Sublethal Hyperthermia Research Articles

Photothermal treatment-based heat stress regulates function of myeloid-derived suppressor cells

Photothermal therapy is an alternative cancer therapy that uses a photothermal agent with light irradiation to induce fatal hyperthermia in cancer cells. In a previous study, we found that ex vivo photothermal (PT) treatment induced expression of heat shock proteins (HSPs), such as HSP70, HSP27, and HSP90, in cancer cells; moreover, immunization with lysates from PT-treated tumor cells resulted in significant tumor growth inhibition in tumor-bearing mice. In this study, we hypothesized that sublethal PT treatment of antigen-presenting cells regulates their immunogenicity. We observed the upregulation of expression of intracellular HSP70 and surface activation markers, such as CD40, CD80, CD86, and MHC class II, in sublethal PT-treated cells. The protumoral activity of myeloid-derived suppressor cells (MDSCs) was reduced by sublethal hyperthermia. Furthermore, poorly immunogenic MDSCs were converted into immunogenic antigen-presenting cells by PT treatment. The differences in immunogenicity between MDSCs untreated or treated with the PT technique were evaluated using the Student’s t-test or Mann–Whitney rank sum test. Collectively, direct hyperthermic treatment resulted in phenotypic changes and the functional regulation of immune cells.

Neuroprotective Effect of Sub-lethal Hyperthermia Preconditioning in a Rat Model of Repeated Closed Head Injury

Repeated mild traumatic brain injury (rTBI), one of the most common forms of traumatic brain injury, is a worldwide severe public health concern. rTBI induces cumulative neuronal injury, neurological dysfunction, and cognitive deficits. Although there are clinical treatment methods, there is still an urgent need to develop preventive approaches for susceptible populations. Using a repeated closed head injury (rCHI) rat model, we interrogate the effect of sub-lethal hyperthermia preconditioning (SHP) on rCHI-induced neuronal injury and behavioral changes. Our study applied the repeated weight-drop model to induce the rCHI. According to the changes of heat shock protein 70 (HSP 70) in the cortex and hippocampus following a single SHP treatment in normal rats, the SHP was delivered to the rats 18 h before rCHI. We found that HSP significantly alleviated rCHI-induced anxiety-like behaviors and impairments in motor abilities and spatial memory. SHP exerts significant neuroprotection against rCHI-induced neuronal damage, apoptosis, and neuroinflammation. Our findings support the potential use of SHP as a preventative approach for alleviating rCHI-induced brain damage.

Sublethal Hyperthermia Transiently Disrupts Cortisol Steroidogenesis in Adrenocortical Cells.

Primary aldosteronism is the most common cause of secondary hypertension. The first-line treatment adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone-producing adenomas, while preserving adjacent normal adrenal cortex. To determine the extent of damage to adrenal cells upon exposure to hyperthermia, the steroidogenic adrenocortical cell lines H295R and HAC15 were treated with hyperthermia at temperatures between 37 and 50°C with the effects of hyperthermia on steroidogenesis evaluated following stimulation with forskolin and ANGII. Cell death, protein/mRNA expression of steroidogenic enzymes and damage markers (HSP70/90), and steroid secretion were analyzed immediately and 7 days after treatment. Following treatment with hyperthermia, 42°C and 45°C did not induce cell death and were deemed sublethal doses while ≥50°C caused excess cell death in adrenal cells. Sublethal hyperthermia (45°C) caused a significant reduction in cortisol secretion immediately following treatment while differentially affecting the expression of various steroidogenic enzymes, although recovery of steroidogenesis was evident 7 days after treatment. As such, sublethal hyperthermia, which occurs in the transitional zone during thermal ablation induces a short-lived, unsustained inhibition of cortisol steroidogenesis in adrenocortical cells in vitro.

