Nephrology Dialysis Transplantation, May 2014

Introduction and Aims: Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been suggested to be involved in the mechanism of renal fibrosis. Previously, we have shown the direct effects of S1P on the fibrotic process in the unilateral ureteral obstruction (UUO) model using nude mice which were characterized by deficit of immune response. To get more insight into roles for S1P and receptor subtype effects in vitro, we performed using antagoists and siRNAs knockdown of receptor subtypes. Methods: Normal rat kidney interstitial fibroblast (NRK-49F) cells were stimulated with exogenous S1P and the expressions (mRNA/Western blotting) of a-SMA, E-cadherin, collagen type 1 (COL1), collagen type 4 (COL4), tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and plasminogen activator inhibitor-1 (PAI1) were examined. To specify the kidney specific signal pathway, antagonists and siRNAs targeted to S1P receptor subtypes were generated. Then, the fibrotic changes of the NRK-49F cells after stimulation by S1P were examined (3 days, 7 days). The growth and migration of cultured cells were quantified by using CL-Quant software to analyze time-lapse images in a Nikon BioStation CT. The real-time images of cell migration were monitored for 2 days. And also NRK-49F cells were stimulated with S1P after the addition of FTY720 (S1P 1, 3, 4, 5 agonist), or DMS (sphingosine kinase inhibitor) were evaluated. Results: S1P stimulated fibrosis of NRK-49F cells in a dose- and time-dependent manner as previously observed, and induced morphological changes (elongation of the cell shape with spindle-like extension, increased migration) of the NRK-49F cells. Migration of NRK49F cells was accelerated and increase in a-SMA, COL1, COL4, TIMP1 and PAI1 expressions and decrease in E-cadherin expression were observed by addition of S1P. Antagonist and siRNA transfection to NRK-49F cells of Sphingosine 1-phosphate receptor-3 (S1PR-3) attenuated cell growth and migration, in addition, the expression of fibrotic markers was also diminished by antagonist and siRNA transfection to NRK-49F cells of S1PR-3. And also in the presence of FTY720 and DMS, fibrosis and migration induced by S1P were suppressed. Conclusions: These results suggest that activation of S1P signaling mediated by S1PR-3 results in chronic pathological fibrosis, such as in chronic kidney disease (CKD).

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Won Seok Yang 0 1 3 4 6 8 9 10 11 13 14 15 16 17 Nam Jeong Han 0 1 2 4 6 7 9 10 11 12 14 17 Joo Mi Lee 0 1 2 4 6 7 9 10 11 12 14 17 Chung Hee Baek 0 1 3 4 6 8 9 10 11 13 14 15 16 17 Su-Kil Park 0 1 3 4 6 8 9 10 11 13 14 15 16 17 0 Sun Woo Kang 1 Steffen Grampp 2 Asan Institute for Life Sciences , Seoul , Republic of Korea 3 Asan Medical Center , Seoul , Republic of Korea 4 Shunji Shiohira 5 UCLA , Los angeles, CA , USA 6 Lucas L Falke 7 University of Western Ontario , London, ON , Canada 8 UMC Utrecht , Utrecht , The Netherlands 9 Nuria Troyano Suárez 10 Gabor Kokeny 11 Adam M Zawada 12 International Laser Centre , Bratislava , Slovakia 13 Saarland University Medical Center , Homburg , Germany 14 Ingrid Lajdova 15 Chonnam National University , Toronto, ON , Canada 16 G. D'Annunzio University , Chieti , Italy 17 Paolo Felaco - CELL SIGNALLING AND APOPTOSIS Introduction and Aims: Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been suggested to be involved in the mechanism of renal fibrosis. Previously, we have shown the direct effects of S1P on the fibrotic process in the unilateral ureteral obstruction (UUO) model using nude mice which were characterized by deficit of immune response. To get more insight into roles for S1P and receptor subtype effects in vitro, we performed using antagoists and siRNAs knockdown of receptor subtypes. Methods: Normal rat kidney interstitial fibroblast (NRK-49F) cells were stimulated with exogenous S1P and the expressions (mRNA/Western blotting) of a-SMA, E-cadherin, collagen type 1 (COL1), collagen type 4 (COL4), tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and plasminogen activator inhibitor-1 (PAI1) were examined. To specify the kidney specific signal pathway, antagonists and siRNAs targeted to S1P receptor subtypes were generated. Then, the fibrotic changes of the NRK-49F cells after stimulation by S1P were examined (3 days, 7 days). The growth and migration of cultured cells were quantified by using CL-Quant software to analyze time-lapse images in a Nikon BioStation CT. The real-time images of cell migration were monitored for 2 days. And also NRK-49F cells were stimulated with S1P after the addition of FTY720 (S1P 1, 3, 4, 5 agonist), or DMS (sphingosine kinase inhibitor) were evaluated. Results: S1P stimulated fibrosis of NRK-49F cells in a dose- and time-dependent manner as previously observed, and induced morphological changes (elongation of the cell shape with spindle-like extension, increased migration) of the NRK-49F cells. Migration of NRK49F cells was accelerated and increase in a-SMA, COL1, COL4, TIMP1 and PAI1 expressions and decrease in E-cadherin expression were observed by addition of S1P. Antagonist and siRNA transfection to NRK-49F cells of Sphingosine 1-phosphate receptor-3 (S1PR-3) attenuated cell growth and migration, in addition, the expression of fibrotic markers was also diminished by antagonist and siRNA transfection to NRK-49F cells of S1PR-3. And also in the presence of FTY720 and DMS, fibrosis and migration induced by S1P were suppressed. Conclusions: These results suggest that activation of S1P signaling mediated by S1PR-3 results in chronic pathological fibrosis, such as in chronic kidney disease (CKD). MP002 ANALYSES OF HYPOXIA INDUCIBLE TRANSCRIPTION FACTOR DNA-BINDING AND HYPOXIC GENE REGULATION IN PRIMARY HUMAN RENAL TUBULAR CELLS Introduction and Aims: The kidneys receive approximately twenty percent of the cardiac output. However, oxygen tensions fluctuate physiologically in the kidney with very low oxygen levels especially in the medulla. Hypoxia inducible transcription factors (HIF) initiate adaptive mechanisms to hypoxia in order to secure cell survival. Animal studies have shown that genetic or preconditional pharmacological HIF stabilisation leads to a better outcome in models of acute kidney injury. The precise mechanisms responsible for the protective effect and the relevance of the HIF system in human kidneys remain unknown. Therefore, the aim of this study was to characterise the hypoxic response and to investigate direct HIF effects in primary human tubular cells. Methods: Healthy human kidney tissue from patients undergoing tumor nephrectomy was used for tissue studies and isolation of primary tubular cells. Primary tubular cells were separated in distal and predominantly proximal tubular cells using different adherence properties of the two cell types. Purity of the cell cultures was surveyed by immunocytochemistry and immunoblotting for N- and E-cadherin as markers for proximal and distal tubular cells, respectively. HIF protein was stabilised by hypoxia (1%) or the hypoxia mimetic dimethyl oxalylglycine (DMOG). Chromatin immunoprecipitation (ChIP) was employed to identify HIF and RNA polymerase 2 DNA-binding sites. Gene expression analysis by Western blotting or qPCR was used for validation of gene regulation. Results: Immunoblots of kidney tissue incubated with DMOG showed increased HIF stabilisation over time. In parallel experiments immunohistochemistry showed the presence of HIF-1α protein in tubular cells. Co-staining revealed the colocalisation of HIF-1α with the 11β-hydroxy steroid dehydrogenase (11β-HSD) in the distal connecting tubule (DCT) and the collecting duct (CD). Therefore, we used distal primary cells to further explore the HIF-system. Immunoblot analysis confirmed stabilisation of HIF and expression qPCR showed the induction of well established HIF-target genes (e.g. PHD3, HIG2) by hypoxia or DMOG. To identify direct transcriptional targets of HIF we employed ChIP experiments using specific antibodies against HIF-1α and HIF-1β subunits and RNA polymerase 2. In hypoxia experiments we found robust enrichment of HIF protein at regulatory DNA elements at known HIF-target gene loci (PHD3, HIG2), which stimulated transcriptional activity at these genes and increased target gene mRNA in cultures of distal tubular cells from different individuals. Conclusions: The importance of the HIF system for cell survival and integrity in regards to hypoxic cell damage is well documented for rodent kidneys. We show that in human kidneys HIF stabilisation appears predominantly in distal tubular cells. Transcriptional activity is conserved at established target genes across several individuals. Therefore, this experimental setting seems suitable for further testing of potential protective mechanisms of the HIF system in human kidneys and to explore new therapeutic options in human kidney disease. MP003 BLOCKING CLASS II HISTONE DEACETYLASE ACTIVITY INHIBITS RENAL FIBROSIS Introduction and Aims: Fibrosis is the final, common pathological outcome of many chronic kidney diseases. Although histone deacetylases (HDACs) have been reported to be involved in renal fibrosis, it is still unclear which class of HDAC is involved in the pathophysiology of renal fibrosis. To investigate which class of HDAC is involved in pathogenic renal fibrosis and evaluate anti-fibrotic effect of the defined HDAC inhibitors. Methods: The enzyme activity of class I and class II was examined on TGF-beta 1-induced epithelial-to-mesenchymal transition (EMT) of the human renal proximal tubular epithelial cell line HK-2. By using the pan-HDAC inhibitor (SB939), class I-specific HDAC inhibitor (MS275), and class II-specific HDAC inhibitor (MC1568), we defined the roles of class I and class II enzymes in EMT. To confirm the role of HDACs in vivo, we used the unilateral ureteric obstruction (UUO) model of renal fibrosis. Results: We found that class II enzyme activity was markedly induced on TGF-beta 1-induced EMT but class I enzyme was not induced. Treatment of pan-inhibitor SB939 strongly inhibited TGF-beta 1-induced upregulation of collagen type I and alpha-SMA. Class II-specific inhibitor MC1568 had the similar effects of SB939, but class I-specific inhibitor MS275 did not have the effects. UUO model with SB939 treatment was markedly inhibited accumulation of alpha-SMA and deposition of collagen type