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(Circulation Research. 2003;92:293.)
© 2003 American Heart Association, Inc.

Cellular Biology

Liposomal Delivery of Heat Shock Protein 72 Into Renal Tubular Cells Blocks Nuclear Factor-B Activation, Tumor Necrosis Factor- Production, and Subsequent Ischemia-Induced Apoptosis

Kirstan K. Meldrum, Arthur L. Burnett, Xianzhong Meng, Rosalia Misseri, Matthew B.K. Shaw, John P. Gearhart, Daniel R. Meldrum

From the Departments of Urology and Surgery (K.K.M., R.M., M.B.K.S., D.R.M.), Indiana University Medical Center, Indianapolis, Ind; Johns Hopkins Hospital (A.L.B., J.P.G.), Baltimore, Md; and the Department of Surgery (X.M.), University of Colorado Health Sciences Center, Denver, Colo.

Correspondence to Daniel R. Meldrum, MD, Dept of Surgery, Cardiothoracic Surgery, Indiana University Medical Center, 545 Barnhill Dr, Emerson 215, Indianapolis, IN 46202. E-mail dmeldrum{at}iupui.edu

	Abstract

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Heat shock protein 72 (HSP72) is a stress-inducible protein capable of protecting a variety of cells from toxins, thermal stress, and ischemic injury. The cytoprotective role and mechanism of action of HSP72 in renal cell ischemic injury remain unclear. To study this, HSP72 was introduced (liposomal transfer) or induced (thermal stress, 43°Cx1 hour) in renal tubular cells (LLC-PK1) with Western blot confirmation. Cells were subjected to simulated ischemia 24 hours after liposomal HSP72 transfer or thermal stress, and the effect of HSP72 on nuclear factor-B (NF-B) activation (electrophoretic mobility shift assay and immunohistochemistry), IB production (Western blot), postischemic tumor necrosis factor- (TNF-) production (RT-PCR), and apoptosis (TUNEL assay) were determined. In separate experiments, the role of TNF- in apoptosis was determined (anti-TNF- neutralizing antibody). Results demonstrated that both liposomal transfer of HSP72 and thermal induction of HSP72 prevented NF-B activation and translocation, TNF- gene transcription, and subsequent ischemia-induced renal tubular cell apoptosis. Furthermore, TNF- neutralization also inhibited ischemia-induced renal tubular cell apoptosis. These results indicate that liposomal delivery of HSP72 inhibits ischemia-induced renal tubular cell apoptosis by preventing NF-B activation and subsequent TNF- production. Further elucidation of the mechanisms of HSP-induced cytoprotection may result in therapeutic strategies that limit or prevent ischemia-induced renal damage.

Key Words: inflammation • reperfusion • kidney

	Introduction

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Ischemia-induced acute renal failure (ARF) remains a significant cause of patient morbidity and mortality.¹ After renal ischemia, lethally injured tubular cells expire by either an apoptotic or necrotic pathway.^2–4 The potential causes of apoptosis during ischemia-induced ARF are multiple and include deficiencies in renal growth factors, loss of cell-cell interactions, and receptor-mediated cytotoxicity (ie, tumor necrosis factor [TNF-]).^5,6 Indeed, recent evidence suggests that TNF- is not only an important mediator of renal ischemic injury,⁷ it is capable of inducing apoptosis in renal tubular cells (LLC-PK1).⁸

Proinflammatory cytokine gene transcription is regulated in part by nuclear factor-B (NF-B) activation. In most cells, NF-B is sequestered in the cytoplasm in an inactive state due to its association with a class of inhibitory proteins termed inhibitory B (IB). During cellular oxidant stress (ie, ischemia, endotoxin), phosphorylation of IB ensues, resulting in rapid degradation of IB and disruption of the NF-B-IB complex.^9,11 NF-B subsequently translocates from the cytoplasm to the nucleus where it binds to B promotor sites and transactivates downstream proinflammatory genes, such as TNF-.¹²