PSUN16 Sublethal Hyperthermia Decreases Cellular Proliferation and Transiently Disrupts Steroidogenesis in Adrenal Cells

Abstract Introduction Primary Aldosteronism is the most common cause of secondary hypertension. First-line treatment; adrenalectomy resects adrenal nodules and adjacent normal tissue, limiting suitability to those who present with unilateral disease. Use of thermal ablation represents an emerging approach as a possible minimally invasive therapy for unilateral and bilateral disease, to target and disrupt hypersecreting aldosterone producing adenomas, while preserving adjacent normal adrenal cortex. Ablation involves heating tissue >50°C to induce cellular necrosis. Outside the core ablation zone, the transitional zone is an area exposed to variable temperatures between 37°C – 50°C. To understand the feasibility of precision ablation in the adrenal gland, we examined the effect of applying these temperatures to adrenocortical cells to identify i) the required temperature to effectively ablate adrenal cells ii) the extent of damage that may occur to surrounding healthy adrenal cells with exposure to transitional zone temperatures. Methods Steroidogenic adrenocortical cell lines, H295R and HAC15, were treated with hyperthermia (high precision water bath) at temperatures of 37, 42, 45, 48 and 50°C and steroidogenesis was subsequently stimulated using forskolin (10µM) and angiotensin II (10nM). Cell death (Propidium iodide staining by flow cytometry), proliferation (xCELLigence real-time cell analysis), protein expression (Western blot/qRT-PCR), and steroid secretion (HPLC-Mass spectrometry) were analysed immediately and 7-days post-treatment. Results Cell death occurred at 48°C and 50°C (p<0.05 vs 37°C control), but not 45°C, or 42°C. Sublethal hyperthermia (42°C and 45°C for 30 minutes) induced a heat shock response demonstrated by upregulation of HSP70 and HSP90. Proliferation was subsequently reduced over 7-days alongside a decrease in aldosterone and cortisol secretion (p<0.05), and reduced expression of steroidogenic enzymes (CYP11B1, CYP11B2, CYP11A1) 18-hours post treatment (p<0.05). Following 7-days sublethal hyperthermia, steroid secretion and steroidogenic enzymatic expression returned to baseline levels. Conclusion Hyperthermia at 48°C and 50°C for 15 minutes is required for sustained cell death at 7-days post treatment. Sublethal hyperthermia, equivalent to that produced in the transitional zone during thermal ablation, produces a short-lived unsustained inhibition of steroidogenesis that recovers 7-days post treatment. Therefore, segmental adrenal sparing ablation is possible with recovery of transitional zone following ablation. This underlines the potential for precision technology development for bilateral adrenal ablation as definitive measure to treat PA caused by APA or Micronodular disease. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Sublethal hyperthermia decreases cellular proliferation and transiently disrupts steroidogenesis in adrenal cells

Sublethal hyperthermia decreases cellular proliferation and transiently disrupts steroidogenesis in adrenal cells

Sublethal hyperthermia enhances anticancer activity of doxorubicin in chronically hypoxic HepG2 cells through ROS-dependent mechanism.

Thermal ablation in combination with transarterial chemoembolization (TACE) has been reported to exert a more powerful antitumor effect than thermal ablation alone in hepatocellular carcinoma patients. However, the underlying mechanisms remain unclear. The purpose of the present study was to evaluate whether sublethal hyperthermia encountered in the periablation zone during thermal ablation enhances the anticancer activity of doxorubicin in chronically hypoxic (encountered in the tumor area after TACE) liver cancer cells and to explore the underlying mechanisms. In the present study, HepG2 cells precultured under chronic hypoxic conditions (1% oxygen) were treated in a 42°C water bath for 15 or 30 min, followed by incubation with doxorubicin. Assays were then performed to determine intracellular uptake of doxorubicin, cell viability, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and total antioxidant capacity. The results confirmed that sublethal hyperthermia enhanced the intracellular uptake of doxorubicin into hypoxic HepG2 cells. Hyperthermia combined with doxorubicin led to a greater inhibition of cell viability and increased apoptosis in hypoxic HepG2 cells as compared with hyperthermia or doxorubicin alone. In addition, the combination induced apoptosis by increasing ROS and causing disruption of MMP. Pretreatment with the ROS scavenger N-acetyl cysteine significantly inhibited the apoptotic response, suggesting that cell death is ROS-dependent. These findings suggested that sublethal hyperthermia enhances the anticancer activity of doxorubicin in hypoxic HepG2 cells via a ROS-dependent mechanism.

