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NDT Advance Access originally published online on January 25, 2007
Nephrology Dialysis Transplantation 2007 22(4):1052-1061; doi:10.1093/ndt/gfl775
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Proximal tubules from caspase-1-deficient mice are protected against hypoxia-induced membrane injury

Charles L. Edelstein1, Thomas S. Hoke1, Hilary Somerset1, Wenfeng Fang2, Christina L. Klein1, Charles A. Dinarello1 and Sarah Faubel1

1University of Colorado Health Science Center, Department of Internal Medicine, Denver Colorado, USA and 2Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital - Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan

Correspondence and offprint requests to: Sarah Faubel, MD, Biomedical Research Building, Room 512A 4200 East 9th Ave, Box C281 Denver CO, 80262 Email sarah.faubel{at}uchsc.edu



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Caspase-1 is a proinflammatory caspase via activation of the cytokine IL-18. We have recently demonstrated that the caspase-1-mediated production of IL-18 plays a deleterious role in ischaemic acute renal failure (ARF) which is independent of neutrophils and CD4+ T cells. The role of caspase-1 in hypoxia-induced membrane injury of proximal tubules (PT) in vitro is unknown.

Methods. Freshly isolated mouse PT exposed to 25 min of hypoxia were used to study the role of caspases, caspase-1 and IL-18 in hypoxia-induced membrane injury. Lactate dehydrogenase (LDH) release into the PT medium was used as a biochemical parameter of cell membrane damage. IL-18 was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting.

Results. PT pre-incubated with the novel pancaspase inhibitor IDN-8050 were protected; LDH release (%) was 35 ± 3 in vehicle-treated hypoxic PT and 21 ± 2 in IDN-8050-treated hypoxic PT (P < 0.01, n = 6). To investigate the mechanism of protection and examine the role of caspase-1 specifically, PT were isolated in parallel from wild-type and caspase-1- deficient (–/–) mice. PT from caspase-1 –/– mice demonstrated less hypoxia-induced membrane injury. LDH release was 37 ± 2 in wild-type hypoxic PT and 28 ± 2 in caspase-1 –/– hypoxic PT (P < 0.01, n = 12). IL-18 was detected in PT by immunoblotting and ELISA. PT pre-incubated with IL-18 binding protein, an inhibitor of IL-18, were not protected.

Conclusions. These studies demonstrate a deleterious effect of the proinflammatory caspase, caspase-1, on PT in vitro in the absence of inflammatory cells and vascular effects.

Keywords: apoptosis; caspase; tubular cells; renal hypoxia



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Caspases are a family of intracellular cysteine proteases which participate in two distinct signalling pathways: (i) activation of pro-inflammatory cytokines via caspase-1, and (ii) induction of apoptotic cell death, predominantly via caspase-3. Pancaspase inhibition protects against ischaemic acute renal failure (ARF) in vivo [1] as well as hypoxia-induced cell death in proximal tubules in vitro [2,3]. We have previously demonstrated that the caspase-1-mediated production of IL-18 plays a deleterious role in ischaemic ARF in mice [1,4] Caspase-1 –/– mice have a defect in the production of mature IL-1ß and IL-18 [5–8] and are protected against ischaemic ARF [4]. Data suggests that this protection is due to a lack of mature IL-18 [4], rather than a lack of IL-1ß [9].

The cellular sources and targets of caspase-1 and IL-18 in ischaemic ARF are unknown. We have demonstrated that IL-18-mediated renal injury occurs in the absence of neutrophils [1] and that neither neutrophils nor CD4+ T cells [10] are a source of caspase-1 or IL-18 in ischaemic ARF. The proximal tubule (PT) itself may be a source of caspase-1 and IL-18. Although cytokines are generally associated with production by leukocytes, endothelial and epithelial cells are also known to produce cytokines. For example, renal proximal tubule cells in culture produce IL-1ß after exposure to Escherichia coli [11]. Increased IL-18 has been found in the urine of mice and patients with ischaemic acute tubular necrosis (ATN), possibly representing release of IL-18 from proximal tubular cells [4,12,13].

