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Factors (HIFs), to upregulate renoprotective genes and protect against injury. Proven methods of preconditioning are cobalt stimulation and transient hypoxia (ischaemic preconditioning), which may offer protection to renal cells against irreversible nephron loss and tolerate ischaemia beyond the accepted critical ischaemia time. Whilst explored in other organs, no study has compared the effects of these two techniques against each other in the kidney. There is emerging evidence that transient ischaemia and cobalt stimulation may act through different mechanisms in conferring renoprotection, with the concept of combination preconditioning offering better protection yet to be explored. METHODS: 24 solitary kidney-model Sprague Dawley rats were divided into groups of 6 undergoing either a) control treatment, b) 30mg/kg subcutaneous cobalt chloride (CoCl2) treatment for 24 and 6 hours pre-ischemia c) intermittent clamping (IC) consisting of 5 minutes renal artery clamping followed by 10 minutes reperfusion over 4 cycles, or d) a combination of both CoCl2 and IC. Following preconditioning, all rats underwent 40 minutes of renal artery clamping and were followed up with serum renal function tests and animal health assessment scores as a measure of renal impairment for 7 days. RESULTS: All rats demonstrated the greatest rise in serum urea and creatinine peaking at 24 hours, with levels return to basal levels by day 7. All preconditioning methods offered biochemical improvement up to 72 hours (mean ⫹/⫺ SEM creatinine in mol/l: control group, 328.3 ⫹/⫺ 46; CoCl2, 76.3 ⫹/⫺ 10.7 p⬍0.005; IC, 76.3 ⫹/⫺ 36.2 p⬍0.01; combination, 271.1 ⫹/⫺ 76). Rats treated with CoCl2 had the lowest rise in serum creatinine at 24 hours (Control 406.8 ⫹/⫺ 62.1; CoCl2, 144.7 ⫹/⫺ 31.5 p⬍0.0001). Whilst the control group had a 50% mortality rate, no rats in the preconditioning groups died (p⬍0.005). CONCLUSIONS: Individual Cobalt treatment offers greater protection against renal damage than IC or a combination of techniques in the kidney. Development of similar hypoxia-mimetic agents that specifically target the prolyl hydroxylation pathway of HIF activation would offer the greatest benefit in renal preconditioning for clinical application. Source of Funding: None

396 PRESENCE OF GRP78 ON THE CELL SURFACE OF KIDNEY CANCER CELLS CORRELATES WITH INCREASED ANTI-GRP78 AUTOANTIBODY TITRES IN KIDNEY CANCER PATIENTS- A POTENTIAL NOVEL DIAGNOSTIC MARKER Richard Austin, Hamilton, Canada; Mario Gonzalez-Gronow, Durham, NC; Sarka Lhotak, Ali Al-Hashimi, Liz Crane, Anil Kapoor, Jehonathan Pinthus*, Hamilton, Canada INTRODUCTION AND OBJECTIVES: GRP78 is an endoplasmic reticulum (ER)-resident molecular chaperone that functions in correct folding of newly synthesized proteins. Translocation of GRP78 to the cell surface allows it to function as a unique signaling receptor to promote cell proliferation and survival. Furthermore, exposure of GRP78 on the surface of cancer cells stimulates the production of anti-GRP78 autoantibodies which correlate with accelerated cancer progression, enhanced metastatic potential and reduced survival. The purpose of these study was to assess the expression of cell surface GRP78 in kidney cancer specimens as well as the presence of antiGRP78 autoantibodies in the serum of patients with kidney cancer. METHODS: Tumor and normal surrounding renal parenchyma as well as serum samples were obtained prospectively from 65 with kidney cancer patients who underwent partial or radical nephrectomy in the past year (2009) in our institution. Immunocytochemistry staining with anti-GRP78 antibodies was performed on all specimens. AntiGRP78 autoantibody titres in the serum were determined using ELISA assay and compared to that of 14 control non-kidney cancer patients. Changes in the activity of tissue factor procoagulant following treatment of CRL-1932 human clear cell renal cancer cells with purified antiGRP78 autoantibodies were measured.