Abstract No. 538 Sublethal hyperthermia enhances the anticancer activity of doxorubicin against acute hypoxic HepG2 cells through ROS-dependent mechanisms

Abstract No. 538 Sublethal hyperthermia enhances the anticancer activity of doxorubicin against acute hypoxic HepG2 cells through ROS-dependent mechanisms

Abstract No. 611 Immunologic ramifications of tumor-specific hyperthermia in a preclinical model of hepatocellular carcinoma

Recent clinical trials have demonstrated that immune checkpoint inhibitors are effective for only ∼20% of hepatocellular carcinoma (HCC) patients. Adjuvant hyperthermia treatment can potentially overcome barriers to immunotherapy by inducing immunogenic cell death; however, existing hyperthermia modalities are poorly optimized for immune stimulation. The purpose of this study was to evaluate the immunologic ramifications of a novel hyperthermia modality that promotes tumor-specific heat generation. An orthotopic HCC model was generated by subcapsular implantation of syngeneic McA-RH7777 hepatoma cells in female Buffalo rats (n=20). 96h following the i.v. administration of 0.5mg/kg indocyanine green (ICG), tumor-specific hyperthermia at sublethal (42–47C) or ablative (> 50C) temperatures was generated by illuminating the tumor (or adjacent liver tissue as control) with a 785nm near infrared laser coupled to a clinical fiberoptic catheter. 72h after treatment, normal and tumor tissue were harvested for immunohistochemistry, multi-color flow cytometry, and transcriptomic analysis. Principal component analysis of RNAseq data revealed distinct expression profiles as a function of thermal dose. In particular, sublethal hyperthermia was found to upregulate pathways associated with immunogenic cell death such as the unfolded protein response. It also suppressed non-immunogenic cell death pathways such as apoptosis and upregulated genes associated with the type I interferon response. Both sublethal and ablative hyperthermia doses were associated with an increase in CD8+ cytotoxic T-cell infiltration within the tumors. Paradoxically, ablative hyperthermia was also associated with an increase in the expression of multiple components of the hepatocyte growth factor/cMet pathway. Tumor-specific hyperthermia is associated with transcriptional and microenvironmental modifications that can improve anti-tumor immunity.

Sub-lethal hyperthermia promotes epithelial-to-mesenchymal-like transition of breast cancer cells: implication of the synergy between hyperthermia and chemotherapy

Thermotherapy has demonstrated a potential to be an effective non-surgical technique to treat breast cancer. Despite its advantages, including low toxicity and high repeatability, thermotherapy is typically required to be applied in combination with other treatments since the residual tumor cells that survive after hyperthermal treatment often cause recurrence. In this study, we confirmed that breast cancer cells tolerate temperature of up to 47 °C by synthesizing a large amount of heat shock proteins. Further changes in the molecular properties of the heat-exposed cells were investigated using western blotting, quantitative reverse transcription polymerase chain reaction, and immunocytochemistry. We found that low-temperature hyperthermia promoted epithelial-to-mesenchymal-like transition (EMT), as observed by the increased mesenchymal marker expression levels while decreasing epithelial markers. Moreover, cell morphology changed from cobblestone-like to a more spindle-like appearance, in addition to significantly enhanced cell motility upon heat treatment. These results all support that sub-lethal hyperthermal stress induces EMT. In addition, we examined changes in the chemo-sensitivity of the heat-treated cells. Addition of a chemo-drugs caused increased cytotoxicity of the heat-treated cells compared to the cells that were not co-treated with heat. Our study demonstrates that thermotherapy alone may cause undesirable EMT, which could be well overcome through a synergistic effect when applied with chemotherapy.