With this background, we hypothesized that PT might be a source of caspase-1 and IL-18, and that caspase-1 and IL-18 contribute to PT injury. In the present study, freshly isolated mouse PT exposed to hypoxia were used to study the direct injurious role of caspases, caspase-1 and IL-18. To study the role of caspases, the novel pancaspase inhibitors, IDN-8050 and OPH-001, were used. Currently, examination of individual caspases in mediating injury is difficult as no caspase inhibitor is specific for only one caspase [14]. Therefore, to specifically study the role of caspase-1, PT were isolated in parallel from wild-type and caspase-1 –/– mice. To study the role of IL-18, IL-18 binding protein (BP) was used. Use of the freshly isolated PT model is appealing as the effects of caspases, caspase-1 and IL-18 on the proximal tubule may be evaluated in the absence of inflammatory cells and vascular effects.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Mice
Adult male C57BL/6 wild-type (Jackson Laboratories) or caspase-1 –/– mice (20–25 g body weight) were used for the isolation of PT. The caspase-1-deficient mice [15], backcrossed ≥8 generations on a C57BL/6 background, were kindly provided by Hiroko Tsutsui of the Department of Immunology & Medical Zoology at Hyogo College of Medicine, Japan. Deficiency of caspase-1 was confirmed by immunoblotting of protein extracts of kidney and spleen for the pro form of caspase-1 (data not shown). All experiments were conducted with adherence to the NIH Guide for the Care and Use of Laboratory Animals. The animal protocol was approved by the Animal Care and Use Committee of the University of Colorado Health Sciences Center.

Preparation of mouse proximal tubules
Proximal tubules were isolated from the kidney cortex using collagenase digestion and Percoll centrifugation as previously described [1,16,17]. We placed 5 ml aliquots of tubule suspension (~1 to 2 mg/ml) were placed in siliconized 25 ml Erlenmeyer flasks for a recovery period, which included gassing with 95% O2/5% CO2 for 5 min on ice. Flasks were then capped with rubber stoppers and kept at room temperature for 5 min, then placed in a shaking water bath at 37°C for 10 min. To create hypoxia, the tubule suspension was gassed for 5 min with 95% N2/5% CO2 at a rate of 3 l/min. After gassing, the flasks were closed and kept in the shaking water bath for the period of hypoxia studied. At the end of the hypoxic period, 1 and 4 ml aliquots of the tubule suspension samples were centrifuged at 3000 g for 1 min. The 1 ml aliquot pellet (containing PT cells) was resuspended in 1.5% Triton; the resuspended pellet and supernatant (medium) were used to determine lactate dehydrogenase (LDH) release. The 4 ml aliquot pellet and medium were used for IL-18 measurements and prepared for immunoblotting.

For all PT experiments, the operator was blinded to treatment groups. For experiments involving caspase-1 –/– mice, PT were isolated in parallel from wild-type and caspase-1 –/– mice as previously described [17], also in a blinded fashion.

After preparation of PT, an aliquot of the suspension was collected for microscopy to determine the purity of the sample for PT; no extra-renal cells such as neutrophils or red blood cells were identified in the PT suspension.

Ischaemia protocol
Ischaemic ARF was induced as previously described [10]. Mice were anesthetized with an intraperitoneal (IP) injection of Avertin (2,2,2-tribromoethanol: Aldrich, Milwaukee, WI). A midline incision was made and the renal pedicles were bilaterally clamped for 22 min with microaneurysm clamps. After 22 min, clamps were removed. Kidneys were observed for restoration of blood flow by the return to their original colour. The abdomen was closed in two layers. At 24 h, kidneys were collected for MPO activity. Kidneys were also collected from normal mice (mice which had not undergone any procedure).

Measurement of LDH release and protein
LDH release was measured to evaluate cell injury, as described previously [18,19]. The percentage of LDH released from tubules was calculated by determining the ratio of LDH in the medium compared with that in the lysed tubule pellet plus the medium. Tubule protein was measured by the Lowry [20] method, using bovine serum albumin as standard.