I. Conclusions: Our results demonstrate that class II HDACs contribute to renal fibrosis and suggest that class II-selective inhibitors have a therapeutic potential for the treatment of renal fibrosis. MP004 SPLEEN TYROSINE KINASE AS A KEY MOLECULE IN HIGH GLUCOSE-INDUCED TOLL-LIKE RECEPTOR-4 SIGNALING IN PROXIMAL TUBULAR EPITHELIAL CELLS. Introduction and Aims: Spleen tyrosine kinase (Syk) is rapidly activated by high glucose and mediates activations of NF-κB and AP-1, and the downstream synthesis of transforming growth factor-β1 (TGF-β1). However, it remains undetermined how high glucose activates Syk. In the present study, we explored the signal pathway from high glucose to Syk activation in human proximal tubular epithelial cells. Methods: HK-2 cells were cultured. Transfections of siRNA, immunoprecipitation and Western blot analysis were used to explore the signal pathway. Results: High glucose stimulated Syk activation within 10 min. Depletion of toll-like receptor-4 (TLR4) by transfection of TLR4-siRNA attenuated high glucose-induced Syk activation, NF-κB p65 nuclear translocation, and TGF-β1 production. In addition, TLR4-neutralizing antibody, TLR4 inhibitor (TAK-242), and depletion of MyD88 (an adaptor molecule of TLR4) by transfection of MyD88-siRNA all attenuated high glucose-induced Syk activation. As an evidence of TLR4 activation,binding of IRAK-1 to MyD88 increased rapidly upon exposure to high glucose. Syk was co-immunoprecipitated with TLR4. Syk bound to TLR4 was activated by high glucose. High-mobility group box-1 (HMGB-1), an endogenous activator of TLR4, was co-immunoprecipitated with TLR4, and the amount of HMGB-1 immunoprecipitated with anti-TLR4 antibody increased as early as 2.5 min after exposure to high glucose, while glycyrrhizin, an inhibitor of HMGB-1, suppressed high glucose-induced Syk activation. Conclusions: In summary, Syk is constitutively associated with TLR4 complex. High glucose induces an immediate extracellular release of HMGB-1, which interacts with TLR4 and activates it. TLR4 in turn activates Syk, in which MyD88 is also implicated. MP005 ROLE OF NF-KB IN UROMODULIN-ASSOCIATED KIDNEY DISEASE Elisabeth Kemter1, Bernhard Aigner1 and Ruediger Wanke2 1Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany, 2Institute of Veterinary Pathology, Center for Clinical Veterinary Medicine, LMU Munich, Munich, Germany Introduction and Aims: Uromodulin-associated kidney disease (UAKD) is a heritable renal disease in humans caused by mutations in the uromodulin (UMOD) gene. Clinical symptoms of UAKD are very heterogeneous and can comprise hyperuricemia, gout, alteration of urine concentrating ability, and histological alterations of the kidneys like glomerular and tubular cysts, interstitial nephritis and fibrosis. UAKD is progressive and can lead to end-stage kidney disease. Clinical symptoms of UAKD result from dysfunction of cells of the thick ascending limb of Henle (TALH), which is caused by maturation and trafficking defect of mutant uromodulin protein retained in the hyperplastic endoplasmic reticulum (ER) of TALH cells. UAKD belongs to the ER storage diseases.So far, pathways involved in the pathophysiology of UAKD in TALH cells are almost unknown. The recently established mouse models UmodA227T and UmodC93F harbor different amino-acid changing mutations in the Umod gene resulting in UAKD. These mutant mice were used to analyze the role of NF-kappaB signaling in UAKD. Methods: The outer medulla of kidneys of UmodA227T homozygous mutant, UmodC93F homozygous mutant, and wild-type mice was prepared and used for protein isolation. After separation of the lysates on SDS-polyacrylamide gels and electro blotting, immunoblots were performed. Kidneys of additional Umod mutant mice and wild-type mice were fixed in 4% paraformaldehyde, embedded in paraffin, and used for immunohistochemical analyses. Results: UmodA227T homozygous mutant and UmodC93F homozygous mutant mice had a higher protein abundance of NF-kappaB2 p100/p52, RelB, phospho-IKKalpha/ beta and TRAF2 in the renal outer medulla compared to age-matched wild-type littermates. Protein abundances of NF-kappaB p65, IKKalpha and TRAF3 did not differ between genotypes. TALH segments of Umod mutant mice exhibited an altered and increased immunostaining of phospho-IKKalpha/beta and RelB. Conclusions: The results of this study provide evidence for the activation of the non-canonical NF-kappaB pathway in TALH cells in our two mouse models of UAKD. MP006 CELLULAR SENESCENCE ILK DEPENDENT IS INDUCED BY THE CONSTITUTIVE ACTIVATION OF THE INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) PATHWAY Introduction and Aims: Integrin linked kinase (ILK) is a component of the multiprotein transmembrane complex which is mediating the integrin-dependent signaling. ILK plays a central role in cellular senescence induced by stressfull stimuli. ILK is up-regulated after stimulation with glycated albumin (GA), oxidants or high phosphate and increases the expression of senescent genes p53 and p16. However, the mechanisms involved in this ILK overexpression remain unclarified. On the other side, the IGF-1 axis, a nutrient sensing pathway, and its downstream intracellular effectors such as AKT, and FOXO, also has an essential role in the aging process.Aims. To analyze the role of IGF-1-AKT pathway stimulation in the cellular senescence induced by ILK overexpression. Methods: Methods. Human vascular smooth muscle cells (HVSMC) and mice proximal tubular cells (MCT) were treated with 100μM GA or the phosphate donor beta glycerophosphate (BGP, 10mM) for 24, 48 and 72 hours. After treatment senescence associated β-galactosisase activity (SA-B-GAL) was assessed with a fluorescent probe by confocal microscopy, and by the expression of p16 and p53 by western blot. ILK expression was analyzed by western blot. IGF-1 R activation was determined by western blot using specific phospho-IGF-1 receptor and IGF-1 receptor antibodies. To analyze the involvement of IGF-1 receptor in ILK overexpression and senescence we used a pharmacological inhibitor of IGF-1R activation, IGF-1R inhibitor 12μM, a stable transfection of cells with a vector encoding for Klotho protein an endogenous inhibitor of the IGF-1 receptor, and addition of Klotho recombinant protein. IGF-1 expression was analyzed by qRT-PCR. AKT and FOXO activity and expression were assessed by western blot. Results: Results. Addition GA and BGP for 24, 48 or 72 hours induced a higher mRNA expression of IGF-1 and also increased the expression of IGF-1 receptor and its activation. AKT activity was increased after GA or BGP addition and FOXO-1 was inactivated in the same conditions. In parallel, treatment with GA or BGP increases expression of ILK and β-galactosidase activity and p53 and P16 expression, in both MCT and HVSMC. Cells transfected with a vector encoding for transmembrane Klotho protein did not overexpressed ILK after treatment with GA or BGP and did not undergo to senescence. On the other hand, inhibition of IGF-1 R activity with the specific inhibitor, also suppressed ILK expression and senescence induced by stressful stimuli. All inhibitors used effectively suppressed IGF-1Receptor activation. Conclusions: Conclusion. We proposed that activation of IGF-1 receptor pathway by stressful stimuli, such as glycated albumin and high phosphate levels, is responsible for the overexpression of ILK in senescent cell. MP007 REDUCTION OF CCN2 EXPRESSION BELOW NORMAL LEVELS EFFECTIVELY REDUCES DAMAGE AND FIBROSIS IN A SEVERE MODEL OF CKD Introduction and Aims: Several papers have described the efficacy of Cyr61/CTGF/ Nov family member 2 (CCN2) targeted therapy during fibrotic disease. Moreover, we showed that a 50% CCN2 reduction by genetic deletion in severe models of CKD was not sufficient to hamper fibrosis. Notably, in these models the Ccn2 expression remained elevated above baseline.Aim: To investigate whether further reduction of CCN2 to below baseline levels is protective in severe and chronic kidney disease. Methods: Methods: Heterozygous constitutional CCN2 knock out mice (+/-) and ROSACre tamoxifen inducible conditional CCN2 full knock out (cKO) mice were used in this study. Appropriate wild type (wt) or corn-oil injected mice (Veh) were used as controls. The ureter of the left kidney was ligated (OBstructed Kidney, OBK). At 7 or 14 days after the UUO and kidneys were analysed using (immuno)histochemistry, RT-qPCR and Western blot analysis. Contralateral kidneys (CLK) were used as internal control. Statistical significance was determined using a one-way ANOVA with a Tukey correction for multiple testing. Results: qPCR analysis revealed that after 7 days, Ccn2 expression was doubled in wtOBK. In the +/-OBKs, Ccn2 increased relative to +/-CLK, but stayed below wtCLK levels. Analysis of PAS and Sirius Red stained slides showed that in the +/-OBKs the kidney morphology was better preserved and less scar tissue accumulated. After 14 days of UUO, Ccn2 expression in cKO-OBKs was reduced by 90% relative to Veh-OBK, the remaining level being well below normal baseline. This was associated with profound reduction of kidney damage and fibrosis in the cKO-OBKs. Conclusions: A 50% reduction of Ccn2 expression to still elevated levels relative to normal controls had no appreciable effect in 14 day UUO. However, reduction of Ccn2 expression to below normal control levels effectively reduced damage and fibrosis even in this severe model of CKD. MP008 TAMOXIFEN DECREASES RENAL FIBROBLAST ACTIVATION VIA MODULATION OF ESTROGEN RECEPTOR-α MEDIATED TGF-β1/SMAD SIGNALING PATHWAY Sung Kwang Park1, Dal Kim1, Ae Sin Lee1, Yu Jin Jung1, Kyoung Hee Yang1, Sik Lee1, Won Kim1, Won Kim1 and Kyung Pyo Kang1 1Chonbuk National University Medical School, Jeonju, Republic of Korea Introduction and Aims: After insult to the kidney, renal fibrotic process is initiated with sustained inflammation, activation of matrix-producing fibroblast and accumulation of extracellular matrix. Modulation of renal fibrogenesis may be one of the promising therapeutic targets for attenuating the progression of chronic kidney diseases.Tamoxifen, known as selective estrogen receptor modulator (SERM), has anti-estrogenic effect on mammary gland and uterus and can be used to treat estrogen receptor (ER) positive breast cancer patients after surgery or radiation therapy. In addition, tamoxifen has been successfully used for treating fibrosclerotic