Characterizing Thermal Augmentation of Convection-Enhanced Drug Delivery with the Fiberoptic Microneedle Device

ABSTRACT Convection-enhanced delivery (CED) is a promising technique leveraging pressure-driven flow to increase penetration of infused drugs into interstitial spaces. We have developed a fiberoptic microneedle device for inducing local sub-lethal hyperthermia to further improve CED drug distribution volumes, and this study seeks to quantitatively characterize this approach in agarose tissue phantoms. Infusions of dye were conducted in 0.6% (w/w) agarose tissue phantoms with isothermal conditions at 15 °C, 20 °C, 25 °C, and 30 °C. Infusion metrics were quantified using a custom shadowgraphy setup and image-processing algorithm. These data were used to build an empirical predictive temporal model of distribution volume as a function of phantom temperature. A second set of proof-of-concept experiments was conducted to evaluate a novel fiberoptic device capable of generating local photothermal heating during fluid infusion. The isothermal infusions showed a positive correlation between temperature and distribution volume, with the volume at 30 °C showing a 7-fold increase at 100 min over the 15 °C isothermal case. Infusions during photothermal heating (1064 nm at 500 mW) showed a similar effect with a 3.5-fold increase at 4 h over the control (0 mW). These results and analyses serve to provide insight into and characterization of heat-mediated enhancement of volumetric dispersal.

Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress

Recently introduced new technologies that enable temperature-controlled laser irradiation on the RPE allowed us to investigate temperature-resolved RPE cell responses. In this study we aimed primarily to establish an experimental setup that can realize laser irradiation on RPE cell culture with the similar temperature distribution as in the clinical application, with a precise time/temperature history. With this setup, we conducted investigations to elucidate the temperature-dependent RPE cell biochemical responses and the effect of transient hyperthermia on the responses of RPE cells to the secondary-exposed oxidative stress. Porcine RPE cells cultivated in a culture dish (inner diameter = 30 mm) with culture medium were used, on which laser radiation (λ = 1940 nm, spot diameter = 30 mm) over 10 s was applied as a heat source. The irradiation provides a radially decreasing temperature profile which is close to a Gaussian shape with the highest temperature in the center. Power setting for irradiation was determined such that the peak temperature (Tmax) in the center of the laser spot at the cells reaches from 40 °C to 58 °C (40, 43, 46, 50, 58 °C). Cell viability was investigated with ethidium homodimer III staining at the time points of 3 and 24 h following laser irradiation. Twenty four hours after laser irradiation the cells were exposed to hydrogen peroxide (H2O2) for 5 h, followed by the measurement of intracellular glutathione, intracellular 4-hydroxynonenal (HNE) protein adducts, and secreted vascular endothelial growth factor (VEGF). The mean temperature threshold for RPE cell death after 3 h was found to be around 52 °C, and for 24 h around 50 °C with the current irradiation setting. A sub-lethal preconditioning on Tmax = 43 °C significantly induced the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, and decreased H2O2-induced increase of intracellular 4-HNE protein adducts. Although sub-lethal hyperthermia (Tmax = 40 °C, 43 °C, and 46 °C) caused a slight increase of VEGF secretion in 6 h directly following irradiation, secondary exposed H2O2-induced VEGF secretion was significantly reduced in the sub-lethally preheated groups, where the largest effect was seen following the irradiation with Tmax = 43 °C. In summary, the current results suggest that sub-lethal thermal laser irradiation on the RPE at Tmax = 43 °C for 10 s enhances cell defense system against oxidative stress, with increasing the GSH/GSSG ratio. Together with the results that the decreased amount of H2O2-induced 4-HNE in sub-lethally preheated RPE cells was accompanied by the lower secretion of VEGF, it is also strongly suggested that the sub-lethal hyperthermia may modify RPE cell functionality to protect RPE cells from oxidative stress and associated functional decrease, which are considered to play a significant role in the pathogenesis of age-related macular degeneration and other chorioretinal degenerative diseases.