Preparation of reagents
The pancaspase inhibitor IDN-8050 was kindly provided by Idun Pharmaceuticals, Inc (San Diego, CA, USA). Quinoline-val-asp(Ome)-CH2-OPH (Q-VD-(Ome)-OPH) (OPH-001) was obtained from Enzyme Systems Products (Livermore, CA, USA). IDN-8050 and OPH-001 are irreversible pancaspase inhibitors that have little effect on non-caspase proteases [1,21,22]. 60 µl of a 500 µl stock solution of IDN-8050 or OPH-001 in DMSO was added to the PT medium 10 min prior to hypoxia to achieve a final concentration of 500 µM. Vehicle-treated PT received the same volume of DMSO only (60 µl) for a final concentration of 0.01% DMSO. DMSO is an antioxidant which can protect against hypoxic injury by other models [23], but at higher concentrations (e.g. 0.4%). Our data suggests that this low concentration of DMSO does not protect against hypoxia-induced membrane injury in our model (See ‘Results’ and Figures 2 and 4, subsequently). Recombinant human IL-18BP was a gift provided by Serono pharmaceutical research institute (SPRI, Geneva) [24]. IL-18BP in saline was added to the tubule medium to reach a final concentration of 1.4 µg/ml.


Figure 2
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  		Fig. 2. PT treated with the pancaspase inhibitor IDN-8050 are protected against hypoxia-induced membrane injury. PT were pre-treated with the pancaspase inhibitor IDN-8050 and exposed to 25 min of either normoxia or hypoxia as described in ‘Methods’. LDH release was determined as a marker for cell death (*P < 0.001 vs normoxia, n = 3–6; **P < 0.01 vs hypoxia + vehicle, n = 6).

 

Figure 4
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  		Fig. 4. PT from caspase-1 –/– mice have less hypoxia-induced membrane injury. PT from wild-type (+/+) and caspase-1 –/– mice were isolated in parallel and exposed to 25 min of either normoxia or hypoxia as described in ‘Methods’ (*P < 0.001 vs normoxia +/+ and normoxia caspase-1 –/–, n = 6–12; **P < 0.01 vs hypoxia +/+, n = 12).

 
Nitric oxide (NO) assay
The media from the proximal tubule preparation was filtered using a 30 kDa molecular weight cut-off filter. Total NO was measured using a commercially available kit which converts nitrate to nitrate and then measures total nitrite using the Greiss reaction (Calbiochem, San Diego, CA, USA).

Western blot analysis
PT pellets were homogenized in radioimmunoprecipitation assay (RIPA) buffer and western blotting was performed using standard protocols as previously described in detail [1]. The protein in the pellet and media was concentrated by TCA precipitation and centrifugation followed by cold acetone washes. Protein loading for pellet and media was 100 or 200 µg per lane. Equal loading of protein was performed for all immunoblots. Mouse anti-caspase-1 mAb (BD PharMingen, San Diego, CA, USA; 1:1000), goat anti-IL-18 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100), or human anti-caspase-3 (H-277) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100) were used. Recombinant murine IL-18 (Peprotech, Rocky Hill, NJ, USA) was used as a positive control for the IL-18 immunoblots. Spleens prepared from wild-type and caspase-1 –/– mice were used as positive and negative controls, respectively, for the caspase-1 immunoblots.

IL-18 ELISA
IL-18 was measured in homogenized PT pellets and PT media using a mouse IL-18 enzyme-linked immunosorbent assay (ELISA) kit (Medical and Biological Laboratories, Nagoya, Japan) according to manufacturer's directions. The detection limit for this assay is 25 pg/ml. IL-18 was examined in PT medium on undiluted samples. PT pellets were homogenized in fresh PT medium solution then centrifuged at 20°C for 15 min; the supernatant was collected for IL-18 measurement and protein determination. IL-18 measurements of the pellet were reported as pg/mg pellet protein (the protein content of hypoxic and normoxic PT was similar).

MPO assay
The presence of neutrophils in PT and kidneys were assessed by MPO activity. PT pellets or half a frozen kidney were homogenized in 1 ml of ice-cold HTAB buffer (50 mM KPO4 buffer, 0.5% HTAB, pH 6.0). Samples were then sonicated on ice for 10 s (model VC500; Sonics & Materials Inc., Danbury, CT, USA), transferred to microcentrifuge tubes and centrifuged at 14 000 g for 30 min at 4°C. A 20 µl of supernatant was transferred into a 96-well plate and 200 µl of 37°C O-dianisidine hydrochloride solution (16.7 mg O-dianisidine, 100 ml: 90% water, 10% 50 mM KPO4 buffer + 0.0005% H2O2 added just prior to combining with samples) was added immediately before reading the optical density at 450 nm and again 30 s later (Benchmark microplate reader, BioRad, Hercules, CA, USA). MPO activity was expressed as change in optical density per minute per milligram of protein in the supernatant.