disorders such as idiopathic retroperitoneal fibrosis, encapsulating peritoneal fibrosis, fibrosing mediastinitis and desmoid tumors. On the basis of above considerations, we investigated the effect of tamoxifen on TGF-β1-induced renal fibroblast activation and their mechanisms. Methods: In vitro experiments were performed using rat renal fibroblast cell line (NRK-49F cells). Cell proliferation and migration were evaluated by XTT assay and wound healing assay. TGF-β1-induced renal fibroblast activation was evaluated by Western blot analysis. Results: Treatment of TGF-β1 increased significantly proliferation and cell migration of NRK 49F cells compared to that of vehicle treated cells. Tamoxifen treatment decreased TGF-β1-induced proliferation of NRK 49F cells in a dose dependent manner. Tamoxifen treatment significantly inhibited TGF-β1-induced cell migration. Tamoxifen decreased TGF-β1-induced α-SMA expression in a dose dependent manner. Treatment of NRK 49F cells with TGF-β1 increased levels of phospho-Smad2 and 3 in a time dependent manner. TGF-β1 treatment increased the levels of phospho-Smad2 and 3 in NRK 49F cells, and tamoxifen significantly decreased the levels of phospho-Smad2 and 3 expressions in a dose dependent manner. To verify the effect of tamoxifen on ERα-mediated TGF-β1/Smad signaling pathway, we treated ERα siRNA in NRK 49F cell. After knock-down of ERα, effect of tamoxifen was abolished. Conclusions: These data suggest that tamoxifen modulates activation of renal interstitial fibroblasts through ERα-mediated TGF-β1/Smad signaling pathway. MP009 ROLE OF INTEGRIN-LINKED KINASE (ILK) IN ENDOTHELIAL DYSFUNCTION ASSOCIATED WITH UREMIA IN CHRONIC KIDNEY DISEASE Andrea García-Jérez1, Alicia Luengo-Rodríguez1, Rafael Ramirez-Chamond1, Julia Carracedo2, Diana Medrano-Andres3, Diego Rodriguez-Puyol3,1 and Laura Calleros1 1Alcala University, Alcala de Henares/Madrid, Spain, 2Reina Sofia University Hospital, Córdoba, Spain, 3Principe de Asturias University Hospital, Alcala de Henares/Madrid, Spain Introduction and Aims: Patients with chronic kidney disease (CKD) have a much higher risk of cardiovascular diseases than the general population. According to the most accepted hypothesis, endothelial dysfunction and damage that is present in almost all patients with CKD, seems to be the start element in the cascade of events that leads to cardiovascular disease. The endothelium of patients with CKD is permanently exposed to uremic toxins. Several uremic toxins, mostly protein- bound compounds such as indoxyl sulfate and p-cresyl sulfate that have poor clearance by conventional dialysis, induces specific endothelial toxicity. However, the molecular mechanism by which the uremic toxins regulate the early stages of endothelial dysfunction remains unclear. Recent studies have demonstrated the important role of integrin-linked kinase (ILK), a multidomain signaling protein that localizes to focal adhesions, in the maintenance of endothelial integrity.In the present study we investigate the involvement of ILK in the mechanism that lead to vascular endothelial dysfunction that occurs in uremia. Methods: Confluent EA.hy926 endothelial cells were incubated for different concentrations and times with indoxyl sulfate and/or p-cresyl sulfate, in the presence of 2,5% of human serum. The protein level of ILK and phospho-serine 9-glycogen synthase kinase 3β (P-GSK-3β) was determined by western blot. ILK kinase activity was also determined by immunoprecipitation in vitro kinase assay. Cell number was assessed using the MTT assay and endothelial cell proliferation was also determined by 5-bromo-2-deoxy-uridine (BrdU) incorporation into cellular DNA. The effect of uremic toxins on cell viability was measured by Tripan blue exclusion. The apoptosis was determined by Anexin-V, flow cytometric analysis of cells with sub-G1 DNA content and by morphology. Results: We performed dose and time-response experiments analyzing the effect of p-cresyl sulfate and indoxyl sulfate on endothelial cells signaling and function. First, we show that exposure of EA.hy926 endothelial cells to indoxyl sulfate (25-100 μg/ml), p-cresyl sulfate (10-100 μg/ml) or both toxins together resulted in a significant increase in the ILK downstream effector GSK-3β phosphorylation, compared to cells exposed to supplemented with human serum control medium. This increase was dose and time-dependent and reached maximum at high doses of both toxins together. Moreover, the ability of uremic toxins to increase ILK activity was confirmed by in vitro immunoprecipitated ILK kinase activity. Next, we observed that both toxins induced a decrease in cell proliferation and viability. Therefore, we evaluated whether these two compounds would produce endothelial cell apoptosis. The indoxyl sulfate, p-cresyl sulfate or both toxins together, induces a sligth increase on the percentage of annexinV-positive cells, hypodiploid apoptotics cells and in apoptotic morphology confirmed by DAPI staining that disclosed fragmented, bright nuclei. Interestingly, the cells displayed a strong increase in apoptosis percentage when the protein ILK was knockdown with small interfering RNA (siRNA). Conclusions: This study demonstrate for the first time the implication of ILK in the endothelial cells apoptotic process induced by uremic toxins, that are difficult to remove by standard dialysis strategies. These results identify a molecular mechanism that could play a protective role in early stages of endothelial dysfunction observed in uremic patients. MP010 1,25-DIHYDROXYVITAMIN D3 ATTENUATES TUMOR NECROSIS FACTOR-AΑ-INDUCED TRANSFORMING GROWTH FACTOR-Β-1 PRODUCTION IN HUMAN AORTIC SMOOTH MUSCLE CELLS Hyun Woo Kim1, Su-Kil Park2, Won Seok Yang2, Sang Koo Lee2, Jai Won Chang2 and Jang Won Seo3 1Jeju National University, School of Medicine, Jeju-si, Republic of Korea, 2Asan Medical Center, Seoul, Republic of Korea, 3Dongtan Hanlym Medical Center, Hwaseong-si, Gyeonggi-do, Republic of Korea Introduction and Aims: Proliferation of vascular smooth muscle cells (VSMCs) is an important event during the development and the progression of atherosclerosis and many VSMCs-derived cytokines such as tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta-1 (TGF-β-1) have an important role in development of atherosclerotic lesions. Although vitamin D has been suggested to play a role in preventing the development of atherosclerotic diseases, the molecular mechanisms associated with TNF-α-induced TGF-β-1 production in VSMCs are unclear. Our study was to evaluate the effect of 1,25-dihydroxyvitamin D3, biologically active form of vitamin D, on the production of TGF-β-1 induced by TNF-α in human aortic smooth muscle cells (AOSMCs). Methods: Human AOSMCs were cultured in smooth muscle growth medium-2 with 5% fetal bovine serum in a humidified incubator maintained at 37°C, with 5% CO2. Effect of 1,25-dihydroxyvitamin D3 on TNF-α-induced TGF-β-1 production, and involvement of ras-related C3 botulinum toxin substrate 1 (Rac1), spleen tyrosine kinase (Syk), and p38 mitogen-activated protein kinase (MAPK) in TNF-α-induced TGF-β-1 production were assessed by Western blot using antibodies directed against TNF-α, TGF-β-1, Rac1, Syk, phsophorylated Syk, p38 MAPK, or phsophorylated p38 MAPK. Involvement of activator protein-1 (AP-1) and nuclear factor-kappa-B (NF-κB) in TNF-α-induced TGF-β-1 production were evaluated with decoy oligodeoxy nucleotides (ODNs) against the AP-1 and NF-κB binding sites and their mismatched ODNs. The production of intracellular reactive oxygen species (ROS) was evaluated with the probe 5-(and-6)-chloromethyl-20-70-dichlorodihydrofluorescein diacetate and involvement of ROS in TNF-α-induced TGF-β-1 production was assessed with using N-acetylcysteine. DNA-binding activities of AP-1 and NF-κB were assessed using chemiluminescent electrophoretic mobility shift assay. Results: TNF-α increased TGF-β-1 production in AOSMCs, whereas transfection with AP-1 and NF-κB decoy ODNs inhibited it, suggesting that increased AP-1 and NF-κB DNA-binding activities are associated with TNF-α-induced TGF-β-1 production. 1,25-dihydroxyvitamin D3 attenuated TGF-β-1 production induced by TNF-α and the inhibitory effect of 1,25-dihydroxyvitamin D3 on TGF-β-1 was mediated by reducing both AP-1 and NF-κB DNA-binding activities. 1,25-dihydroxyvitamin D3 attenuated Rac1 activation triggered by TNF-α, and reduced TNF-α-induced intracellular ROS accumulation. 1,25-dihydroxyvitamin D3 that downregulated TNF-α-induced ROS also inhibited Syk activation, but not p38 MAPK. 1,25-dihydroxyvitamin D3 had no effect on either TNF-α-induced I-kappa-B-alpha degradation or NF-κB nuclear translocation. Conclusions: 1,25-dihydroxyvitamin D3 attenuated TNF-α-induced TGF-β-1 production through inhibitions of activation of Rac1, generation of ROS, and activation of Syk that consequently regulated NF-κB as well as AP-1 in AOSMCs. These findings suggest that 1,25-dihydroxyvitamin D3 could have an effect on amelioration of atherosclerosis through its anti-oxidant and anti-inflammatory properties. MP011 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ AGONIST ATTENUATES INDOXYL SULFATE-INDUCED ENDOTHELIAL ACTIVATION BY SUPPRESSION OF ERK1/2 AND P38 MAPK Chien-Te Lee1, Chia-An Chou1, Yueh-Ting Lee1 and Hwee-Yeong Ng1 1Kaohsiung Chang-Gung Memorial Hospital, Kaohsiung, Taiwan Introduction and Aims: Indoxyl sulfate is one of the protein-bound uremic toxins that exert deleterious effects on cardiovascular system. Clinical studies have identified serum levels of indoxyl sulfate predicts long-term outcome of patients with chronic kidney disease (CKD). Rosiglitazone (RGZ) is an insulin sensitizer used for glycemic control in type 2 diabetes. This drug is also known as cardiovascular protective agent due to its pleiotropic effects. Whether RGZ can improve indoxyl sulfate-induced endothelial damage has not been investigated. Methods: We undertook an in vitro study to examine the effects of RGZ in indoxyl sulfate-induced endothelial injury. The cultured human umbilical vein endothelial cells (HUVECs) were exposed to two concentrations of RGZ (5μM and 10μM) and then treated with indoxyl sulfate (100 μM and 1000 μM) for 48 hours. Gene expression of adhesion molecules such as intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), Monocyte chemotactic protein-1 (MCP-1) were examined. Protein abundance of NADPH oxidase 4 (NOX4) and