁺ (Fig. 3). The present data strongly suggest that calpains mediate extracellular Ca²⁺ influx and subsequent cell death.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   The effects of SJA7019 and SJA7029 on antimycin A-induced RPT 45Ca2+ influx. RPTs were exposed to antimycin A (AA, 10 µM) or diluent DMSO (CON, <0.25% total volume) for 30 min. SJA7019 (100 µM) or SJA7029 (100 µM) was added 30 min before antimycin A. 45Ca2+ was added 15 min after antimycin A, and 45Ca2+ content was determined 15 min later. Data are means ± S.E., n = 5 to 6. Bars with different letters are significantly different from one another; P < 0.05.

Time Course of Basal RPT Calpain Inhibition by SJA7209. The time-dependent inhibitory effect of SJA7029 (100 µM) on basal RPT calpains was determined 5, 10, and 30 min after its administration using FITC-casein zymography. SJA7029 inhibited RPT µ- and m-calpain maximally 5 min after its administration, with the inhibitory effect becoming less when the incubation was increased (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Time course of basal RPT µ- (A) and m- (B) calpain inhibition by SJA7029. RPTs were treated with SJA7029 (100 µM) or diluent DMSO (control, <0.1% total volume) for 5, 10, or 30 min. At the corresponding time points, aliquots of RPTs were removed and processed for FITC-casein zymography (20 µg). Results are expressed as percentage of controls. Data are means ± S.E., n = 4. Bars with different letters are significantly different from one another; P < 0.05.

Post-Treatment with SJA7209 Protects RPTs against Antimycin A-Induced Influx of Extracellular Ca²⁺ and Cell Death. Previous work from this laboratory demonstrated that antimycin A exposure results in immediate release of endoplasmic reticulum Ca²⁺ stores and an increased cytosolic free Ca²⁺ ([Ca²⁺]_f) and calpain activity (Harriman et al., 2002). The above experiment demonstrates that calpains mediate influx of extracellular Ca²⁺. However, the relative time window of calpain-mediated influx of extracellular Ca²⁺ is unknown. A post-treatment experiment was performed to approach this question since the above experiment showed that SJA7029 inhibited basal RPT calpain activity as early as 5 min after its administration. SJA7029 (100 µM) was added 10 min after antimycin A exposure, and the uptake of extracellular ⁴⁵Ca²⁺ was determined. Post-treatment with SJA7029 blocked antimycin A-induced uptake of extracellular Ca²⁺ and LDH release (Fig. 5, A and B). The results demonstrate that calpain-mediated influx of extracellular Ca²⁺ and other detrimental intracellular events occur 10 min after antimycin A exposure, downstream of ATP depletion, endoplasmic reticulum Ca²⁺ release, Na⁺ influx, K⁺ efflux, and loss of plasma membrane potential.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   The effect of post-treatment of SJA7029 on antimycin A-induced RPT 45Ca2+ influx (A) and cell death (B). RPTs were exposed to antimycin A (AA, 10 µM) or diluent DMSO (CON, <0.25% total volume) for 30 min. SJA7029 (100 µM) was added 10 min after antimycin A. 45Ca2+ was added 15 min after antimycin A, and 45Ca2+ content was determined 15 min later. LDH release was determined 30 min after antimycin A addition. Data are means ± S.E., n = 4. Bars with different letters are significantly different from one another; P < 0.05.

SJA7029 Allows the Recovery of Mitochondrial Respiration and Active Na⁺ Transport in RPTs Subjected to Hypoxia/Reoxygenation. Although these results demonstrate that calpain inhibitors prevent cell death/lysis produced by multiple insults, the prevention of cell death/lysis may not necessarily reflect true cytoprotection. Therefore, basal and ouabain-sensitive QO₂ were measured as markers of mitochondrial function and active Na⁺ transport in rabbit RPTs after hypoxia/reoxygenation. One hour of hypoxia resulted in extensive cell death, and the reoxygenation period did not increase cell death further (Fig. 6A). Addition of the calpain inhibitor SJA7029 (30 µM) at the onset of hypoxia decreased LDH release following the hypoxia/reoxygenation periods (Fig. 6A). RPTs subjected to hypoxia/reoxygenation displayed impaired mitochondrial function and active Na⁺ transport indicated by decreases in basal QO₂ and ouabain-sensitive QO₂ (Fig. 6, B and C). The presence of SJA7029 improved basal QO₂ and ouabain-sensitive QO₂. These results demonstrate that SJA7029 is a true cytoprotectant.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   The effect of SJA7029 on cell death, basal QO2, or ouabain-sensitive QO2 in RPTs subjected to hypoxia/reoxygenation. RPTs were subjected to 1 h of hypoxia/1 h of reoxygenation. SJA7029 (30 µM) or diluent DMSO (CON, <0.2% total volume) was added immediately before the onset of hypoxia. After 1 h of hypoxia, aliquots of RPTs were removed for LDH release analysis (A, black bars). After 1 h of hypoxia and 1 h of reoxygenation, aliquots of RPTs were removed for LDH release (A, striped bars), basal QO2 (B), or ouabain-sensitive QO2 measurements (C). Basal and ouabain-sensitive QO2 results are expressed as percentage of controls. Data are means ± S.E., n = 6. Bars with different letters are significantly different from one another; P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous work has demonstrated a critical role for increased cytosolic [Ca²⁺]_f and calpain activity in the process of renal cell injury/death (Wilson and Hartz, 1991; Elliget et al., 1991; Kribben et al., 1994; Edelstein et al., 1995, 1996, 1997a,b; Waters et al., 1997; Harriman et al., 2002). Recent studies by Chatterjee et al. (2001) and McDonald et al. (2001) show that calpain inhibitor 1 ameliorates multiple organ failure produced by hemorrhagic shock and renal failure induced by ischemia/reperfusion, indicating that calpains can be important therapeutic targets in the treatment of acute renal failure.