Statistical analyses
Multiple group comparisons were done using analysis of variance (ANOVA) with post-test according to Newman–Keuls. A P-value <0.05 was considered statistically significant. Values are expressed as means ± SEM.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
PT preparation
After isolation, an aliquot of the PT preparation was collected and examined by microscopy for purity; representative images of PT preparations are shown in Figure 1. To further assess the purity of the PT preparation and evaluate for neutrophil contamination, we measured MPO activity, a biochemical marker of neutrophils. Because ischaemia reperfusion injury is known to cause renal neutrophil infiltration, we used kidneys 24 h post-ischaemic reperfusion as a control. MPO activity was determined for ischaemic kidney, normal kidney and proximal tubules. Normal kidney had significantly less MPO activity compared with ischaemic kidney, and MPO activity was virtually absent in the PT preparation as shown in Figure 1. To determine the duration of hypoxia which resulted in hypoxia-induced membrane injury, PT were exposed to normoxia, and 15, 20 and 25 min of hypoxia. As shown in Figure 1, hypoxia-induced membrane injury occurs at 20 and 25 min, but not at 15 min.


Figure 1
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  		Fig. 1. Freshly isolated proximal tubules (PT). PT were isolated as described in ‘Methods’. An aliquot of each PT preparation was analysed by microscopy; images at 10x (A) and 40x (B) demonstrate successful isolation of PT from whole kidneys without the presence of other cell types. Figures are representative images of PT preparations. (C) MPO activity (a biochemical marker for neutrophils) was determined in ischaemic kidneys (n = 6), normal kidneys (n = 10) and PT preparations (n = 10) (*P < 0.001 vs ischaemic kidney; **P < 0.01 vs normal kidney and P < 0.001 vs ischaemic kidney). (D) Increasing time of hypoxia increases cell membrane damage (*P < 0.001 vs normoxia and 15 and 25 min hypoxia **P < 0.001 vs normoxia and 15 min hypoxia and P < 0.01 vs 20 min hypoxia, n = 5–6).

 
PT pre-treated with the pancaspase inhibitor IDN-8050 are protected against hypoxia-induced membrane injury
To examine the role of caspases in hypoxia-induced membrane injury in PT, isolated PT were pre-incubated with the pancaspase inhibitor IDN-8050 for 10 min and then exposed to 25 min of hypoxia. IDN-8050 protected against hypoxia-induced membrane injury (Figure 2). LDH release (%) was 10 ± 2 in normoxic PT, 35 ± 3 in vehicle-treated hypoxic PT and 21 ± 2 in IDN-8050-treated hypoxic PT (P < 0.01 vs vehicle-treated hypoxic PT, n = 6).

Protection against hypoxia-induced membrane injury with IDN-8050 is not dependent on NO
To determine if pancaspase inhibition protected against hypoxia-induced membrane injury via inhibition of NO, we examined NO production. NO production was the same after IDN-8050 and vehicle treatment. NO (pmol/µg) was 22 ± 6 in normoxia, 54 ± 13 in vehicle-treated hypoxia, and 63 ± 2 in IDN-8050-treated hypoxia (P < 0.01 vs. normoxia; P = NS vs vehicle-treated hypoxia, n = 6.)

Caspase-1 is present in PT isolated from wild-type mice and absent in PT isolated from caspase-1 –/– mice
To determine the presence of caspase-1 in PT isolated from wild-type mice and confirm its absence in PT isolated from caspase-1 –/– mice, immunoblotting for caspase-1 was done. As shown in Figure 3A, caspase-1 is present in PT isolated from wild-type mice and absent in PT isolated from caspase-1 –/– mice. Caspase-1 was also examined in hypoxic PT and the pro and active forms were identified (Figure 3B).


Figure 3
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  		Fig. 3. Immunoblots for caspase-1 in PT. Homogenates of PT were prepared for immunoblotting as described in ‘Methods’ and immunoblotted for caspase-1. (A) The immunoblot of PT isolated from wild-type and caspase-1 –/– mice demonstrates that caspase-1 is present in the PT of wild-type mice and absent in PT from the caspase-1 –/– mice. The immunoblot is representative of three separate experiments. (B) The immunoblot of hypoxic PT from wild-type mice demonstrates the presence of the pro (45 kDa) and active (20 kDa) forms of caspase-1. The immunoblot is representative of at least three separate experiments.