nuclear factor (NF) κB were analyzed. The alterations of ERK 1/2 and p38 MAPK signal pathways were also investigated. Results: Our results revealed that indoxyl sulfate significantly up-regulated ICAM-1, VCAM-1 and MCP-1. Indoxyl sulfate (1000μM) also increased the abundance of NOX4 and NFκB (182 ± 7%, p < 0.05) and NOX4 (1000 μM: 151 ± 7%, p < 0.05). The phosphorylation of ERK1/2 and P38 MAPK were increased significantly with high concentration of indoxyl sulfate (100μM: 106 ± 5% and 132 ± 6%; 1000μM: 178 ± 4% and 221 ± 7%, p < 0.01). Both low and high dose RGZ attenuated the activation of adhesion molecules and reduced the increase of NFκB and NOX4 (5μM: 135 ± 6% and 94 ± 8%, p > 0.05; 10μM: 104 ± 7% and 102 ± 8%, both p > 0.05). The activation of ERK1/2 and p38 MAPK pathways was reversed by RGZ (5μM: 126 ± 5% and 127 ± 6%, p > 0.05; 10μM: 94 ± 5% and 131 ± 7%, both p > 0.05). Conclusions: We concluded that RGZ ameliorated indoxyl sulfate-induced endothelial injury via suppression of ERK1/2 and p38 MAPK signal pathways. ii | Abstracts MP012 ALBUMIN-INDUCED APOPTOSIS OF TUBULAR CELLS IS MODULATED BY BASP1 Maria D Sanchez-Niño1, Beatriz Fernandez-Fernandez2, Maria Vanesa Perez-Gomez2, Jonay Poveda2, Ana B Sanz2, Pablo Cannata-Ortiz2, Jesus Egido2, Rafael Selgas1 and Alberto Ortiz2 1Idipaz, Madrid, Spain, 2IIS-FJD, Madrid, Spain Introduction and Aims: Albuminuria promotes tubular injury and cell death and is associated with faster progression of chronic kidney disease (CKD) to end-stage renal disease. However, the molecular mechanisms regulating tubular cell death in response to albuminuria are not fully understood. Brain abundant signal protein 1 (BASP1) was recently shown to mediate glucose-induced apoptosis in tubular cells. We have studied the role of BASP1 in albumin-induced tubular cell death. Methods: BASP1 expression was studied in experimental puromycin aminonucleoside-induced nephrotic syndrome in rats and in human nephrotic syndrome. The role of BASP1 in albumin-induced apoptosis was studied in cultured human HK2 proximal tubular epithelial cells. Results: Puromycin aminonucleoside induced proteinuria and increased total kidney BASP1 mRNA and protein expression. Immunohistochemistry localized the increased BASP1 to tubular cells. BASP1 expression colocalized with deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) staining for apoptotic cells. Increased tubular BASP1 expression was observed in human proteinuric nephropathy by immunohistochemistry, providing evidence for potential clinical relevance. In cultured tubular cells, albumin induced apoptosis and increased BASP1 mRNA and protein expression at 6-48h. Confocal microscopy localized the increased BASP1 expression in albumin-treated cells mainly to the perinuclear area. A peripheral location near the cell membrane was more conspicuous in albumin-treated apoptotic cells, where it colocalized with actin. Inhibition of BASP1 expression by a BASP1 siRNA protected from albumin-induced apoptosis. Conclusions: In conclusion, albumin-induced apoptosis in tubular cells is BASP1-dependent. This information may be used to design novel therapeutic approaches to slow CKD progression based on protection of tubular cells from the adverse consequences of albuminuria. MP013 ACTIVATION OF GPR40 ATTENUATES APOPTOSIS AND EMT INDUCED BY TNF-α IN RAT PROXIMAL TUBULAR CELLS Seong Kwon Ma1, In Jin Kim1, Chang Seong Kim1, Eun Hui Bae1 and Soo Wan Kim1 1Chonnam National University Medical School, Gwangju, Republic of Korea Introduction and Aims: G-protein-coupled receptor 40 (GPR40) plays diverse functions including regulation of physiological effects of fatty acids, insulin secretion and inflammation. However, the pathophysiological roles of GPR40 in the pathogenesis of kidney diseases have not yet been established. The present study investigated the changes in the expression of GPR40 in the obstructed kidney of mice with unilateral ureteral obstruction (UUO). Furthermore, we also investigated the effects of GPR40 activation on the apoptosis and epithelial-mesenchymal transition (EMT) induced by tumor necrosis factor (TNF)-α in rat proximal tubular (NRK52E) cells. Methods: UUO was induced in C57BL/6J mice for 2 weeks. NRK52E cells were cultured with TNF-α in the absence or presence of GW9508, a selective GPR40 agonist. Results: The protein expression of GPR40 was decreased in the obstructed kidney of mice. The expression of Bax was increased but that of Bcl-2 was decreased. The expression of transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF) and α-smooth muscle actin (SMA) was increased. In NRK52E cells, the pretreatment of GW9508 attenuated the decreased cell viability by TNF-α treatment. TNF-α treatment increased the number of cells with condensed nuclei, which was ameliorated by GW9508 pretreatment. TUNEL stain also showed that the pretreatment of GW9508 ameliorated apoptosis induced by TNF-α treatment. TNF-α treatment increased the protein expression of Bax/Bcl-2, TGF-β1, CTGF and α-SMA, which was ameliorated by GW9508 pretreatment. TNF-α treatment activated the generation of reactive oxygen species (ROS) and the expression of Src/epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK), which was counteracted by the pretreatment of GW9508. Conclusions: Thus, activation of GPR40 attenuates the apoptosis and EMT induced by TNF-α in NRK52 cells through the inhibition of ROS generation, Src/EGFR/ERK signaling pathway, pro-apoptotic proteins, and pro-fibrotic proteins. MP014 EGR1 AND EGR2 IN MYOCARDIAL FIBROSIS OF TGF-β TRANSGENIC MICE AND THEIR ASSOCIATION WITH TIMP-1 1Semmelweis University, Budapest, Hungary Introduction and Aims: Recently we found strong association between cardiac TIMP-1 expression and the severity of myocardial fibrosis in TGF-ß1 transgenic mice on different genetic backgrounds (B6 and CBAxB6 F1) [NDT 2012, 27 (S2); ii328]. In the present study, we aimed to investigate the strain dependent molecular regulation of cardiac TIMP-1 and its association with profibrotic transcription factors EGR1 and EGR2. Methods: Cardiac samples of 14-days old male TGF-ß1 transgenic B6-TGFß (n=8) and CBAxB6-TGFß F1 (n=9) mice, and wild type B6 (n=6) and CBAxB6 F1 controls (n=6) were analyzed for mRNA and protein expression. Results: Compared to wild type and B6-TGFß samples, CBAxB6-TGFß hearts showed 4-fold higher TIMP-1 gene expression (relative expression:, B6: 1.5±0.6; CBAxB6: 1.4 ±0.3; B6-TGFß: 1.6±0.7; CBAxB6-TGFß: 6.3±2.6; mean±SD, p<0.001) which was associated with increased EGR1 (B6: 1.3±0.5; CBAxB6: 1.3±0.4; B6-TGF-ß: 1.0±0.7; CBAxB6-TGFß: 2.4±0.4, p<0.01). and EGR2 mRNA expression (B6: 0.9±0.2; CBAxB6: 0.8±0.5; B6-TGFß: 0.7±0.4; CBAxB6-TGFß: 2.3±0.3, p<0.05). The fibrotic CBAxB6-TGFß hearts depicted significantly increased number of EGR1 positive cardiomyocytes, and also increased number of EGR2 positive cells as compared to controls and B6-TGFß mice. Ingenuity Pathway Analysis revealed new associations between EGR molecules and TIMP-1, in which STAT3 (B6: 0.8±0.1; CBAxB6: 1.2±0.2; B6-TGFß: 1.1±0.1; CBAxB6-TGFß: 1.9±0.6; p<0.05) might play a significant role. Conclusions: We conclude that EGR2 might contribute to the development of myocardial fibrosis. Based on our data we suggest that EGR1 and EGR2 might contribute to the strain dependent regulation of TIMP-1 in this model. MP015 KIM-1 VALUES IN URINE OF CLEAR CELL RENAL CARCINOMA PATIENTS ARE ASSOCIATED WITH TUMOR SIZE, TNM STAGE, FUHRMAN GRADUS AND THERAPY RESPONSE Vojvodic Danilo , Milovic Novak , Cerovic Snezana , Stanojevic Ivan, Drivanstanojevic@yahoo. Com., Kovacevic Bozidar, and Mirjana Mijuskovic1,1,1,1 1Faculty of Medicine, Military Medical Academy, Belgrade, Serbia Introduction and Aims: KIM-1 is suggested to play a key role in the carcinogenesis and progression of renal cell carcinoma. KIM-1 staining is upregulated in proximal tubule-derived renal cell carcinoma (RCC) including clear renal cell carcinoma (cRCC). Attention is currently focused on the potential use of the urinary level of KIM-1(uKIM-1) in making an early diagnosis and determination of the histologic characteristics of renal cell carcinoma. This study was designed to prospectively examine uKIM-1 level before, 7th day and 30th day after removal of cRCC. Methods: A total of 39 patients, pts (25 male and 14 female, 37-77 years, median 55) were enrolled in the study based on pre-operative imaging studies and post-operative diagnosis of cRCC. At the time of operation all pts had GFR (CKP-EPI formula) higher than 60 ml/min/1.73 m2. Urinary KIM-1 (ELISA, TIM-1/KIM-1/HAVCR, R&D Systems Inc, Minneapolis, MN, USA) were measured pre-operative (39 pts) and during follow-up (7th day-36 pts and 30th day-23 pts) after removal of renal tumors. Results: KIM1 concentration was significantly elevated in RCC patients urine samples comparing to healthy controls but not to other none celar cell renal carcinoma patients. KIM1 values were significantly associated with large tumor mass, higher TNM stage and Fuhrman grade, comparing to levels found in samples of patients with smaller tumor mass, and lower TNM and Fuhrman ( p=0.0007, p=0.0001, p=0.0007, respectively). Highest average concentration of KIM-1 in urine samples was before surgery (1.1±0.3 ng/ml). Average concentration of KIM 1 was significantly decreased in both, the first (0.3±0.3, p= 0.0070) and the second control (0.1±0.9, p<0.0000) compared to pre-operative values. Completely nephrectomised patients had continous fall of urinary KIM1, while partial nephrectomy induced even KIM1 increase on first interval control. Anyway, both surgical procedures induced KIM1 reduction to a levels identical in healthy controls. Conclusions: Our prospective study showed significant reduction in uKIM after nephrectomy, suggesting that urine KIM-1 may serve as a valuble biomarker for diagnosis of cRCC and therapy monitoring. MP016 INFLAMMATORY SLAN+ CELLS IN CKD5 AND HEALTHY CONTROLS - IN VITRO GENERATION OF SLAN+ MONOCYTES Christof Ulrich1, Henrike Berger1, Bogusz Trojanowicz1, Felix Kohler1, Anna Wolf1, Eric Seibert1, Roman Fiedler1, Silke Markau1, Marcus Glomb1 and Matthias Girndt1 1Martin-Luther-University Halle, Halle, Germany Introduction and Aims: Monocyte heterogeneity (CD14++16-(Mo1), CD14++CD16+ (Mo2), CD14dim CD16+ (Mo3)) is influenced by inflammatory diseases. CD16+ monocytes expressing large amounts of inflammatory cytokines are elevated in chronic kidney disease. Their