In the kidney, HSP72 has been demonstrated to protect renal tubular cells against heat stress and toxins^12,13 and recently to prevent ischemia-induced apoptosis.^3,15,16 The mechanisms by which heat shock and induction of HSP72 exert a cytoprotective effect in the kidney are not entirely understood. The proapoptotic properties of TNF- and its role in renal ischemia-reperfusion (IR) injury suggest that heat shock-induced ischemic cytoprotection involves HSP-mediated inhibition of NF-B activation and TNF- gene transcription. Indeed, in nonrenal cells, the heat shock response and HSP70 have been demonstrated to inhibit cytokine-mediated NF-B activation and inflammatory mediator production.^17–21 By using an in vitro model of renal tubular cell ischemia, the purposes of the this study were to determine (1) the quantification of heat shock induction versus liposomal delivery of HSP72, (2) the effect of HSP72 on ischemia-induced NF-B activation and IB production, (3) the effect of HSP72 on ischemia-induced TNF- mRNA production, (4) the effect of HSP72 on ischemia-induced apoptosis, and (5) the role of TNF- in ischemia-induced apoptosis.

	Materials and Methods

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Cell Culture
The renal tubular epithelial cell line LLC-PK1 was cultured in Medium 199 (LTI, Gaithersburg, Md) supplemented with 10% fetal bovine serum (FBS). The cells were passaged weekly by trypsinization (0.25% trypsin, 0.02% EDTA) after formation of a confluent monolayer and were placed in serum-free media (1% FBS) 24 hours before stimulation.

Simulated Ischemia
After formation of a confluent epithelial monolayer, the cells were washed twice with PBS and the monolayer was immersed in mineral oil, simulating ischemic conditions by restricting cell exposure to oxygen and nutrients and preventing metabolite washout.^2,22

Thermal Induction of HSP72
After formation of a confluent epithelial sheet, the cells were immersed in a 43°C water bath for 1 hour. Twenty-four hours after thermal stress, the cells were either collected for Western blot analysis or exposed to 1 hour of simulated ischemia and 24 hours of substrate replacement (reimmersion in media).

Liposomal Delivery of HSP72
Recombinant human HSP72 was introduced into renal tubular epithelial cells by liposomal delivery. The liposomal suspension was prepared with HSP72 or vehicle²³ and added to the cell culture media (10 µg/mL) 24 hours before simulated ischemia.

Western Blotting
Western blot analysis was performed on control samples, samples exposed to thermal stress, and samples exposed to the liposomal solution (prepared with either HSP72 or vehicle). Each 100-mm confluent plate of cells was washed twice with PBS, scraped, and centrifuged. The supernatant was removed and the cell pellet was lysed using a 2% SDS solution. The protein extracts (200 µg/lane, HSP72; 20 µg/lane, Iß) were electrophoresed on a 10% SDS-polyacrylamide gel (HSP72) or 4% to 20% SDS gradient gel (IB) and transferred to a nitrocellulose membrane. Immunoblotting for HSP72 was performed as previously described.³ For IB immunoblotting, the membrane was incubated in 5% dry milk for 1 hour followed by incubation with a polyclonal anti-IB antibody (1:200; Santa Cruz) for 2 hours. The membrane was then washed 2 times for 5 minutes in PBS, incubated with a peroxidase-conjugated secondary antibody (1:10 000) for 2 hours, and again washed 2 times for 5 minutes in PBS. The membranes were developed using enhanced chemiluminescence (Amersham Pharmacia Biotech Inc).