 
Proximal tubules isolated from caspase-1 –/– mice have less hypoxia-induced membrane injury
We have previously determined that caspase-1 activity increases in hypoxic PT which is reduced with pancaspase inhibition [3]. Therefore, to investigate a specific caspase involved in the protection seen in the IDN-8050-treated PT, the role of caspase-1 was examined. PT from caspase-1 –/– and wild-type mice were isolated in parallel and exposed to 25 min of either normoxia or hypoxia. Percent LDH release was 8 ± 3 in normoxic wild-type PT and 10 ± 2 in normoxic caspase-1 –/– PT (P = NS); 37 ± 2 in hypoxic wild-type PT (P < 0.001 vs normoxic wild-type PT, n = 6–12); and 28 ± 2 in hypoxic caspase-1 –/– PT (P < 0.01 vs hypoxic wild-type PT, n = 12) (Figure 4). Thus, PT isolated from caspase-1-deficient mice were protected against hypoxia-induced membrane injury.

IL-18 is present in PT as determined by immunoblotting
Because caspase-1 activates the pro form of IL-18 to the mature (active) form, we examined the PT media and pellets for the pro and mature forms of IL-18. Immunoblots demonstrated the presence of both pro (24 kDa) and mature IL-18 (18 kDa) in normoxic and hypoxic PT (Figure 5). To determine if pancaspase inhibition would affect IL-18 content, IL-18 was examined in the hypoxic PT cells treated with pancaspase inhibition. As shown in Figure 5B, IL-18 was not decreased in the hypoxic PT with pancaspase inhibition.


Figure 5
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  		Fig. 5. Immunoblots for IL-18. Immunoblots of normoxic and hypoxic wild-type PT were performed. (A) The immunoblot for the pro (24 kDa) and mature (18 kDa) forms of IL-18 demonstrates the presence of IL-18 in normoxic and hypoxic PT. (B) The immunoblot demonstrates that the mature form of IL-18 is the same in PT with normoxia, hypoxia plus pancaspase inhibition with OPH-001, and hypoxia. (C) IL-18 was present in the medium of hypoxic PT, and reduced in the medium with hypoxia plus pancaspase inhibition with OPH-001.

 
To determine if hypoxia affected IL-18 release, IL-18 was examined in the media of normoxic and hypoxic PT by immunoblotting. As shown in Figure 5C, IL-18 was detected in the hypoxic media, which was not detected with pancaspase inhibition.

The concentration of IL-18 is reduced in hypoxic PT and increased in the hypoxic PT medium
ELISA was used to quantify the amount of the IL-18 in the PT (Figure 6). IL-18 (pg/mg PT protein) was 20 ± 1 in normoxic PT and was 16 ± 1 in hypoxic PT (P < 0.01 vs normoxic PT, n = 6). The reduction in IL-18 in the hypoxic PT suggested that IL-18 was being released into the medium. Therefore, IL-18 in the medium was determined. IL-18 (pg/mg PT protein) was 2 ± 0 in normoxic medium and 7 ± 2 in hypoxic medium (P < 0.05, n = 6–9) confirming IL-18 release during hypoxia. Thus, isolated PT contain IL-18 and release IL-18 into the media under hypoxic conditions.


Figure 6
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  		Fig. 6. PT contain IL-18 which is released under hypoxic conditions. IL-18 was determined using ELISA as described in ‘Methods’. IL-18 was higher in the normoxic PT compared with hypoxic PT (*P < 0.05 vs PT normoxia, n = 6). IL-18 in the hypoxic PT medium was increased compared with normoxic PT medium (*P < 0.05 vs PT medium normoxia, n = 6–9).

 
To determine if pancaspase inhibition affected the release of IL-18 from hypoxic PT, IL-18 was examined by ELISA in the media of hypoxic PT pre-treated with pancaspase inhibition; IL-18 was not detected in the media of hypoxic PT treated with pancaspase inhibition (n = 3). To determine if IL-18 content and release was affected by caspase-1 deficiency, IL-18 was determined in the PT and media from caspase-1 –/– PT; IL-18 by ELISA (pg/mg) was 5 ± 4 in normoxic caspase-1 –/– PT and 3 ± 1 in hypoxic caspase-1 –/– PT (P = NS, n = 5–10) and was not detected in the media of either normoxic or hypoxic caspase-1 –/– PT (n = 3).