role in cardiovascular mortality is widely acknowledged. Within CD14dimCD16+ cells, a cell population (20 -70%) expresses 6-sulpho LacNAc (SLAN +) as a result of glycosylation of CD162. The functions of these cells are controversially discussed. Some authors link SLAN+ to inflammatory dendritic cells - particularly relevant to dermal diseases. Patients on maintenance hemodialysis very often face cutaneous abnormalities including pruritus, atopic dermatitis and xerosis.We investigated the presence of SLAN+ monocytes in chronic kidney disease stage 5 patients and control subjects. The development and the fate of these cells were studied in vitro - following the hypothesis that uremic conditions will favor the development of this cell type. Methods: 31 healthy subjects (age 53±6), recruited from hospital staff as well as 28 hemodialysis patients (age 62±10) were enrolled in the study. For in vitro studies PBMC’s of healthy subjects (N=6) were incubated (24-72h) under uremic (autologous vs. pooled uremic sera) and glycemic conditions. SLAN positivity was analyzed by flow cytometry and by Western blot technique. Results: Ex vivo analysis confirmed SLAN+ monocytes to exclusively be part of Mo3. SLAN+ cells (% of total monocytes) were more frequent in CKD5 patients in comparison to controls (4.7±2.7 vs. 2.9±2.2, p=0.006). In contrast to our hypothesis, SLAN positivity was not increased by uremic culture conditions. But, while in uremia SLAN+ cells mainly can be defined by low CD14 expression (Mo3) - in medium supplemented with autologous serum, the phenotype of SLAN +cells changed towards Mo2. Interestingly, the frequency of SLAN+ monocytes was 2- to 3-fold increased when culture medium was supplemented with high concentrations of different mono-/ disaccharides. Conclusions: Our data confirm the enrichment of inflammatory monocytes in CKD5 patients - in this case, we found significantly elevated numbers of SLAN+Mo3. The in vitro generation of these cells could not simply be reached by mimicking uremic, inflammatory conditions. In contrast, changing the sugar composition of the culture medium, the frequency of SLAN+ monocytes significantly increased. These results focus the interest on metabolic complications of CKD patients. MP017 THE BENEFICAL EFFECT OF VITAMIN D3 ON CALCIUM ENTRY VIA PURINERGIC P2X7 RECEPTORS IN CKD PATIENTS Introduction and Aims: Increased intracellular calcium concentration can be toxic to the cells. Already an early-stage chronic kidney disease (CKD) is associated with increased free cytosolic calcium concentration ([Ca2+]i) and intracellular calcium reserves, resulting in impairment of cellular functions/structure [1, 2]. The purinergic P2X7 receptors, which are responsible for multiple processes including calcium influx participate on these disturbances. Activation with ATP results in opening of cation-specific channels followed by forming non-specific pores that allow the passage of low-molecular-mass solutes (< 900 Da). The aim of this study was to examine the effect of vitamin D3 supplementation on the functionality of P2X7 channels and pores and the expression of P2X7 receptors. Methods: The study involved 16 non-diabetic patients with stage 2-3 CKD, all of them supplemented with cholecalciferol 7 000 - 14 000 IU/week for 6 months. Cytosolic Ca2 + measurements were performed by Fluo-3 fluorimetry in isolated peripheral blood mononuclear cells (PBMCs). To determine the P2X7 receptors function, a highly selective antagonist (AZ11645373) and the most potent agonist (BzATP) were used. The function of P2X7 pores was measured by ethidium uptake at basal conditions and after stimulation or inhibition. The expression of surface P2X7 receptors was evaluated by flow cytometry using the antibody to this receptor (anti-P2X7 extracellular). To visualize P2X7 receptors, the fluorescence microscopy was used. Results: Vitamin D3 supplementation reduced [Ca2+]i from 120 ± 1.6 to 106 ± 1.1 nmol/l, (P < 0.001) in PBMCs of CKD patients. The application of P2X7 receptors antagonist on PBMCs before vitamin D3 supplementation reduced [Ca2+]i (120 ± 1.6 v.s. 112 ± 2.2 nmol/l, P < 0.001), but had no effect after vitamin D3 supplementation. Moreover, the effect of an antagonist on stimulated PBMCs was significantly decreased after vitamin D3 supplementation (36.4 ± 7.1 v.s. 17.5 ± 2.9 nmol/l, P < 0.01). These results demonstrate the inhibitory effect of vitamin D3 supplementation on calcium entry through P2X7 receptor channels. The effect of vitamin D3 supplementation on the permeability of P2X7 pores at basal conditions and after the application of the agonist or antagonist has not been demonstrated. The expression of surface P2X7 receptors was reduced by 55 %. Conclusions: Vitamin D3 supplementation has a beneficial effect on [Ca2+]i already in early stages of CKD. The decreased calcium entry via P2X7 channels and the reduction of P2X7 receptor expression participate on this effect. References1 Lajdova I. et al.: Nephrol Dial Transplant 24: 3376-81, 20092 Lajdova I. et al.: Kidney Blood Press. Res 35: 48-57, 2012Supported by grant VG SZU 19-90-07,"NanoNet 2" (ITMS 26240120018) andAPVV-0242-11. MP018 THE REGULATION OF THE NITRIC OXIDE SYSTEM CAN MODULATE THE KLOTHO EXPRESSION IN KIDNEY VIA TWIST 2 AND E-CADHERIN Seung Ok Choi1, Jae Seok Kim1, Byoung Geun Han1 and Jae Won Yang1 1Yonsei University Wonju College of Medicine, Wonju, Republic of Korea Introduction and Aims: The klotho was originally identified as an anti-aging peptide, and was subsequently found to have various biologic actions. Animal experiments clearly showed that renal klotho was lacking in acute kidney injury from a variety of causes including ischemia/reperfusion injury. We investigated whether there was the relationship between NO (nitric oxide) system and klotho expression in kidney, and also investigated TWIST (basic helix-loop-helix transcription factors)-1 and 2, E-cadherin as a possible pathway of such relationship. Methods: The 10th weeks Sprague-Dawley rats (N= 24, 200g, male) were divided into four groups. Next, we fed low salt diet to control group (N=6), and gave drinking water mixed with L-NAME 1 mg/mL to L-NAME group (N=6), and udenafil 5 mg by subcutaneous injection to Udenafil group (N=6), L-NAME and udenafil to the L-NAME / Udenafil group (N=6) for 4 weeks. On the 28th day, we collected blood and urine samples, and took the kidneys from rats. And then, we measured the level of serum creatinine, urine nitrate/nitrite, urine cGMP by ELISA, and conducted immunohistochemical staining, RT-PCR for klotho, TWIST 1 and 2, E-cadherin. Results: The serum creatinine and urine nitrate/nitrite level did not show statistical differences between four groups. On the other hand, between control, L-NAME, Udenafil and L-NAME / Udenatil groups, there were significant differences in urine cGMP (2.59±0.88, 1.79±0.99, 1.20±0.52, 0.69±0.59 pmol/well, p=0.0087), klotho mRNA expression (0.98±0.01, 0.30±0.11, 0.68±0.15, 0.54±0.26, p=0.0017), TWIST-2 mRNA expression (1.90±1.65, 139.27±114.87, 10.33±8.42, 20.19±12.25, p=0.0163), and E-cadherin mRNA expression (0.64±0.32, 1.57±0.97, 1.24±1.27, 13.82±3.04, p=0.0029). Therefore, the blocking of NO system decreased klotho expression with TWIST-2 increase, while the induction of NO system increased klotho expression with E-cadherin increase. Conclusions: This study demonstrates that the regulation of NO system can modulate the klotho expression in kidney via TWIST-2 and E-cadherin pathway. MP019 MIR-146A INHIBITOR ATTENUATES LPS-INDUCED SUPPRESSION OF NRK-52E CELL SURVIVAL VIA INCREASING MEK/ERK1/2 ACTIVITY Shanying Liu1, Jun Lv1, Ruiming Chang1, Fang Su1 and Weiwen Liang1 1Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Introduction and Aims: MicroRNA-146a has been recognized as a negative regulator of inflammatory response by targeting TLR4-TRAF6 in immune and non-immune cells. However, its role in the regulation of cell survival in the presence of LPS remains unclear. In this study we investigated the effect of miR-146a on LPS-induced suppression of renal tubule epithelial NRK-52E cell survival and the involvement of MEK/ERK1/2. Methods: MiR-146a mimics and inhibitor have been transfected into NRK-52E cells 24 hours before LPS treatments, respectively. Cell survival was evaluated by cck-8 assay. Protein levels of TRAF6, total and phosphorylated ERK1/2, and tubulin were measured by Western blotting. Results: LPS reduced viability of NRK-52E cell at 1μg/ml at 3 days. Transfection of miR-146a mimics augmented LPS-induced suppression of NRK-52E cell survival as compared with negative controls. However, in the presence of miR-146a inhibitor, NRK-52E cell survival was enhanced upon LPS challenge in association with increases in TRAF6 protein and phosphorylation of ERK1/2. When phosphorylation of ERK1/2 was blocked by a selective inhibitor for MEK1/2, U0126, survival of NRK-52E cells exposed to LPS was further reduced. Conclusions: MiR-146a inhibitor prevents LPS-induced suppression of NRK-52E cell survival by enhancing activation of MEK/ERK1/2. MP020 MODULATION OF MONOCYTE SUBSETS BY IMMUNOSUPPRESSANTS Introduction and Aims: Heterogeneity of human monocytes subsets - which comprise classical CD14++CD16-, intermediate CD14++CD16+ and nonclassical CD14+CD16 ++ monocytes - has widely been acknowledged. Intermediate monocytes have been found to mediate inflammation in CKD. Subsequently, immunomodulation of monocyte subsets has been proposed. We aimed to analyze the impact of immunosuppressive medications which are routinely used in nephrology - e.g. steroids and mTOR inhibitors - on intermediate monocytes. Methods: In experimental analyses we tested the effect of rapamycin (100 nM) and dexamethasone (250 nM) on the differentiation of monocyte subsets from CD34+ hematopoetic stem cells. We next validated clinical implications of in vitro findings in our HOMe ALONE study, which analyzed monocyte subset distribution in 159 stable renal allograft recipients ≥ 12 months after transplantation. Among all study participants, 27 patients received mTOR inhitibors, and 114 patients received steroids. Co-medication comprised calcineurin inhibitors and / or mycophenolate mofetil in all patients. Results: In vitro, differentiation of monocyte subsets from hematopoetic stem cells was significantly reduced after both rapamycin (3.7 fold reduction of intermediate monocytes) and dexamethasone (2.2 fold