Nuclear Protein Extract
Nuclear extracts were prepared from cell cultures exposed to media alone, media supplemented with 100 µL of mineral oil, media supplemented with empty liposomes, 10 minutes of simulated ischemia alone, or 10 minutes of simulated ischemia in the presence of heat shock, empty liposomes, or liposomal HSP72. Cellular monolayers were washed twice with PBS and subsequently immersed in 800 µL of lysis buffer (10 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 0.1 mmol/L EGTA, 0.1 mmol/L EDTA, 1 mmol/L DTT, and 0.5 mmol/L PMSF) for 15 minutes. Fifty microliters of NP-40 was then added to each monolayer to induce cellular lysis. Cell lysates were collected, vortexed, and centrifuged for 30 seconds at 12 000g. The nuclear extract for each sample was then prepared.⁹

Electrophoretic Mobility Shift Assay for NF-B Activation
The electrophoretic mobility shift assay (EMSA) was performed as described by Nakshatri et al.¹⁰ For supershift studies, 1 µL of a polyclonal antibody to p65 NF-B (Upstate Biotechnology) was added to the DNA binding reaction cocktail and incubated for 10 minutes. Binding of the antibody to the appropriate transcription factor was indicated by a supershift in the EMSA.

Immunolocalization of NF-B
Cells were plated in 4-well chamber slides at 1.5x10⁴ cells per well and grown in 1 mL of Medium 199 supplemented with 10% FBS until a confluent monolayer was obtained. The cells were exposed to media alone, 10 minutes of simulated ischemia alone, or 10 minutes of simulated ischemia in the presence of heat shock or liposomal HSP72. After stimulation, the cells were fixed in a cold mixture (1:1) of methanol and acetone for 10 minutes and subsequently a 4% paraformaldehyde solution for 15 minutes. The slides were washed twice in PBS and incubated in 10% goat serum for 40 minutes at room temperature. After washing once in PBS, the slides were incubated with rabbit anti-human p65 (1:100; Chemicon) for 90 minutes at 37°C. The slides were then washed with PBS 3 times for 5 minutes each time and incubated with Cy-3-conjugated goat anti-rabbit IgG (1:50; Zymed) for 60 minutes at 37°C. After washing again with PBS 3 times for 5 minutes each time, the cell nuclei were counterstained with bis-benzimide (10 µg/mL) for 30 seconds.

TNF- RT-PCR
Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess TNF- gene expression. The cells were seeded at 2.0x10⁵ on 100-mm culture plates and grown to confluence. The cells were exposed to media alone, media supplemented with 100 µL of mineral oil, media supplemented with empty liposomes, 1 hour of simulated ischemia alone, or 1 hour of simulated ischemia in the presence of heat shock, empty liposomes, or liposomal HSP72. Total RNA was extracted from each culture dish of cells (3 per time point) using Trizol (Gibco BRL) after washing the cells twice with PBS. The RNA was then isolated by precipitation with chloroform and isopropanol. Two micrograms of the isolated RNA was subjected to RT-PCR.² The amplified products were separated on a 2% agarose gel and stained with ethidium bromide.²

Quantitative TNF- PCR
cDNA from each sample was used to detect real-time RT-PCR products for TNF- using SYBR Green and ABI PRISM 7700 sequence detection system (PE Applied Biosystems). The PCR cycling conditions were performed for all of the samples as follows: 3 minutes at 95°C for AmpliTaq Gold activation and 40 cycles for the melting (95°C, 15 seconds) and annealing/extension (60°C, 30 seconds) steps. TNF- and GAPDH primers for quantitative RT-PCR were designed using the PRIMER express program (PE Applied Biosystems). All QRT-PCR experiments were performed twice in duplicates in one 96-well plate for TNF- and GAPDH. By using the comparative CT method (PE Applied Biosystems), resulting CT values were evaluated and normalized with GAPDH. This value was then averaged for each duplicate.

Quantification of Apoptosis
Cells were plated in 4-well chamber slides at 1.5x10⁴ cells per well and grown in 1 mL of Medium 199 supplemented with 10% FBS until a confluent monolayer was obtained. The cells were exposed to media alone, media supplemented with 100 µL of mineral oil, media supplemented with empty liposomes, 1 hour of simulated ischemia and 24 hours of substrate replacement alone, or 1 hour of simulated ischemia and 24 hours of substrate replacement in the presence of heat shock, liposomal HSP72, or TNF- neutralization (2 µg/mL of goat anti-porcine TNF- antibody [R&D Systems; concentration based on neutralization dose [ND₅₀] for this antibody as defined by the manufacturer). Four treatment chambers in each experimental group were quantified for apoptosis using a kit from Boehringer Mannheim. The cell nuclei in each well were then counterstained with bis-benzimide (10 µg/mL) for 30 seconds to ensure a constant cell density across all treatment groups examined. The number of apoptotic nuclei were counted in 3 nonoverlapping x320 microscope fields per well and averaged. Furthermore, the characteristic morphological features of apoptosis (ie, nuclear condensation) were observed among cells in each well examined. The entire protocol was then repeated in triplicate.