IL-18BP in hypoxia-induced membrane injury
To determine if the mechanism of protection in the caspase-1 –/– PT was due to a lack of IL-18, wild-type PT were incubated with IL-18BP (1.4 µg/ml) or vehicle (saline) and exposed to 25 min of hypoxia. IL-18 BP is a naturally occurring inhibitor of IL-18. LDH release (%) was 9 ± 1 in normoxic PT (n = 6), 45 ± 2 in vehicle-treated hypoxic PT (n = 12) (P < 0.001 vs normoxic PT); and 41 ± 2 in IL-18BP-treated hypoxic PT (n = 12) (P = NS vs vehicle-treated hypoxic PT) (Figure 7). IL-18BP was also added to PT isolated from caspase-1 –/– PT. LDH release (%) was 9 ± 1 in normoxic PT (n = 2), and 39 ± 4 in vehicle-treated hypoxic wild-type PT (n = 3) (P < 0.001 vs normoxic PT) and 39 ± 4 in IL-18BP-treated hypoxic caspase-1 –/– PT (n = 3) (P = NS vs vehicle-treated hypoxic PT). Therefore, IL-18BP did not affect hypoxia-induced membrane injury in PT isolated from wild-type or caspase-1 –/– mice.


Figure 7
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  		Fig. 7. IL-18BP in hypoxia-induced membrane injury. PT from wild-type (WT) mice were incubated with IL-18 binding protein (BP) (1.4 µg/ml) or vehicle (saline) and exposed to 25 min of hypoxia. *P < 0.001 vs WT normoxic PT and NS vs WT hypoxic PT + IL-18BP, n = 12).

 
Active caspase-3 is reduced with pancaspase inhibition
We have previously demonstrated that pancaspase inhibition with OPH-001 protects against hypoxia-induced membrane injury to a similar degree as IDN-8050 [1]. To determine if other caspases might play a role in the protection against hypoxia-induced membrane injury with pancaspase inhibition, immunoblotting for caspase-3 was performed. Caspase-3 was determined in hypoxic PT and hypoxic PT pre-treated with the pancaspase inhibitor OPH-001. The 20 kDa active form of caspase-3 was reduced in the PT treated with pancaspase inhibitor (Figure 8).


Figure 8
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  		Fig. 8. Immunoblot for active caspase-3. Immunoblotting for caspase-3 was performed in hypoxic PT and hypoxic PT pre-treated with the pancaspase inhibitor OPH-001. The 20 kDa active form of caspase-3 was reduced in the PT treated with a pancaspase inhibitor. The immunoblot is representative of at least three separate experiments.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we have demonstrated that caspases in general and, caspase-1 in particular, contribute to hypoxia-induced proximal tubule membrane injury in vitro. PT treated with the pancaspase inhibitor IDN-8050 had a 40% reduction in LDH release. PT cells contain caspase-1 and PT isolated from caspase-1 –/– mice had a 24% reduction in LDH release. PT cells contain IL-18 and data suggest that IL-18 is released from PT during hypoxia, however, wild-type PT pre-incubated with IL-18BP were not protected against hypoxia-induced membrane injury. These data are relevant in regard to the pathogenesis of proximal tubular injury in ischaemic ARF in vivo and are the first demonstration of a direct role of caspase-1 in mediating hypoxia-induced membrane injury of PT in vitro.

Caspases are a family of intracellular cysteine proteases which are typically divided into two categories based on their primary functions: pro-inflammatory and pro-apoptotic [25]. Currently, 14 members of the caspase family have been identified, caspases 1–14. The role of caspases in mediating ischaemic injury in vivo is well established. The protective effect of pancaspase inhibitors has been demonstrated in models of cerebral [26,29], cardiac [30] and renal [31] ischaemia. In these models, tissue protection is associated with a reduction in necrosis, apoptosis and inflammation.