reduction, respectively) treatment. In our clinical validation cohort, patients on steroid medication had higher cells counts of classical (steroid: 639 ± 202 cells/μl; no steroid: 462 ± 182 cells/μl; p < 0.001) and intermediate (steroid: 37 ± 22 cells/μl; no steroid: 28 ± 16 cells/μl; p < 0.01) but lower cell counts of nonclassical monocytes (steroid: 58 ± 29 cells/μl; no steroid 76 ± 41 cells/ ii | Abstracts μl; p < 0.05) compared to patients not on steroid medication. mTOR medication had no significant impact on cell counts of monocyte subsets. Conclusions: mTOR medication affected in vitro differentiation of monocyte subsets in the experimental setting, but had questionable clinical consequence. In contrast, steroids modulated differentiation and distribution of monocyte subsets both in vitroand in vivo. Given the clinical implications of monocyte heterogeneity in CKD, further studies will have to delineate the molecular pathways by which immunosuppressive medication modulate monocyte subsets. MP021 PHOSPHOTRIESTERASE-RELATED PROTEIN SENSED PROTEINURIA AND CONFERRED RENAL TUBULAR CELL ACTIVATION IN MEMBRANOUS NEPHROPATHY Jin-Shuen Chen1, Chao-Wen Cheng2, Li-Cheng Chang3 and Chung-Ze Wu4 1Tri-Service General Hospital, Taipei, Taiwan, 2Graduate Institute of Clinical Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan, 3School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, 4Division of Endocrinology and Metabolism, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan Introduction and Aims: Membranous nephropathy (MN) is a common cause of nephrotic syndrome that may progress to end-stage renal disease (ESRD). The formation of MN involves the in situ formation of subepithelial immune deposits and leads to proteinuria; however, the underlying mechanism of how MN leads to ESRD remains unclear. Methods: The iTRAQ and cDNA microarray technologies were applied to identify the differential renal protein and mRNA expression changes in a cationic bovine serum albumin-induced MN mouse model. Results: Seven genes had shown a consistent expression pattern in both mRNA and protein levels between MN and control groups; among these, phosphotriesterase-related protein (PTER) was selected for further validation. PTER was significantly increased in the MN mouse model and clinical cases, and mainly expressed in the renal tubular cells. The expression of PTER was increased in a concentration- and time-dependent manner in the in vitro proteinuria tubular cell model. Specifically knockdown the expression of PTER by RNA interference had diminished proteinuria-induced inflammatory cytokines production. Conclusions: Our findings revealed the PTER can sense proteinuria in the progression of MN, induce tubular cell activation and lead to ESRD. MP022 EVALUATION OF EXPRESSION AND FUNCTION OF AT1 AND AT2 RECEPTORS IN NUCLEAR MEMBRANE OF MESANGIAL CELLS Antônio S Novaes1, Fernanda T Borges1 and Mirian A Boim1 1Federal University of São Paulo, São Paulo, Brazil Introduction and Aims: Currently is discussed the biological relevance of the intracellular renin-angiotensin system (RAS). The presence of receptors in the intracellular compartment may mediate nonclassical effects of Ang II, such as, growth, proliferation, and regulation of gene expression of AngII target genes such as the pro-inflammatory and pro-fibrotic. The objectives of this study were to evaluate the presence and the ability of AngII to bind to these receptors present in the nuclear membrane of human mesangial cells (HMC). Methods: The presence of AT1 and AT2 receptors in the nuclear membrane HMC was evaluated in intact cells (immunofluorescence) and in isolated nuclei (western blot). The ability of Ang II to bind to its receptors was analyzed under increased AngII concentrations in both inside and/or outside of the cell. AngII binding to nuclear receptors, was evaluated under following conditions: Control: HMC and isolated nucleus HMC incubated with secondary antibody conjugated with fluorochrome; HMC and isolated nuclei labeled with anti-AT1 and anti-AT2 antibodies; HMC pre pretreated with losartan (AT1 blocker) and PD-123319 (AT2 blocker); HMC and isolated nuclei exposed to AngII labeled with fluorochrome-conjugated FITC. Results: Nuclear extract from HCM expressed AT1 and AT2 receptors proteins. These receptors were observed by immunostaining. The presence of AT1 and AT2 blockers hindered Ang II to bind the receptors in both intact HMC and in isolated nuclei. The addition of exogenous labeled Ang II resulted in co-localized staining compatible with both receptors in isolated nuclei and HCM indicating that the exogenous Ang II was able to bind to nuclear receptors. Conclusions: These results showed that HMC present nuclear AT1 and AT2 receptors. Endogenous and exogenous AngII is able to bind nuclear receptors. The actions of Ang II mediated by activation of these receptors are being investigated. Introduction and Aims: Sustained proteinuria and tubulointerstitial damage have been closely linked to progressive renal failure. Upon protein endocytosis, tubular epithelial cells are thought to produce mediators that promote inflammation, tubular degeneration, and fibrosis. Previous studies demonstrated that genes encoding membrane transporters in renal tubular cells were up- or down- regulated by proteinuria.P-glycoprotein (Pgp) is a glycoprotein involved in the ATP-dependent transmembrane efflux of a wide range of compounds leading to a decrease in drug concentration within the cell. In the kidney Pgp is mainly expressed in the proximal tubule. Albumin is able to reduce expression and function of Pgp in HK-2 proximal tubular cells. Bradykinin (BR) is involved in inflammation, and increasing clinical evidence suggests that BR could play a role in the beneficial effect of ACE inhibitors in diabetic nephropathy. BR inhibited morphological alterations in HK-2 cells exposed to albumin.The aim of this study was to investigate the effect of BR on Pgp expression in HK-2 cells cultured in presence of albumin. Methods: Tubular cells were cultured in presence of albumin (15 mg/ml) and BR (20 nM) in culture medium for 72 hours. Pgp protein expression was assessed by Western Blot (WB). Gene expression was evaluated by RT-PCR. Results: After albumin exposure a reduction in Pgp expression to 47% respect to controls (Western blot) was found. BR added to the medium partially restored Pgp expression to 67% of controls. RT-PCR did not show significant differences in gene expression between cells treated with BR and controls. Conclusions: Tubular cells exposed to albumin show a reduction in the expression of Pgp with ensuing impairment in the membrane transport function. The Pgp transport impairment induced by albumin may increase the sensitivity of tubular cells to chemicals leading to progression of renal damage observed in proteinuric diseases. The albumin down-regulation of Pgp can be reversed by BR providing further knowledge on the pathophysiology of tubular injury induced by proteinuria. The results of this study suggest that kinin system may make tubular cells less sensitive to protein damage and provide additional information on ACEI positive influence in proteinuric kidney diseases. MP024 IRON STIMULATION ENHANCED CALCIFICATION IN VASCULAR SMOOTH MUSCLE CELLS Sayuri Hamahata1, Yasuyuki Nagasawa1, Mutsuki Kawabe1, Aritoshi Kida1, Mana Yahiro1, Masayoshi Nanami1, Yukiko Hasuike1, Takahiro Kuragano1, Keiji Nakasho1, Hideki Ohyama1 and Takeshi Nakanishi1 1Hyogo College of Medicine, Nishinomiya, Japan Introduction and Aims: In CKD patients, atherosclerosis is one of important key factors which determine their prognosis. It was reported the calcification induced by TNF-alpha was related with iron in HUVEC cells by our group (Nanami-M, et al, Arterioscler Thromb Vasc Biol,2005). The feature of the atherosclerosis in CKD patients was called as Moenckeberg's arteriosclerosis which was seen in vascular media. To reveal the relationship between calcification in vascular media and iron stimulation using vascular smooth muscle cells. Methods: The aorta smooth muscle cells were cultured for three weeks. At day 0, we changed the usual culture medium to calcification medium, and TNF-alpha and iron were added to the calcification medium. Calcification in each condition was confirmed by Alizarin staining. And to reveal early mechanism to enhance the calcification by iron and TNF-alpha stimulation, we compared the gene expression profile between each condition in day 1 and day 3 using microarray analysis. Results: We confirmed both iron TNF-alpha stimulation enhanced calcification by Alizarin Staining. Moreover iron TNF-alpha stimulation at the same time enhanced calcification more strongly than single stimulation (shown below).The gene expression which increases more than 45 times one day after iron stimulation was shown in below. Each condition showed different gene expression gene profiles which shown in below partially. MP023 BRADYKININ INCREASES P GLYCOPROTEIN EXPRESSION IN HUMAN TUBULAR CELLS EXPOSED TO ALBUMIN Gianfranco Tramonti1, Nadia Romiti2 and Elisabetta Chieli2 1UO Nefrologia Trapianti e Dialisi, Pisa, Italy, 2Patologia Generale, Pisa, Italy MP024 MP024 Change of gene expression by microarray analysis Gene Description calcium-binding protein hypothetical protein olfactory receptor non-coding RNA colony stimulating factor 3 ZNF539 protein hypothetical protein fructose-1,6-bisphosphatase 2 cramped-like (Drosophila) Gene Smpbol Introduction and Aims: It is well-known that patients with chronic kidney disease (CKD), even if they have either obesity or diabetes, exhibit severe insulin resistance. In dialysis patients, cardiovascular mortality rate is approximately 30 times higher than that of general population. The insulin resistance might be involved in such a high mortality of this population. Although uremic toxins can be involved in these mechanisms, little is known so far. Previously, we demonstrated that p-cresol, one of uremic toxins, inhibited proliferation and differentiation, and induced apoptosis in preadipocytic 3T3-L1 cells and that decreased number of mature adipocytes was attributable to reduced glucose uptake in the presence and absence of insulin (Artificial Organs, in press). However, p-cresol generated by the intestinal flora is mostly conjugated to be p-cresylsulfate, so that p-cresol exists at very low concentrations even in CKD patients. In addition, a