Cell Viability
Cells were seeded at 5.0x10⁵ cells per 60-mm culture plate and grown in 3 mL of media until a confluent monolayer was obtained. By using the treatment groups described above, a cell suspension in PBS was prepared from each culture dish. Trypan Blue (0.4%) was added to each cell suspension and the mixture was allowed to stand at room temperature for 5 minutes. The number of viable cells was counted in each sample and expressed as a percentage of the total cell count.

Statistical Analysis
Data are presented as mean±SEM. Differences at the 95% confidence level were considered significant. The experimental groups were compared using ANOVA with post hoc Bonferroni-Dunn (StatView 4.5, Berkeley, Calif).

	Results

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HSP72 Expression
HSP72 induction by thermal stress and introduction by liposomal transfer was confirmed by Western blot analysis. Control samples expressed low levels of HSP72, whereas samples exposed to thermal stress or liposomal delivery of HSP72 demonstrated a marked increase in HSP72 expression (Figure 1).

View larger version (15K): [in this window] [in a new window]   Figure 1. Western blot demonstrating increased expression of HSP72 in cells exposed to thermal stress (43°Cx1 hour [3]) or liposomal HSP72 (4) in comparison to controls (untreated cells [1]; cells exposed to empty liposome [2]).

Effect of HSP72 on Ischemia-Induced NF-B Activation
Simulated ischemia-induced NF-B activation was assessed by both EMSA and NF-B subunit p65 subcellular immunolocalization in the presence or absence of heat shock or liposomal HSP72 delivery. The EMSA demonstrated little NF-B activation in control samples; however, significant NF-B binding and activation (p65 supershift) was evident after 10 minutes of simulated ischemia (Figure 2). Cellular pretreatment with either heat shock or liposomal HSP72 prevented the observed increase in simulated ischemia-induced NF-B activation.

View larger version (40K): [in this window] [in a new window]   Figure 2. Gel photographs depicting NF-B DNA binding after 10 minutes of simulated ischemia, in the presence or absence of heat shock or liposomal HSP72 delivery. NF-B DNA binding (gel 1) was not detected in samples containing radiolabeled probe alone (1), competitor (cold) oligonucleotide (2), untreated cells (3), cells exposed to media supplemented with mineral oil (4), or cells exposed to media supplemented with empty liposomes (5). In contrast, 10 minutes of simulated ischemia in the presence (8) or absence (6) of empty liposomes induced significant NF-B DNA binding (gel 1) and activation (gel 2, p65 supershift band). Cellular pretreatment with heat shock (7) or liposomal HSP72 (9) prevented the observed increase in ischemia-induced NF-B DNA binding.

Subcellular immunolocalization of the p65 subunit of NF-B demonstrated a cytoplasmic distribution in control samples (Figure 3). After 10 minutes of simulated ischemia, nuclear translocation of NF-B became evident. Cellular pretreatment with either heat shock or liposomal HSP72 prevented simulated ischemia-induced nuclear translocation of NF-B. These results indicate that ischemia-induced NF-B activation is prevented by heat shock or liposomal HSP72 pretreatment.

View larger version (92K): [in this window] [in a new window]   Figure 3. Photographs (magnification x320) demonstrating the subcellular localization of NF-B (p65 subunit) and the effects of heat shock and liposomally delivered HSP72 on 10 minutes of simulated ischemia. A, Untreated cells. Nuclear stain (blue) and Cy-3-conjugated anti-p65 (red) demonstrate a cytoplasmic distribution of NF-B. B, Cells exposed to 10 minutes of simulated ischemia. NF-B demonstrates a nuclear redistribution in response to ischemia. C, Cells exposed to 10 minutes of ischemia after heat preconditioning. Heat shock prevents ischemia-induced nuclear translocation. D, Cells exposed to 10 minutes of ischemia after liposomal delivery of HSP72. HSP72 prevents ischemia-induced nuclear translocation.