Monitoring Nanoparticle-Mediated Cellular Hyperthermia with A High-Sensitivity Biosensor

To develop and apply a heat-responsive and secreted reporter assay for comparing cellular response to nanoparticle (NP)- and macroscopic-mediated sublethal hyperthermia. Reporter cells were heated by water bath (macroscopic heating) or iron oxide NPs activated by alternating magnetic fields (nanoscopic heating). Cellular responses to these thermal stresses were measured in the conditioned media by secreted luciferase assay. Reporter activity was responsive to macroscopic and nanoparticle heating and activity correlated with measured macroscopic thermal dose. Significant cellular responses were observed with NP heating under doses that were insufficient to measurably change the temperature of the system. Under these conditions, the reporter response correlated with proximity to cells loaded with heated nanoparticles. These results suggest that NP and macroscopic hyperthermia may be distinctive under conditions of mild hyperthermia.

Combination Therapy with Local Radiofrequency Ablation and Systemic Vaccine Enhances Antitumor Immunity and Mediates Local and Distal Tumor Regression

PurposeRadiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression.Experimental DesignMurine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA+) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA+ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM).Results In vitro, sublethal hyperthermia of MC38-CEA+ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4+ T-cell immune responses to CEA and eradicated both primary CEA+ and distal CEA– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.

Increasing the rate of heating: A potential therapeutic approach for achieving synergistic tumour killing in combined hyperthermia and chemotherapy

Purpose: A synergistic cancer cell killing effect of sub-lethal hyperthermia and chemotherapy has been reported extensively. In this study, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted in order to investigate the role of heating rate in achieving a synergistic effect. The mode of cell death, induction of thermal tolerance and P-glycoprotein (P-gp) mediated MDR following two different rates of heating were studied.Materials and methods: Doxorubicin (DOX) was used as the chemotherapy drug. A rapid rate hyperthermia was achieved by near infrared laser (NIR) excited indocyanine green (ICG) dye (absorption maximum at 808 nm, ideal for tissue penetration). A slow rate hyperthermia was provided by a cell culture incubator.Results: The potentiating effect of hyperthermia to chemotherapy can be maximised by increasing the rate of heating. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of DOX. Meanwhile, the rapid rate laser-ICG hyperthermia at 43°C caused cell necrosis whereas the slow rate incubator hyperthermia at 43°C induced mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified.Conclusions: This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

Non-damaging Retinal Phototherapy: Dynamic Range of Heat Shock Protein Expression

Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse. One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage. A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters. Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.

The role of endogenous opioids in the protective effects of local sublethal hyperthermia against the progression of burn injury

The aim of the present study was to evaluate the role of endogenous opioids in local sublethal hyperthermia-induced protection against burn injury. Second-degree burn wounds were induced on the back of Balb/c mice. Progression of burn injury and expression of heat shock protein (HSP)-70 were evaluated after 24 h. Both inhibition of HSP synthesis and blocking opioid receptors before applying local sublethal hyperthermia decreased the protective effects of sublethal hyperthermia against the progression of burn injury. Blocking opioid receptors attenuated induction of HSP-70 by sublethal hyperthermia.

Post heat shock tolerance: a neuroimmunological anti-inflammatory phenomenon

We previously showed that the progression of burn-induced injury was inhibited by exposing the peripheral area of injured skin to sublethal hyperthermia following the burn. We called this phenomenon post-heat shock tolerance. Here we suggest a mechanism for this phenomenon. Exposure of the peripheral primary hyperalgesic/allodynic area of burned skin to local hyperthermia (45°C, 30 seconds), which is a non-painful stimulus for normal skin, results in a painful sensation transmitted by nociceptors. This hyperthermia is too mild to induce any tissue injury, but it does result in pain due to burn-induced hyperalgesia/allodynia. This mild painful stimulus can result in the induction of descending anti-nociceptive mechanisms, especially in the adjacent burned area. Some of these inhibitory mechanisms, such as alterations of sympathetic outflow and the production of endogenous opioids, can modify peripheral tissue inflammation. This decrease in burn-induced inflammation can diminish the progression of burn injury.