recent study has shown that the serum concentration of p-cresylsulfate predicts mortality in CKD patients of each stage. Thus, we examined the effects of p-cresylsulfate in adipocytes. The purpose of this study is to investigate effects of uremic toxin p-cresylsulfate on proliferation, apoptosis, differentiation and glucose uptake in adipocytes. Methods: We cultured preadipocyte cell line 3T3-L1 cells and were differentiated with 500μM IBMX, 250nM dexamethasone, 10μg/ml Insulin after 90% confluency. Treatment with p-cresylsulfate was performed in various concentrations (2-200 μM). Cell proliferation was determined by cell count and Brd-U antibody detection method. The maturity of adipocyte was investigated by oil red-O staining and by real-time PCR to see the mRNA expression of PPARγ. Apoptosis was measured by ELISA kit. We also examined glucose uptake in the presence and absence of insulin using radiolabeled 2-deoxyglucose. Results: In the cell count experiment, significant change was not observed in 2-200 μM of p-cresylsulfate treatment during proliferation. Brd-U antibody detection showed similar results. Moreover, the apoptotic cell number was not increased, and mRNA expression of PPARγ, a main regulator of the differentiation was not changed by treatment with 2-200μM of p-cresylsulfate. In oil red-O staining at day 7, no significant difference was found in the number of mature adipocytes after treatment with p-cresylsulfate. On the other hand, a fold increase in 3H-labeled 2-deoxyglucose glucose uptake by insulin stimulation over the baseline was significantly decreased by 200μM of p-cresylsulfate in mature 3T3-L1 adipocytes. To investigate an involvement of inflammatory cytokines in reduced insulin sensitivity, we performed real-time PCR and showed increased expressions of TNFα and MCP-1 after treatment with p-cresylsulfate. When cells were pretreated with neutralizing antibody against TNFα, decreased response of glucose uptake by p-cresylsulfate was recovered to the control level. These findings suggest that increased expression of TNFα and MCP-1 induced by p-cresylsulfate is involved in the development of insulin resistance in mature 3T3-L1 adipocytes. Conclusions: Unlike p-cresol, p-cresylsulfate did not affect normal cell cycle, apoptosis, and differentiation of preadipocyte into mature adipocyte. However, high concentration of p-cresylsulfate developed insulin resistance through induction of cytokine/chemokine expression and most probably through an inhibition of insulin signaling. These findings indicate that the accumulation of uremic toxins may induce insulin resistance, and eventually lead to poor prognosis in chronic dialysis patients. MP026 SIRT1 ACTIVATION PROTECTS HHE-INDUCED OXIDATIVE STRESS IN M1 CELLS local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. The aldehyde products of lipid peroxidation such as 4-hydroxy-2-hexenal (HHE) might be responsible for the tubular injury. The present study was aimed to investigate the effects of Sirt1 on renal medulla and its signaling mechanisms. Methods: The protein expression of Sirt-1 and AQP2 was determined by semiquantitative immunoblotting in vivo (M-1 cells) and in vitro (DOCA-salt hypertensive rats model). The protein expression of NOX4, NF-κB, mitogen activated protein kinase (MAPK), COX-2 was determined by semiquantitative immunoblotting after M-1 cells were treated with 10 μM of HHE and co-treatment with NF-κB inhibitor (Bay 117082), N-acetyl-L-cysteine (NAC), or resveratrol (Sirt-1 activator). Results: HHE decreased the expression of Sirt-1, AQP 2 and 3 in in vitro, while it increased the expression of p38 MAPK, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), NOX4, p47phox and COX2. HHE induced NF-κB activation and IκB-α degradation. Increased nuclear NF-κB activation, NOX4, p47phox, MAPK and COX2 was attenuated by the treatment of Bay 117082, NAC, or resveratrol. Conclusions: HHE and DOCA-salt decreased sirt-1 expression in M1 cells and DOCA-salt-induced hypertensive rat model. Sirt-1 activation by resveratrol, NAC, Bay attenuates inflammation proteins and ROS generation in M1 cell. MP027 HYPERPHOSPHATAEMIA INCREASES ENDOTHELIAL CELL SIZE, GRANULARITY AND RATE OF PROLIFERATION Kathryn K Stevens1, Dianne Z Hillyard1, Christian Delles1 and Alan G Jardine1 1University of Glasgow, Glasgow, United Kingdom Introduction and Aims: Elevated serum phosphate is an independent risk factor for cardiovascular disease although the mechanism of action is unclear. This study looks at the effect of elevated phosphate concentration on human umbilical vein endothelial cell (HUVECs) growth and proliferation. Methods: HUVECs were cultured in standard or high phosphate media (0.5mM and 3mM) from the outset. Cells were counted by two independent, blinded observers and then photographed and cell length and area measured. Cell proliferation was measured using the MTT assay in the presence of standard and high phosphate media alone or in combination with FGF-23, Klotho and LNAME (eNOS inhibitor). Western blot was performed for phospho and total eNOS. Nitric oxide (NO) was measured by the Griess reaction. FACscan was used to assess granularity of the cells. Differences between groups were calculated in SPSS using the Student’s t test or a 2 way ANOVA with post hoc analysis as appropriate. Results: HUVECs grown in high phosphate medium are more granular ( p=0.007) and proliferate more rapidly than cells in standard medium ( p<0.001). The morphology of the cells is different with a bigger cell area. MTT assays in cells grown in standard phosphate concentration medium, treated with L-NAME proliferate at a similar rate to cells grown in high phosphate medium alone ( p<0.001). FGF-23 and Klotho do not affect the rate of cell proliferation. There is less expression of Phospho and total eNOS in HUVECs grown in high phosphate concentration medium and less NO produced ( p<0.05). Conclusions: NO is known to inhibit cell growth. HUVECS, grown in high phosphate medium, are bigger, more granular and proliferate more rapidly. This is likely to be secondary to an increase in oxidative stress resulting in reduced nitric oxide production and therefore removing growth inhibition. There is less NO by the Griess reaction and Western blot confirms that less eNOS is expressed in cells grown in high phosphate concentration medium. These experiments were designed to mimic the chronic uraemic state and may offer anexplanation for elevated serum phosphate as a cardiovascular risk factor. Introduction and Aims: Background. Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to MP027 ii | Abstracts MP028 BCL-XL AND MCL1 EXPRESSION IN NON MELANOMA SKIN CANCERS IN RENAL TRANSPLANT RECIPIENTS Michael Burke1,2, Christudas Morais3, Peter Soyer4, Sudipta Sinnya4, Clay Winterford5, Kimberley Oliver1, Duncan Lambie6,7, Christine Staatz8, Robert Carroll9,10, Scott Campbell1,2 and Nicole Isbel1,2 1Princess Alexandra Hospital, Brisbane, Australia, 2School of Medicine, University of Queensland, Brisbane, Australia, 3Centre for Kidney Disease Research, School of Medicine, University of Queensland, Translational Research Institute, Brisbane, Australia, 4Dermatology Research Centre, School of Medicine, the University of Queensland, Translational Research Institute, Brisbane, Australia, 5Qimr Berghofer Medical Research Institute, Brisbane, Australia, 6Diamantina Institute, University of Queensland, Brisbane, Australia, 7IQ Pathology, Brisbane, Australia, 8School of Pharmacy, University of Queensland, Brisbane, Australia, 9Central Northern Adelaide Renal Transplantation Service, Adelaide, Australia, 10Adelaide University, Adelaide, Australia Introduction and Aims: Non Melanoma Skin Cancer (NMSC) frequently occurs as a complication of immunosuppression in Renal Transplant Recipients (RTRs). Usage of the immunosuppressant sirolimus, but not tacrolimus, has been shown to reduce the frequency of NMSC in RTRs. Mcl-1 and Bcl-xL are anti-apoptotic members of the BCL-2 protein family and are important in cell survival and carcinogenesis. This study investigated differences in expression of Mcl-1 and Bcl-xL in NMSC and normal epidermis in RTRs treated with sirolimus and tacrolimus and non-transplant patients who were not immunosuppressed. Methods: NMSCs (squamous cell and intra epithelial carcinomas) were obtained from the tissue blocks of 10 RTRs treated with sirolimus, 11 RTRs treated with tacrolimus and 10 non transplant patients. These sections were deparaffinised and underwent immunohistochemical staining with Mcl-1 and Bcl-xL antibodies. Digital quantitative image analysis was performed with an Aperio cytoplasmic algorithm. The percentage of cells positively expressing Mcl-1 and Bcl-xL in NMSC and normal- adjacent epidermis in the three subject groups were estimated and compared. Statistical analyses were performed based on Wilcoxon and Kruskal Wallis Tests. Results: There was no difference in the expression of Mcl-1 (n=31,p=0.32) or Bcl-xL (n=31,p=0.43) in NMSC areas between sirolimus, tacrolimus or non immunosuppressed groups. Subgroup analyses showed increased Mcl-1 expression in NMSC compared with normal epidermis; in sirolimus (n=10,p=0.01) and tacrolimus (n=11, p=<0.01) treated patients, but no difference in non-immunosuppressed patients (n=10,p=0.1). There was no difference in Bcl-xL expression in NMSC compared with normal epidermis in sirolimus (n=10,p=1), tacrolimus (n=11,p=0.58) or non immunosuppressed patients (n=10,p=0.57). Conclusions: Expression of Mcl-1 is increased in NMSCs compared with normal epidermis in RTRs treated with sirolimus and tacrolimus. When comparing only NMSC samples, expression of Mcl-1 did not differ depending on immunosuppressant treatment. Increased expression of anti-apoptotic Mcl-1 proteins has been identified in lymphoma and lung cancer and may be a vital step in the development of NMSC in RTRs. Increased expression of Mcl-1 occurred in patients receiving the immunosuppressant and chemotherapeutic medication sirolimus and so may contribute to NMSC chemoresistance through the inhibition of apoptosis. Drugs targeting anti-apoptotic Bcl-2 family members have been developed for non-skin malignancies and may have a role in the prevention or treatment of NMSCs in RTRs. MP029 ERYTHROPOIETIN AND PRIMED LEUKOCYTE: MODULATION OF GENE EXPRESSION PROFILE Introduction and Aims: Anaemia of chronic kidney disease is associated, in a considerable proportion