Effect of HSP72 on IB Production
The expression of IB after cellular ischemia with or without heat shock or liposomal HSP72 delivery was evaluated with Western blot analysis (Figure 4). IB degradation was evident in cells exposed to simulated ischemia compared with control samples. Liposomal delivery of HSP72 did not prevent the observed degradation in IB after simulated ischemia.

View larger version (10K): [in this window] [in a new window]   Figure 4. Western blot demonstrating degradation of IB in cells exposed to simulated ischemia (4) compared with control samples (1 through 3). Liposomal delivery of HSP72 did not prevent the observed degradation in IB after simulated ischemia (5).

Effect of HSP72 on Ischemia-Induced TNF- mRNA Expression
Simulated ischemia-induced TNF- mRNA expression was assessed in the presence or absence of heat shock or liposomal HSP72 delivery. Control samples demonstrated little TNF- mRNA induction whereas TNF- expression increased in response to simulated ischemia (Figures 5A and 5B). Prior cellular exposure to heat shock or liposomal HSP72 prevented the observed increase in ischemia-induced TNF- mRNA production. Quantitative PCR analysis of TNF- mRNA expression as a percentage of GAPDH mRNA is shown in Figure 5B. In samples exposed to media alone, media supplemented with mineral oil, and media supplemented with empty liposomes, TNF- mRNA expression represented 5.4±0.2%, 22±3%, and 1.3±0.3% of GAPDH mRNA, respectively. Cells exposed to simulated ischemia in the presence or absence of empty liposomes demonstrated a significant increase in TNF- expression (8083±833% [P<0.05 versus untreated cells] and 5041±2125% of GAPDH mRNA, respectively, whereas cells exposed to heat shock or liposomal HSP72 before simulated ischemia demonstrated a significant reduction in TNF- mRNA expression [17±6.5% and 1999±84% of GAPDH, respectively, P<0.05 versus ischemic cells]).

View larger version (34K): [in this window] [in a new window]   Figure 5. A, Gel photograph depicting TNF- and GAPDH mRNA bands in various treatment groups. B, Quantitative PCR analysis of TNF-/GAPDH mRNA expression in the various treatment groups. Cells exposed to media alone (untreated), media supplemented with mineral oil, or media supplemented with empty liposomes did not demonstrate an increase in TNF- mRNA expression. In contrast, 1 hour of ischemia alone stimulated increased TNF- mRNA production. This response was ameliorated by prior cellular exposure to either heat stress or liposomal HSP72.

Quantification of Apoptosis in Response to Simulated Ischemia
Cells exposed to media alone, media supplemented with mineral oil, or media supplemented with empty liposomes did not undergo apoptosis (1.1±0.49, 0.4±0.3, and 2.3±1.4 apoptotic nuclei/high-powered field [hpf], respectively). In contrast, simulated ischemia induced a significant degree of apoptosis (72±6.1 apoptotic nuclei/hpf, P<0.05 versus untreated cells, Figures 6 and 7). Cells exposed to media alone, media supplemented with mineral oil, and media supplemented with empty liposomes demonstrated 90±2.1%, 84±2.8%, and 83±3.2% viability respectively, whereas cells exposed to simulated ischemia demonstrated 81±5.4% viability (not significantly different from controls).

View larger version (15K): [in this window] [in a new window]   Figure 6. Graph depicting the number of apoptotic nuclei per high-powered field (x320) in each treatment group. Thermal preconditioning, liposomal delivery of HSP72, and TNF- neutralization all prevented ischemia-induced apoptosis.