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RESULTS: Prominent GRP78 immunostaining was observed on the cell surface of RCC biopsies. Normal perinuclear immunostaining for GRP78 was observed in adjacent cells from normal renal tissue. Anti-GRP78 autoantibody titres were significantly elevated (p⬍0.01) from the blood of RCC patients, compared to age-matched controls. The addition of purified anti-GRP78 autoantibodies to cultured human RCC cells caused a significant elevation in tissue factor procoagulant activity, an established indicator of cell surface GRP78 activation. CONCLUSIONS: Our findings demonstrate the presence of GRP78 on the surface of RCC cells and correlates with an increase in anti-GRP78 autoantibody titres in blood of kidney cancer patients but not in healthy controls. The diagnostic and prognostic potential of serum anti-GRP78 autoantibodies should be further explored. Source of Funding: None

397 HYPOXIA INDUCES NOX-DEPENDENT ACTIVATION OF NFKB AND SECRETION OF IL-6 AND IL-8 IN RENAL CELL CARCINOMA John Fitzgerald*, Bijaya Nayak, Karthigayan Shanmugasundaram, William Friedrichs, Dipen J. Parekh, Sunil Sudarshan, Karen Block, San Antonio, TX INTRODUCTION AND OBJECTIVES: Renal cell carcinoma (RCC) is associated with inflammation. Inflammation is caused by a variety of pathogenic and environmental factors and is classified as pathogen-derived or nonpathogen-derived (sterile inflammation). Inflammation is associated with oxidative stress, upregulation of hypoxia inducible factor 1-alpha, and several pro-inflammatory gene products through activation of NFkB, together suggesting that hypoxia may be involved in “sterile inflammatory” processes. Primary cells involved in the release of cytokines include lymphocytes and macrophages. However, epithelial cells have also been implicated in cytokine release, although the stimuli to release and molecular mechanisms by which they are released remain unclear. The objectives of this study were to 1) identify renal epithelial-dependent nonpathogen-derived- pro-inflammatory gene products 2) characterize the microenvironment necessary and sufficient for their release (with emphasis on hypoxia) and 3) determine the cellular mechanisms and biological consequences of cytokine secretion in RCC. METHODS: Normal renal and RCC cell lines were subjected to hypoxia for short and long time points in the presence or absence of the Nox inhibitor, DPI and reactive oxygen species (ROS) scavenger, NAC. Elisa analysis was used to detect cytokines (CD40L, INF-gamma, IL-1a, IL-1b, IL-6, IL-8, IL-17,TNF-alpha or MCP-1) Lucigenin-enhanced chemiluminescence analysis was used to detect Nox oxidasederived ROS and Western blot analysis was used to detect activation of signaling pathways. RESULTS: Hypoxia specifically induces IL-6 and IL-8 and solely in RCC cell lines at 48-72 hrs compared to normoxic controls concomitant with NFkB activation. Pretreatment of cells with DPI or NAC, blocked hypoxia-induced NFkB activation and reduced IL-8 secretion. Protein expression of the Nox isoform, Nox4, is upregulated at early timepoints of hypoxia. In addition, IL-6 and IL-8 enhance RCC cell migration and invasion. CONCLUSIONS: We have identified for the first time that IL-6 and IL-8 are specifically secreted by RCC cells under hypoxic conditions. Our data suggest that Nox4-derived ROS mediate IL-8 secretion through activation of the NFkB pathway. Importantly, hypoxia-dependent secretion of IL-6 and IL-8 enhances RCC migration and invasion. Circulating plasma levels of IL-6 and IL-8 in patients with RCC correlate with metastasis and poor outcomes. Together, our data suggest additional mechanisms by which hypoxia may alter tumor RCC phenotype. Source of Funding: Supported by the National Institutes of Health (R01 CA131272, KB; K08 CA138774, SS) the Veterans Administration Career Scientific Award (K.B.), and the

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University of Texas Health Science Center at San Antonio Institute for Integration of Medicine and Science mentored career development award (D.J.P. and K.B.).

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399 DEVELOPMENT AND CHARACTERIZATION OF CLINICALLY RELEVANT TUMOR MODELS FROM PATIENTS WITH RENAL CELL CARCINOMA Jose Karam*, Xiu-Ying Zhang, Pheroze Tamboli, Houston, TX; Vitaly Margulis, Dallas, TX; Hua Wang, E. Jason Abel, Stephen Culp, Christopher Wood, Houston, TX