Freshly isolated RPTs have been used as an in vitro model for investigation of acute renal cell injury/death, an integral pathophysiological component of acute renal failure. In a series of studies we have documented that dissimilar calpain inhibitors are cytoprotective against diverse toxic insults that include hypoxia, mitochondrial inhibition, oxidative stress, quinone, and alkylating agent (Schnellmann et al., 1994; Waters et al., 1997; Schnellmann and Waters-Williams, 1998; Harriman et al., 2000; Liu et al., 2001). However, many current calpain inhibitors exhibit lack of selectivity for µ- and m-calpains, low potency, and limited membrane permeability in cellular systems. For example, high concentrations (300 µM to 1 mM) of catalytic site-directed peptide inhibitors were needed to protect RPTs from cell death (Waters et al., 1997; Harriman et al., 2000). At these high concentrations, the nonspecific actions may occur (Mellgren, 1997). Although the Ca²⁺-binding site-directed inhibitor PD150606 is more potent than the peptide inhibitors in RPTs, PD150606 also has the potential of inhibiting other EF-hand-containing proteins (Wang et al., 1996).

Recently, a group of novel nonpeptidyl catalytic site-directed calpain inhibitors including SJA7019 and SJA7029 were synthesized and identified as potent in vitro calpain inhibitors (see Table 1). Using purified calpains, the IC₅₀ values of these two SJA compounds are approximately 0.1 µM, in the IC₅₀ range (0.0057-1.8 µM) for many peptidyl catalytic site-directed calpain inhibitors (Li et al., 1993, 1996; Harriman et al., 2001). The presence of a methoxy group on the phenyl ring of SJA7019 increases its potency approximately 2-fold compared with SJA7029 using purified calpains. Neither SJA compound distinguishes between µ- and m-calpains.

The calpain isozyme-inhibitory properties of SJA7019 and SJA7029 were determined in freshly isolated rabbit RPTs using FITC-casein zymography. The results demonstrate that both compounds equally inhibit RPT µ- and m-calpains with approximate IC₅₀ values of 30 µM and are less potent when compared with purified calpain activity assays, suggesting that limited cell membrane permeability, biotransformation, and/or efflux may occur. However, compared with typical peptidyl catalytic site-directed calpain inhibitors (e.g., calpain inhibitor 1 and 2, and peptidyl -keto amide inhibitors of calpains), the SJA compounds are approximately 10-fold more potent at RPT calpain inhibition (Waters et al., 1997; Harriman et al., 2000).

Another difference between the SJA compounds and peptidyl catalytic site-directed inhibitors of calpain is the rapid onset of action of the SJA compounds. This is an important feature of these two novel calpain inhibitors, since the clinical use of calpain inhibitors would require a rapid onset of action. The rapid onset of action would suggest that the SJA compounds have a fast rate of calpain association and do not exhibit membrane permeability problems. Another feature of the SJA compounds revealed in the present study is that calpain-inhibitory effect decreases as incubation time increases. The exact mechanism for this phenomenon is not clear. However, it may result from a slow rate of inactivation of calpains in RPTs. Consequently, the nonbound SJA compounds would be available for biotransformation or efflux.

The cytoprotective effects of the SJA compounds were determined in freshly isolated rabbit RPTs exposed to the mitochondrial inhibitor antimycin A. The results demonstrate that the SJA compounds are cytoprotective, and the cytoprotective effects correlate closely with their calpain-inhibitory effects, supporting our previous hypothesis that calpains play a critical role in the process of renal cell injury/death. The finding that the SJA compounds rapidly inhibit RPT calpains allowed us to explore some of the calpain-mediated intracellular events in acute renal cell injury/death. In this model, the addition of antimycin A results in the immediate cessation of respiration followed by ATP depletion and endoplasmic reticulum Ca²⁺ release over the next 10 min (Gullans et al., 1982; Harriman et al., 2002). Previous data demonstrated that calpain inhibitors prevented antimycin A-induced influx of extracellular Ca²⁺ and cell death, suggesting that calpains mediate influx of extracellular Ca²⁺ and other detrimental intracellular events leading to cell death (Waters et al., 1997). However, the relative time frame of calpain-mediated influx of extracellular Ca²⁺ and other detrimental intracellular events remained undetermined. SJA7029, added 10 min after antimycin A exposure, prevented influx of extracellular Ca²⁺ and cell death, suggesting that calpain-mediated influx of extracellular Ca²⁺ and other detrimental intracellular events do not immediately follow the sequence of release of ER Ca²⁺ stores/initial rise in cytosolic [Ca²⁺]_f/early calpain activation produced by antimycin A exposure, but that calpain-mediated extracellular Ca²⁺ influx and other detrimental effects occur after a lag period.

The finding that SJA7029 protects against hypoxia-induced cell death and allows the return of RPT cellular functions after injury demonstrates that SJA7029 is a true cytoprotectant. These results have led to the hypothesis that calpains may mediate the mitochondrial dysfunction and impair active Na⁺ transport during acute renal cell injury/death. This hypothesis is under investigation.

In summary, SJA7019 and SJA7029, two novel nonpeptidyl calpain inhibitors, are more potent than peptidyl catalytic site-directed inhibitors in RPT calpain inhibition and cytoprotection. SJA7029 protects RPTs against hypoxia-induced cell death and allows recovery of RPT cellular functions after hypoxic injury. In RPTs exposed to antimycin A, calpains mediate influx of extracellular Ca²⁺ and other detrimental intracellular effects after 10 min of toxicant exposure. These results suggest potential benefits of utilization of calpain inhibitors in the management of acute renal failure.