In the present study, we examined the effect of pancaspase inhibition on hypoxia-induced injury of freshly isolated mouse PT. Our results using the novel pancaspase inhibitor, IDN-8050, corroborate previous reports demonstrating a protective effect of pancaspase inhibition in hypoxic PT injury [2,3]. In addition to caspases, PLA2 [32], NO [33–35], and calpain [36] have been shown to mediate hypoxia-induced membrane injury in PT. To determine if pancaspase inhibition might have an effect on one of these known mediators of hypoxia-induced membrane injury, we examined NO production and found that NO was not affected by pancaspase inhibition.

The pancaspase inhibitors used in the present study are specific for caspases and do not cross-react with other cysteine proteases such as calpain [1,21,22]. To evaluate the mechanism of protection against hypoxia-induced membrane injury regarding caspases, we investigated the role of one specific caspase, caspase-1. We have previously demonstrated that caspase-1 activity increases in hypoxia-induced membrane injury of PT which is reduced by pancaspase inhibition [3].

Caspase-1 is a proinflammatory caspase which converts the pro forms of IL-1ß and IL-18 to their mature (active) forms. The pathogenic role of caspase-1 and IL-18 as mediators of ischaemic injury in in vivo models of heart, brain and kidney injury is well described [4,37–44]. The mechanism of protection in many of these studies was hypothesized to be due to reduced neutrophil recruitment to the ischaemic tissue. Evidence suggests, however, that caspase-1 may mediate cell death independent of its proinflammatory and neutrophil recruitment effects. For example, a direct role of caspase-1 in mediating cell death was determined in an in vitro model of hypoxia-induced neuronal cell death using cells derived from caspase-1 –/– mice [15]. A role of caspase-1 was also identified in an in vitro model of cardiac ischaemia [42] which examined cardiac contractile force and cell viability using an ischaemia reperfusion model of human atrial myocardium [42]. The specific role of caspase-1 in hypoxia-induced membrane injury of PT has not been previously examined.

We examined the role of caspase-1 using PT isolated from caspase-1 –/– mice. The advantage of using knockout mice in this model is that the role of one specific caspase may be examined. It is known, for example that the ‘specific’ caspase-1 inhibitors with the YVAD substrate may also inhibit the function of caspases –4, –5, –12 and –13 [46–48]; these caspases as well as caspase-1 are members of the group I caspases which have the common function of cytokine processing leading to inflammation and a preference for the substrates YVAD. We found that caspase-1 was present in isolated PT from wild-type mice and that PT isolated from caspase-1 –/– mice had less hypoxia-induced membrane injury as indicated by a 24% reduction in LDH release. Because inflammatory cells are absent in this model, these results suggest a direct role of caspase-1 in mediating hypoxia induced cell death of PT.

To investigate the mechanism of protection seen in PT isolated from caspase-1 –/– mice, we examined IL-18. Caspase-1 converts the pro form of IL-18 to the active form which then exits the cell, thus, caspase-1 –/– mice lack the ability to form mature IL-18 [5–8]. We were able to determine that wild-type PT contain IL-18 by both immunoblotting and ELISA. The presence of IL-18 can be attributed to PT cells as the purity of the preparation was examined by microscopy (no extra-renal cells were observed) and with an MPO assay which showed virtually no neutrophil activity. A final assurance that neutrophils were not responsible for the identification of IL-18 comes from the fact that IL-18 levels in normal polymorphonuclear cells are about 1.2 x 10–5 pg/cell [49]. For IL-18 to be affected by neutrophil contamination, neutrophils would have had to exist in large quantities easily identifiable under the microscope. We were surprised, however, to find that the amount of pro and active form, as identified by immunoblotting, was similar for normoxic and hypoxic PT, as well as hypoxic PT treated with pancaspase inhibition. These data suggest that both pro and active forms of IL-18 are present in isolated proximal tubules.

Using quantitative ELISA we determined that the amount of IL-18 contained in the hypoxic PT was significantly less than in normoxic PT. The reduction of IL-18 in hypoxic PT suggested that IL-18 was being released from the hypoxic PT cells which was confirmed by the detection of IL-18 in the media. To test whether IL-18 was being released from hypoxic PT and contributing to membrane injury, PT were pre-incubated with IL-18BP. Hypoxic PT pre-treated with IL-18BP were not protected against hypoxia-induced membrane injury. IL-18BP is a soluble decoy receptor that inhibits the biological functions of IL-18 in both in vitro and in vitro models of injury [50]. It does not enter the cell but binds to IL-18 preventing it from acting at its receptor on the surface of cells. IL-18BP binds IL-18 with a high affinity and at equimolar ratios inhibits 50–70% of IL-18 [51]. Thus, the dose used in our study was more than sufficient to effectively inhibit the action of IL-18. The lack of protection with IL-18BP suggests that caspase-1 may mediate hypoxia-induced membrane injury of PT independent of IL-18 production. Caspase-1 also activates the pro to the active form of IL-1ß. IL-1ß has been previously examined in hypoxia-induced membrane injury of PT and been found not to play a role [3].