of patients, with blunted response/resistance to erythropoietin (EPO). It is well known that inflammation, by the action of pro-inflammatory cytokines, inhibits erythropoiesis. The aim of the study was to investigate the effect of erythropoietin on gene expression in human primary cultured leukocytes. Methods: Microarray experiments were performed on ex vivo Peripheral Blood Mononuclear Cells (PBMCs) pooled from ten healthy donors, primed or not with TNF-α, and then treated with recombinant human erythropoietin α (EPO-α). Real-time PCR experiments were used to validate expression of the molecular targets considered as most relevant for inflammation. Results: Data analysis suggested that EPO-α treatment mainly modulated genes involved in cell movement and cell-cell interaction in primed PBMCs. Notably, EPO-α exerts anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokine IL-8 and its receptor CXCR2; by contrast, EPO-α further increases the expression of genes related to promotion of inflammation encoding for IL-1β and CCL8, and induces de novo synthesis of IL-1α, CXCL1 and CXCL5 in primed PBMCs. MAPK p38α reducing activity is involved in modulation of IL-1β and IL-8 expression. Conclusions: In conclusion, we highlighted the anti-inflammatory effect of EPO; however, our findings also suggest a plausible in vivo scenario, in which EPO itself may cause a positive correlation between elevated levels of some pro-inflammatory mediators and EPO-resistance. In vivo measurement of the molecular targets underlined may help to clarify markers involved in hypo-responsiveness to EPO. MP030 SHEAR STRESS AFFECTS THE EXPRESSION OF SIRTUIN 1 IN MOUSE PODOCYTES Florian Thilo1, Andreas Zakrzewicz2 and Martin Tepel3 1Charite, Berlin, Germany, 2Institute of Physiology, Berlin, Germany, 3Odense University Hosptial, Odense, Denmark Introduction and Aims: An early feature of diabetic nephropathy is hyperfiltration, leading to fluid flow shear stress in podocytes, a critical component of the blood-urine barrier. Sirtuin 1 has been shown to be part of a critical pathway in podocyte injury. We tested the hypothesis that Sirtuin is increased in podocytes due fluid flow shear stress. Methods: RT-PCR was used to analyze the expression of Sirtuin 1 mRNA in mouse podocytes under shear stress compared to podocytes cultivated under static conditions. Results: After 5 h of regular laminar shear stress from 0.25 - 6 dyn/cm² the expression of SIRT1 mRNA was significantly changed compared to podocytes grown under static conditions (normalized ratio, 0.85 ± 0.32 vs. 2.32 ± 0.32 arbitrary units; p < 0.05; each n = 4 for 6 dyn/cm² and normalized ratio, 0.21 ± 0.12 vs. 1.18 ± 0.26 arbitrary units; p < 0.05; each n = 4 for 0.25 dyn/cm²). In contrast, after 24 h of regular laminar shear stress of 1.5 dyn/cm², the expression of SIRT1 was not significantly changed in podocytes in comparison to podocytes grown under static conditions (normalized ratio, 0.48 ± 0.30 vs. 0.07 ± 0.04 arbitrary units; p = 0.34; each n = 4). To analyze whether the alteration in the expression of SIRT1 mRNA expression is PI-3-kinase dependent, we performed experiments with cells which were pretreated with the PI3-kinase inhibitor LY-294002. In the presence of LY-294002, expression of SIRT1 was not significantly altered in comparison to experiments in the absence of LY-294002 (normalized ratio, 0.20 ± 0.02 vs. 0.25 ± 0.09 arbitrary units; p = 1.0; each n = 4). These data reveal that the alterations in SIRT1 mRNA expression are not PI-3-kinase dependent. Conclusions: Expression of sirtuin 1 is differentially regulated by shear stress in podocytes. MP031 SYNDECAN-4 MRNA EXPRESSION IN ENDOTHELIAL CELLS IN RESPONSE TO SHEAR STRESS Florian Thilo1, Andreas Zakrzewicz1 and Martin Tepel2 1Charite, Berlin, Germany, 2Odense University Hosptial, Odense, Denmark Introduction and Aims: Diabetic vessel disease is associated with endothelial dysfunction. It has been shown that shear stress differentially regulates Syndecan-1 expression, but not Syndecan-4 mRNA expression in endothelial cells. Now we investigated whether changes in shear stress profiles might influence the expression of syndecan 4 in endothelial cells. Methods: We analyzed the expression of Syndecan-4 mRNA in an established in vitro dynamic flow system, in which HUVECs are exposed to two well defined types of mechanical stimulation, considered to have direct pathophysiological relevance to atherosclerotic disease in vivo. Specifically, we compared the effect of an athero-protective flow profile with shear stress ranging from 0 to 40 dyn/cm² and a mean shear stress of 20 dyn/cm², and an athero-prone bidirectional flow profile ranging from 8 to -5 dyn/cm² with a mean shear stress near 0 dyn/cm² on Syndecan-4 mRNA expression. Results: Syndecan-4 mRNA expression was significantly decreased after 24 h exposure to an athero-prone flow profile compared to an athero-protective flow profile (normalized ratio, 9 ± 2 vs. 100 ± 29 arbitrary units; p < 0.05; each n = 4-5). In contrast to athero-prone shear stress, regular laminar shear stress of up to 6 dyn/cm² was chosen, as it can be present at every part of the vascular tree and represents the typical average level in human blood vessels well between venous (≈ 1 dyn/cm²) and arterial conditions (≈ 10 dyn/cm²). After 24 h of laminar shear stress the expression of Syndecan-4 mRNA was not significantly changed compared to control conditions (103 ± 3 vs. 100 ± 4 arbitrary units, p = 0.69). Conclusions: Syndecan-4 is differentially regulated in endothelial cells in response to specific pulsatile shear stress wave forms. As it is increased by an atheroprotective shear stress profile, this could even increase the responsibility of endothelial cells to blood flow. This gain mechanisms might be hampered in diabetics. MP032 LPS UPREGULATES THE EXPRESSION OF PERIOSTIN THROUGH MEK/ERK1/2-TGF-BETA1 PATHWAY IN RAT RENAL TUBULAR EPITHELIAL CELLS Shanying Liu1, Yan Li1, Weiwen Liang1, Fang Su1 and Biyun Wang1 1Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Introduction and Aims: Periostin has recently been recognized as an important biomarker of progression of chronic kidney diseases. However, the mechanism regulating its expression remains unclarified. This study was to explore the effects of LPS on expression of periostin in renal tubular epithelial cells. Methods: Cultured renal tubule epithelial cell line NRK-52E and primary rat proximal renal tubule cells were treated with LPS and TGF-beta1 at different concentration and for certain time courses, in the presence or absence of specific inhibitor for MEK1/2, U0126 or TGF-beta receptor blocker, SB525344. mRNA of periostin and TGF-beta1 was measured by RT-PCR. Protein level of periostin was measured by Western blot. Results: LPS induced increases in mRNA of TGF-beta1 at 8, 24 hours, and periostin at 24 and 48 hours, repectively. TGF-beta1 induced an increase in periostin mRNA, peaked at 8 hours, and protein at 48 hours. Pretreatment with U0126 blocked LPS-induced mRNA expression of TGF-beta1 and periostin. When TGF-beta receptor was blocked by SB525334, LPS failed to increase the mRNA and protein levels of periostin in NRK-52E and rat primary renal proximal tubule cells. Conclusions: LPS promotes the expression of periostin through MEK/ERK1/ 2-TGF-beta1 pathway in rat renal tubule epithelial cells.

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Shunji Shiohira, Takumi Yoshida, Hidekazu Sugiura, Miki Nishida, Kosaku Nitta, Ken Tsuchiya, Steffen Grampp, Margarete Goppelt-Strübe, Kai U Eckardt, Joahnnes Schödel, Sun Woo Kang, Yeonghoon Kim, Su-Kil Seo, Taehee Kim, Seongmoon Ong, Won Seok Yang, Nam Jeong Han, Joo Mi Lee, Chung Hee Baek, Su-Kil Park, Elisabeth Kemter, Bernhard Aigner, Ruediger Wanke, Nuria Troyano Suárez, Gema Olmos Centenero, Ines Mora, Mercedes Griera, Jose Luis Cano, Paloma Martin, Javier Zamora, Maria Piedad Ruiz-Torres, Lucas L Falke, Andrew Leask, Karen Lyons, Tri Q Nguyen, Roel Goldschmeding, Sung Kwang Park, Dal Kim, Ae Sin Lee, Yu Jin Jung, Kyoung Hee Yang, Sik Lee, Won Kim, Won Kim, Kyung Pyo Kang, Andrea García-Jérez, Alicia Luengo-Rodríguez, Rafael Ramirez-Chamond, Julia Carracedo, Diana Medrano-Andres, Diego Rodriguez-Puyol, Laura Calleros, Hyun Woo Kim, Su-Kil Park, Won Seok Yang, Sang Koo Lee, Jai Won Chang, Jang Won Seo, Chien-Te Lee, Chia-An Chou, Yueh-Ting Lee, Hwee-Yeong Ng, Maria D Sanchez-Niño, Beatriz Fernandez-Fernandez, Maria Vanesa Perez-Gomez, Jonay Poveda, Ana B Sanz, Pablo Cannata-Ortiz, Jesus Egido, Rafael Selgas, Alberto Ortiz, Seong Kwon Ma, In Jin Kim, Chang Seong Kim, Eun Hui Bae, Soo Wan Kim, Gabor Kokeny, Martina Böo´´Si, Krisztina Fazekas, László Rosivall, Miklós M Mózes, Vojvodic Danilo Vojvodic.Danilo@gmail.Com, Milovic Novak Lazina@yubc.Net, Cerovic Snezana Profajler@yahoo.Com, Stanojevic Ivan, Drivanstanojevic@yahoo.Com., Kovacevic Bozidar, Bozociti@yahoo.Com, Mirjana Mijuskovic, Christof Ulrich, Henrike Berger, Bogusz Trojanowicz, Felix Kohler, Anna Wolf, Eric Seibert, Roman Fiedler, Silke Markau, Marcus Glomb, Matthias Girndt, Ingrid Lajdova, Viera Spustova, Adrian Oksa, Dusan Chorvat, Alzbeta Marcek Chorvatova, Seung Ok Choi, Jae Seok Kim, Byoung Geun Han, Jae Won Yang, Shanying Liu, Jun Lv, Ruiming Chang, Fang Su, Weiwen Liang, Adam M Zawada, Kyrill S Rogacev, Johanna Hundsdorfer, Urban Sester, Danilo Fliser, Gunnar H Heine, Jin-Shuen Chen, Chao-Wen Cheng, Li-Cheng Chang, Chung-Ze Wu, Antônio S Novaes, Fernanda T Borges, Mirian A Boim, Gianfranco Tramonti, Nadia Romiti, Elisabetta Chieli, Sayuri Hamahata, Yasuyuki Nagasawa, Mutsuki Kawabe, Aritoshi Kida, Mana Yahiro, Masayoshi Nanami, Yukiko Hasuike, Takahiro Kuragano, Keiji Nakasho, Hideki Ohyama, Takeshi Nakanishi, Sayuri Tanaka, Shozo Yano, Toshitsugu Sugimoto, Eunhui Bae, Kathryn K Stevens, Dianne Z Hillyard, Christian Delles, Alan G Jardine, Michael Burke, Christudas Morais, Peter Soyer, Sudipta Sinnya, Clay Winterford, Kimberley Oliver, Duncan Lambie, Christine Staatz, Robert Carroll, Scott Campbell, Nicole Isbel, Paolo Felaco, Mirko Pesce, Antonia Patruno, Vittorio Sirolli, Lorenza Speranza, Luigi Amoroso, Sara Franceschelli, Mario Bonomini, Florian Thilo, Andreas Zakrzewicz, Martin Tepel, Florian Thilo, Andreas Zakrzewicz, Martin Tepel, Shanying Liu, Yan Li, Weiwen Liang, Fang Su, Biyun Wang. CELL SIGNALLING AND APOPTOSIS, Nephrology Dialysis Transplantation, 2014, iii330-iii338, DOI: 10.1093/ndt/gfu161