Intrinsic thermal resistance of the canine brain

Hyperthermia above a critical threshold results in multisystemic changes that include neurological manifestations of heat stroke. It is unknown if the latter represents an intrinsic thermal sensitivity of the CNS or whether injury is secondary to physiological responses of non-CNS origin. To address this issue, the present work examined functional, structural, and biochemical changes in the CNS of dogs subjected to a thermal dosage immediately below that which induces disseminated intravascular coagulation with secondary multiple organ injury. The experimental approach is previously reported, inducing a 42.5°C, 90 min, whole body hyperthermia while preventing other physiological responses to treatment, including respiratory alkalosis and significant reductions in mean arterial pressure. Functional analyses included neurologic examinations and brainstem auditory evoked potential recordings in the post-treatment interval in both hyperthermic and euthermic control populations. Biochemical and structural analyses examined the expression of 70-kDa heat shock proteins, cytokines, markers of astroglial and microglial injury/activation, evidence of vascular endothelial damage, and evidence of neuronal and axonal injury in brain between 0.5 h and 8 days from the end of the treatment. The only significant change associated with treatment was induction of the major inducible 70-kDa heat shock protein, this being most prominent in the cerebellum with maximal expression at 6 h and a return to baseline by 8 days. Collectively, from these results we suggest that the canine brain is intrinsically resistant to sublethal hyperthermia such that when CNS lesions occur, they do so in the presence of other physiological derangements.

Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors

Abstract Transient sublethal hyperthermia followed by recovery from heat stress, referred to as heat shock preconditioning, exerts a protective effect on ischemia/reperfusion-induced injury in many systems. This effect is considered to be correlated to heat shock proteins (HSPs) and might be a critical factor in kidney graft function and survival. This study was designed to examine the impact of heat shock preconditioning on kidney isograft function and survival in a model utilizing non-heart-beating (NHB) donors. Four groups of male Lewis rats (n= 10/group) subjected either to whole body hyperthermia (groups A and C) or to sham anesthesia (groups B and D) were allowed 24 h recovery. Thereafter, 20 min of warm ischemia (A/B), and in a separate set of experiments 40 min of warm ischemia (C/D), were induced by suprarenal aortic cross clamping before renal procurement. After 24-h preservation with University of Wisconsin solution at 4 °C, orthotopic kidney transplantations were performed to syngeneic bilaterally nephrectomized recipients. Tissue specimens were taken to determine HO-1/HSP32, 72, and 90 induction by Western blot analysis. Renal function was measured by means of serum creatinine and creatinine clearance on days 0,3, and 7 as well as urine volume, protein content, and creatinine levels daily. HO-1/ HSP32 and HSP72 were found to be expressed constitutively. Moreover, heat shock strongly induced renal HSP72 and HSP32/HO-1, and to a lesser extent HSP90, expression. For recipients of group A grafts, the graft survival rate was 10/10, whereas it was 7/10 (70%) in recipients of group B grafts (log rank p < 0.05). Following 40 min of warm ischemia, 6/10 (60%) recipients survived, whereas all sham treated animals died with anuria within 6 days (log rank p= 0.01). Heat shock preconditioning strongly improved graft viability and reduced functional impairment. Creatinine clearance (CRC) on day 3 post Tx was 0.43 ± 0.24 ml/min in preconditioned animals (group A) and 0.07 ± 0.09 ml/min (p < 0.001) in sham preconditioned (group B), whereas it was 0.91 ± 0.33 ml/min and 0.03 ± 0.02 ml/min (p < 0.00001) on day 7 post Tx. Following 40 min NHB time, CRC in survivors of preconditioned graft recipients (group C) was 0.32 ± 0.2 ml/ min (day 3 post Tx) and 0.23 ± 0.08 ml/min (day 7 post Tx) and was significantly better than CRC of group B (p < 0.01 and p < 0.00001, respectively). CRCs prior to NHB procedures were comparable in all animals ranging between 1.31 and 1.72 ml/min. Serum creatinine as well as proteinuria were significantly increased after transplantation in both groups but recovered within 5 days inrecipients of preconditioned grafts, whereas kidneys from donors without HP did not recover function. Histologial alterations were also diminished following HP. Hyperthermic preconditioning induces strong and long lasting HO-1/HSP32, HSP72, and HSP90 expression in rat kidneys. HP increases survival following transplantation and improves renal graft function including proteinuria, volume output, and creatinine clearance. HSP induction might be used to develop novel approaches in clinical transplantation.