View larger version (31K): [in this window] [in a new window]   Figure 7. Photographs (magnification x320) demonstrating renal tubular cell apoptosis (TUNEL assay) and the effects of heat preconditioning and liposomal delivery of HSP72 on 1 hour of simulated ischemia. The nuclear stain (blue) and apoptosis stain (TUNEL=green) are shown for each image. One hour of simulated ischemia induced a significant degree of apoptosis; however, both heat preconditioning and liposomal delivery HSP72 pretreatment prevented ischemia-induced apoptosis.

Effect of HSP72 on Simulated Ischemia-Induced Apoptosis
Cellular exposure to heat stress or liposomal HSP72 prevented simulated ischemia-induced apoptosis in renal tubular cells as shown in Figures 6 and 7 (15±6.7 and 13±5.3 apoptotic nuclei/hpf, P<0.05 versus ischemic cells, respectively). Cell viability in these treatment groups was not significantly different from controls at 78±4.2% and 83±3.2%.

Effect of TNF- Neutralization on Simulated Ischemia-Induced Apoptosis
TNF- was neutralized by applying anti-porcine TNF- antibody to cells 24 hours before simulated ischemia and during substrate replacement. TNF- neutralization successfully prevented simulated ischemia-induced apoptosis (10.3±5 apoptotic nuclei/hpf, P<0.05 versus ischemic cells), with cell viability not significantly different from controls (82±4%).

	Discussion

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Significant renal dysfunction can occur after short periods of renal ischemia.⁷ The loss of renal tubular epithelium associated with ischemia-induced ARF is attributed to both apoptotic and necrotic cell death, and renal recovery is largely dependent on the protection of renal tubular cells from death.⁶ Whereas prolonged renal ischemia results in overwhelming cellular ATP depletion and necrotic cell death, less severe ischemia triggers cell death by apoptosis, a process uniquely suited to molecular modulation and possible inhibition.⁴ The mechanisms of ischemia-induced renal dysfunction and cell death are multiple; however, recent evidence suggests that TNF- is an important mediator of this process.^7,9 TNF- gene transcription is primarily regulated by NF-B activation. In animal models of renal IR injury, NF-B activation precedes renal TNF- production⁹; however, both mediators have been implicated in the pathophysiology of ischemic renal injury.^24–26

The heat shock response has been demonstrated to protect a variety of cells, including renal tubular cells, from ischemic injury.^3,15,16,27 This response is mediated, in part, through the production of heat shock proteins such as HSP72; however, the mechanisms by which heat shock and HSP72 exert a cytoprotective effect in the kidney are unclear. In nonrenal cells, the heat shock response and HSP70 have been demonstrated to inhibit cytokine-mediated NF-B activation and inflammatory mediator production.^17–21 Although the role of TNF- in renal IR injury and its proapoptotic properties in renal tubular cells have been well established, the effect of heat shock and HSP72 on ischemia-induced NF-B activation and TNF- production has not been evaluated. This study therefore constitutes the initial demonstration that heat shock and HSP72 inhibit ischemia-induced renal tubular cell apoptosis by preventing NF-B activation and subsequent TNF- production.

Increased HSP72 expression was demonstrated by Western blot analysis after cell exposure to thermal stress or liposomal transfer of HSP72. Upon confirming HSP72 expression, the effect of heat shock and liposomally delivered HSP72 on ischemia-induced NF-B activation was evaluated. Ten minutes of simulated ischemia induced a significant increase in NF-B DNA binding, activation of p65 NF-B (supershift), and nuclear translocation of p65, confirming activation of NF-B in renal tubular cells after simulated ischemia. Similar to observations in nonrenal cells,^17–20 renal cell pretreatment with either heat shock or liposomally delivered HSP72 prevented simulated ischemia-induced NF-B activation. Although it has been well established in the literature that heat shock and HSP70 prevent ischemia-induced NF-B activation by preventing IB kinase activation and subsequent IB phosphorylation and degradation,^17–20 Feinstein et al¹⁹ have shown that HSP70 transfection into cells before injury prevents NF-B activation without preventing IB degradation. They propose that transfected HSP70 may compete with NF-B for the nuclear pore complex, or as a molecular chaperone, HSP70 may interact with NF-B upon its initial dissociation from IB and prevent subsequent NF-B translocation and activation. Our observation that liposomally delivered HSP72 prevents NF-B activation without preventing IB degradation is supported by this study, and further, suggests an additional mechanism by which HSP72 prevents NF-B translocation and activation.