It is important to note that the protection seen in the caspase-1 –/– mice (24% reduction in LDH release), was less than that seen with pancaspase inhibition (40% reduction in LDH release) indicating that other caspases must be important in hypoxia-induced membrane injury of PT. To evaluate the potential role of other caspases, we examined the effect of pancaspase inhibition on caspase-3 activation. Caspase-3, known as the ‘executioner’ caspase, is the major pro-apoptotic caspase and is activated by ‘initiator’ caspases: caspase-8 and caspase-9 [52]. We found that the active form of caspase-3 was reduced in hypoxic PT treated with pancaspase inhibition. This is consistent with our in vitro and in vivo observations regarding the protective effect of pancaspase inhibition in hypoxia-induced membrane injury of PT and ischaemic ARF, respectively [1]. In ischaemic ARF, pancaspase inhibition was associated with a reduction in renal caspase-3 which was associated with a reduction in renal necrosis.

Our data demonstrate that the PT is a potential source of caspase-1 and IL-18 in ischaemic ARF, but it should be recognized that, in vivo, other cell types e.g. endothelial cells and macrophages may also contribute to IL-18 production and that other mechanisms of injury are likely responsible for caspase-1 and IL-18-mediated injury during ischaemic ARF. The observation that PT contain IL-18 has particular relevance in vivo as the value of IL-18 as a urinary biomarker of acute kidney injury in patients is currently being investigated. We have demonstrated that urine IL-18 is increased in patients with acute tubular necrosis (ATN) and delayed graft function compared to other renal diseases such as nephrotic syndrome, urinary tract infection and pre-renal ARF [12]. In addition, urine IL-18 precedes the increase in serum creatinine in critically ill patients with acute respiratory distress syndrome [13]. In light of the data in the present study, it appears that the proximal tubule may be one of the sources of increased urine IL-18 in ischaemic ARF and thus may be a specific marker of acute kidney injury.

It is important to recognize that freshly isolated PT exposed to hypoxia represents a model of necrotic, rather than apoptotic, cell death [3]. In a previous study, PT were stained with the DNA-specific dyes Hoechst 33342 and propidium iodide (PI); the hypoxic tubules displayed extensive necrosis characterized by loss of membrane integrity and PI staining and apoptotic bodies were not identified [3]. Because caspases are generally regarded as either pro-apoptotic or proinflammatory, our results regarding the role of caspases and caspase-1 in particular in mediating necrotic PT cell death are particularly notable. Necrotic cell death is a response that appears to occur rapidly, as membrane injury occurs after only 20 min of hypoxia.

The freshly isolated PT model is preferred over other in vitro methods of studying the effects of hypoxia on PT for several reasons: (i) the biochemical properties of the in vivo state are maintained, (ii) structural integrity is preserved, (iii) sensitivity to hypoxia is preserved and (iv) polarization and differentiation is maintained [53]. In contrast, cultured PT cells are less sensitive to oxygen deprivation because of a change from oxidative metabolism to glycolysis [54]. In addition, cultured cells develop significant structural changes and simplification [55]. For these reasons, our results have particular relevance in vivo and suggest that caspases, and caspase-1 in particular, may mediate necrotic cell death in ischaemic ARF.

In summary, (i) pancaspase inhibition protects against hypoxia-induced membrane injury of PT, (ii) mouse PT contain caspase-1 and IL-18 and (iii) lack of caspase-1 attenuates hypoxia-induced PT injury. The results regarding caspase-1 are remarkable in that they identify a role of a proinflammatory caspase in hypoxia-induced PT membrane injury in vitro, in the absence of inflammatory cells and vascular effects.



   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by NIH grants 1 K08 DK65022-03 to S.F. and 1 R01 DK56851 to C.L.E.

Conflicts of interest statement. None Declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 10.12.05
Accepted in revised form: 29.11.06


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