Hyperthermia-induced HSP expression correlates with improved rat renal isograft viability and survival in kidneys harvested from non-heart-beating donors.

Transient sublethal hyperthermia followed by recovery from heat stress, referred to as heat shock preconditioning, exerts a protective effect on ischemia/reperfusion-induced injury in many systems. This effect is considered to be correlated to heat shock proteins (HSPs) and might be a critical factor in kidney graft function and survival. This study was designed to examine the impact of heat shock preconditioning on kidney isograft function and survival in a model utilizing non-heart-beating (NHB) donors. Four groups of male Lewis rats (n = 10/group) subjected either to whole body hyperthermia (groups A and C) or to sham anesthesia (groups B and D) were allowed 24 h recovery. Thereafter, 20 min of warm ischemia (A/B), and in a separate set of experiments 40 min of warm ischemia (C/D), were induced by suprarenal aortic cross clamping before renal procurement. After 24-h preservation with University of Wisconsin solution at 4 degrees C, orthotopic kidney transplantations were performed to syngeneic bilaterally nephrectomized recipients. Tissue specimens were taken to determine HO-1/HSP32, 72, and 90 induction by Western blot analysis. Renal function was measured by means of serum creatinine and creatinine clearance on days 0, 3, and 7 as well as urine volume, protein content, and creatinine levels daily. HO-1/HSP32 and HSP72 were found to be expressed constitutively. Moreover, heat shock strongly induced renal HSP72 and HSP32/HO-1, and to a lesser extent HSP90, expression. For recipients of group A grafts, the graft survival rate was 10/10, whereas it was 7/10 (70 %) in recipients of group B grafts (log rank p < 0.05). Following 40 min of warm ischemia, 6/10 (60 %) recipients survived, whereas all sham treated animals died with anuria within 6 days (log rank p = 0.01). Heat shock preconditioning strongly improved graft viability and reduced functional impairment. Creatinine clearance (CRC) on day 3 post Tx was 0.43 +/- 0.24 ml/min in preconditioned animals (group A) and 0.07 +/- 0.09 ml/min (p < 0.001) in sham preconditioned (group B), whereas it was 0.91 +/- 0.33 ml/min and 0.03 +/- 0.02 ml/min (p < 0.00 001) on day 7 post Tx. Following 40 min NHB time, CRC in survivors of preconditioned graft recipients (group C) was 0.32 +/- 0.2 ml/min (day 3 post Tx) and 0.23 +/- 0.08 ml/min (day 7 post Tx) and was significantly better than CRC of group B (p < 0.01 and p < 0.00001, respectively). CRCs prior to NHB procedures were comparable in all animals ranging between 1.31 and 1.72 ml/min. Serum creatinine as well as proteinuria were significantly increased after transplantation in both groups but recovered within 5 days in recipients of preconditioned grafts, whereas kidneys from donors without HP did not recover function. Histological alterations were also diminished following HP. Hyperthermic preconditioning induces strong and long lasting HO-1/HSP32, HSP72, and HSP90 expression in rat kidneys. HP increases survival following transplantation and improves renal graft function including proteinuria, volume output, and creatinine clearance. HSP induction might be used to develop novel approaches in clinical transplantation.