TNF- mRNA production was assessed after cell exposure to simulated ischemia, in the presence or absence of heat shock or liposomal HSP72 pretreatment. An increase in TNF- mRNA expression was observed in response to 1 hour of simulated ischemia; however, both heat shock and liposomally delivered HSP72 inhibited ischemia-induced TNF- production. This finding elucidates TNF- as a marker of renal cell injury in this model of simulated ischemia and supports previous observations that the heat shock response can inhibit inflammatory mediator production.^17–21,28

The effect of HSP72 on ischemia-induced renal tubular cell death was investigated by quantifying apoptosis in response to simulated ischemia, in the presence or absence of heat shock or liposomally delivered HSP72. Indeed, whereas simulated ischemia induced significant cellular apoptosis, prior cellular exposure to either heat shock or liposomal HSP72 prevented ischemia-induced apoptosis. The cytoprotective effect of heat shock and HSP70 in ischemia-induced renal cell injury has been controversial.^13–16,29 Turman et al¹⁴ have demonstrated that transfected inducible HSP70 does not protect LLC-PK1 cells from hypoxic injury (loss of cotransfected luciferase activity); however, our results clearly indicate that both heat preconditioning and liposomal delivery of HSP72 prevent ischemia-induced apoptosis. The discrepancy between these studies may be related to differences in the mechanism of HSP70 overexpression (transfection versus liposomal delivery) or the model of cellular injury (hypoxia versus ischemia).

TNF- neutralization also conferred a similar degree of protection against ischemia-induced apoptosis, illustrating that HSP72 inhibits ischemia-induced renal tubular cell death by preventing NF-B activation, TNF- production, and subsequent TNF--dependent apoptosis. The association of ischemia-induced NF-B activation with TNF- production and TNF--dependent apoptosis indicates that NF-B is a proapoptotic signal in this cellular model of ischemia. Interestingly, activation of NF-B is largely considered to be protective against apoptosis^30–33 and further has been demonstrated to suppress TNF--dependent apoptosis in several renal cell lines.^34–36 The role of NF-B in renal ischemic injury is most likely complex, triggering cell protection or death based on a multitude of factors. Indeed, Sugiyama et al³⁴ recently demonstrated that although NF-B inhibition sensitized glomerular mesangial cells to TNF-induced apoptosis, it did not affect apoptosis induced by other inflammatory mediators (ie, IL-1, Fas ligand, lipopolysaccharide, or reactive oxygen species). Providing further support for an injurious role in ischemia, NF-B inhibition has been shown to prevent simulated ischemia-induced renal cell apoptosis³⁷ and neutrophil infiltration and apoptosis associated with in vivo renal IR injury.^25,26

Ischemia-induced ARF is associated with both necrotic and apoptotic renal cell death. Apoptosis, as a gene-directed process, is amenable to modulation and possible therapeutic inhibition. The data in this study demonstrate that HSP72 can prevent ischemia-induced renal tubular cell apoptosis by inhibiting NF-B activation, TNF- production, and ischemia-induced renal cell apoptosis. As we develop a greater understanding of the heat shock response and the mechanisms by which HSPs prevent renal tubular cell injury, therapeutic interventions may emerge that minimize renal dysfunction and accelerate recovery from ARF.

	Acknowledgments

 
This research was supported by a grant from the National Kidney Foundation. The authors would like to thank Chaeyong Jong, PhD, Sanjin Lee, PhD, C. Maximillian Schmidt, MD, and Michele Yip-Schneider, PhD, for their technical assistance and support.

	Footnotes

 
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received June 4, 2002; revision received October 25, 2002; accepted January 14, 2003.

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