398 EXPRESSION OF RICTOR IS ASSOCIATED WITH METASTASIS AND POOR PROGNOSIS OF RENAL CELL CARCINOMA Xusheng Chen*, Chunxiang Li, Hua Guo, Fei Zhang, Xin Yao, Ning Zhang, Tianjin, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Rictor is a key member of mTORC2, which is crucial to various carcinoma. We measured Rictor expression levels in tumor tissues and six RCC cell lines and evaluated its significance and prognostic use in renal cell carcinoma (RCC). Then we evaluated the change of cell migration, invation and adhesion in RCC cell with downregulation of Rictor. METHODS: Expression of Rictor was examined through immunohistochemistry in a tissue microarray constructed from 217 renal cancer patients to assess the correlation between Rictor expression level and RCC progression. We subsequently analyzed Rictor expression levels in several RCC cell lines through western blotting. Rictor expression in ACHN cell line was down-regulated by SiRNA and the change of cell migration, invision and adhesion wan examined. RESULTS: Rictor positive staining was noted in 74% of RCC patients with 86% in CCRCC and 62 % in PRCC respectively, whereas staining of Rictor expression was remarkably weak in most normal tubules.(FigA). Further statistical analysis showed that Rictor expression was detected in 24 out of 31 long metastasis cases (p ⫽ 0.000) and in 10 out of 14 lymph node metastasis cases (p ⫽ 0.003) (Table1). Rictor expression is significantly higher in RCC cell lines with high metastatic potential (ACHN°¢SN12C°¢HTB47) than in primary RCC cell lines(786-O and 769-P )(Fig2A). The Rictor expression level was significantly higher in cases with a symptomatic presentation,as well as in higher TNM stage and higher Furhman grade compared with those with lower stage and lower grade. (Table1) Patients with positive Rictor expression showed lower probability of disease-free survival compared with patients with negative Rictor expression. Knockdown of Rictor by small interference RNA inhibits cancer cell migration and invision and impaired EGF-induced cell adhesion. CONCLUSIONS: The Rictor expression may correlate with cancer migration and metastasis, and the detection of expression levels provides useful prognostic information for cancer-specific survival in patients with RCC. Down-regulation of Rictor inhibits cancer cell migration and invision impaired EGF-induced cell adhesion,so Rictor is a potential target for the treatment of RCC metastasis.

INTRODUCTION AND OBJECTIVES: Animal models are instrumental in understanding disease pathophysiology and mechanisms of therapy action and resistance in vivo. We sought to establish and characterize a panel of mouse models of renal cell carcinoma (RCC) derived from patients undergoing radical nephrectomy. METHODS: Renal cell carcinoma mouse models were established from four patients with distinct histologies of renal cell carcinoma. Tumor tissues obtained during surgery were implanted into subcutaneous space of female BALB/c nude mice and serially passaged into new mice. Tumors obtained directly from patients and those derived from serial passages in nude mice were characterized by histology, Short Tandem Repeat (STR) fingerprinting, VHL gene sequencing, and Single Nucleotide Polymorphism (SNP) analysis for each of the four histologies. Tumor bearing mice were treated with sunitinib or everolimus and growth was compared between different histologies and different tumor passages. Primary cell cultures were also derived from patient tumors and were transfected with a lentiviral vector carrying the luciferase gene. Bioluminescence imaging (BLI) of mice was performed after intraperitoneal injection of luciferin in 2 of the models. RESULTS: Four subcutaneous xenograft mouse models were developed: MDA⫺RCC⫺48, MDA⫺RCC⫺55, MDA⫺RCC⫺62, and MDA⫺RCC⫺80, representing papillary type 1, papillary type 2, clear cell, and clear cell with sarcomatoid features renal cell carcinoma, respectively. Tumor growth was dependent on the histologic type, size of implanted tumor chip, and possibly the passage number. We confirmed that the mouse tumors accurately and faithfully represent their respective original patient tumors as STR fingerprints were matching, histology on H&E staining was comparable, and SNP profiles and VHL mutation status were conserved with multiple passages. BLI results were commensurate with the subcutaneous xenograft growth patterns and enabled sequential and non⫺invasive monitoring of growth in an orthotopic implantation setting. Mice treated with sunitinib and everolimus exhibited an initial response to these agents, followed by a later stage of resistance to these agents, which mimics the clinical observations in patients with RCC. CONCLUSIONS: We developed four mouse xenograft models of RCC with clear cell and papillary histologies, with stable histologic and molecular characteristics. We are currently using these models to identify mechanisms of action, molecular correlates of response, and resistance to novel targeted therapies. Source of Funding: None

400 PNEUMOPERITONEAL INFLUENCE IN RENAL SCHEMIA: HEMOXIGENASE-1 ROLE AS A RENAL PROTECTOR Wilson Molina*, Marcelo Wroclawski, Niels Saraiva-Camara, Alvaro Pacheco e Silva, Sao Paulo, Brazil

Source of Funding: None

INTRODUCTION AND OBJECTIVES: Laparoscopic (Lap) Partial Nephrectomy (PN) has emerged as a good minimally invasive treatment option for small renal masses. Renal warm ischemia time (WIT) remains an important factor that impairs outcome in patients that undergo LapPN. Physiological alterations during pneumoperitoneum were well described but more data is needed to understand renal ischemia during Lap surgery. Hemoxigenase-1 (HO-1) is an enzyme produced during acute phase of inflammatory response, such as ischemia-reperfusion (IR) cell damage. Over expression of HO-1 may prevent renal damage during PN. METHODS: 32 female pigs (Sus Scrofa domestica) were used. All underwent open right nephectomy to create single functional renal