deposition. Immunocytochemistry was used to assess the presence of osteogenic markers. Apoptosis was investigated analyzing: 1) gene expression of early genes of apoptosis (Bax, Bcl-2) by RT-qPCR; 2) caspase activation by In Cell Western (Caspase- 9, Caspase-3); 3) DNA fragmentation by TUNEL assay. AT2R and PPARc were asses by RT-qPCR. RESULTS: We successfully induced calcification of HK-2 grown in osteogenic medium, with calcium and phosphate deposition increasing overtime. These calcified cells exhibited characteristic signs of caspase-dependent apoptosis. We also found that calcifying HK-2 cells displayed osteogenic markers (Runx2, ALP, ON and OP). The cells showed that apoptosis started within 5 days of induction, when the first small Ca and P crystals became apparent and preceded the appearance of osteogenic markers. Expression of AT2R and PPARc was up-regulated in standard condition over the time, whereas in osteogenic condition we found a down-regulation of AT2R and an increase in PPARc from 1 to 5 days, reaching a plateau at 15 days. CONCLUSIONS: Our findings suggest that an osteogenic process was underway at 15 days, and might have persisted afterwards; the presence of apoptotic process was associated with the calcification process. AT2R stimulation has been reported to alter the apoptosis rate, and this effect could be PPARc dependent. This pathway appears active in standard condition where up-regulation of AT2R inhibited apoptosis pathway up-regulating Bcl-2 via PPARc, thereby preventing Ca deposition and calcification. In osteogenic condition, instead, AT2R was downregulated, thus allowing apoptosis and consequently calcification. The same mechanism was seen in vascular calcification where AT2R overexpression was seen to inhibit calcification. However, AT2R down regulation did not determine as a consequence a down regulation of PPARc, probably due to the effect of other signaling than PPARc by AT2R. Further investigation is needed to reveal the detailed mechanism of the AT2R-induced effect on calcification in osteogenic condition. FP072 THE IMPACT OF CHRONIC KIDNEY DISEASE ON CHOLESTEROL CRYSTAL EMBOLISM IN AUTOPSY CASES Emi Furukawa 1 , Satoru Kishi 1 , Kazuyuki Yahagi 1 , Yoshiyasu Ogura 1 , Yuto Takenaka 1 , Toyohiro Hashiba 1 , Hiroko Ambe 1 , Kengo Mayumi 1 , Satoshi Furuse 1 , Masaya Mori 1 , Naobumi Mise 1 1 Mitsui Memorial Hospital, Chiyoda, Japan INTRODUCTION: Cholesterol crystal embolism (CCE) sometimes lacks typical clinical manifestations, which prevents timely diagnosis. We sought to determine risk factors of CCE. METHODS: This retrospective cohort study comprised 344 consecutive autopsy cases between 2009 and 2016. CCE was diagnosed when cholesterol crystal deposition was detected at one or more tissue/organ other than major arteries. Univariate and multiple logistic regression analyses were performed to determine the factors that influenced the development of CCE. RESULTS: CCE was present in 32 cases (9%), and diagnosed before death only in one case. Kidney was the most frequent target organ, and renal CCE was detected in 18 cases (56%). In univariate logistic regression analysis, hypertension (OR=3.99, 95% CI[Confidential Interval]=1.90-9.64, p<0.01), dyslipidemia (OR=2.27, 95% CI=1.02- 5.08, p=0.04), chronic kidney disease (CKD) (OR=5.45, 95% CI=1.64-18.42, p<0.01), transcatheter vascular intervention (OR=4.28, 95% CI=1.90-9.64, p=0.03) and cardiovascular surgery (OR=2.52, 95% CI=1.09-5.81, p=0.03) were significantly associated with CCE. Multiple regression models, adjusting for coronary risk factors, cardiovascular disease and these variables, demonstrated that CKD was independently associated with CCE (Table 1). CONCLUSIONS: Subclinical CCE was not rare, and detected in 9% of our autopsy cases. CKD was an independent risk factor of CCE. FP073 METHYL-CPG-BINDING PROTEIN 2 BINDS TO GREMLIN-1 PROMOTER AND REPRESSES ITS RENAL EXPRESSION: POTENTIAL EPIGENETIC REGULATION OF A KEY PLAYER IN DIABETIC NEPHROPATHY DEVELOPMENT Vanessa Marchant 1 , Sergio Mezzano 1 , Bredford Kerr 1 1 Universidad Austral de Chile, Valdivia, Chile INTRODUCTION: Gremlin-1 (Grem1) is a protein highly expressed in the kidney of both diabetic nephropathy patients and experimental models. We previously described that tubular overexpression of Grem1 aggravates renal lesions induced by hyperglycemia. Besides, other studies have shown that the suppression of Grem1 expression has a protective effect on diabetic nephropathy development. This evidence demonstrates that Grem1 is a key player in the development of diabetic nephropathy. However, the molecular mechanism that mediates the induction of Grem1 has not been studied. Otherwise, emerging evidence supports a role for epigenetic regulation in the pathogenesis of diabetic nephropathy, but the epigenetic regulation of Grem1 transcription has not been explored. The aim of this work was to assess the role of methyl-CpG binding protein 2 (Mecp2), a key epigenetic regulator, in the control of renal expression of Grem1 and its association with diabetic nephropathy. METHODS: We used BTBR ob/ob mice as an experimental model of diabetic nephropathy. The renal expression of Grem1 was evaluated in kidney from 4, 8, 12, and 16-week-old mice by RT-qPCR. The renal expression levels of Mecp2 were determined in 16-week-old mice by Western Blot. Additionally, Grem1 expression was analyzed in Mecp2-null mutant mice by RT-qPCR. The DNA sequence of Grem1 was analyzed with bioinformatics tools (Methprimer, EMBOSS Cpgplot and Dbcat) to identify potential methylable CpG islands. Chromatin immunoprecipitation (ChIP) using anti- Mecp2 antibody was performed to assess the binding of Mecp2 to the Grem1 CpG island previously identified. RESULTS: We observed a progressive increase in Grem1 expression in the renal tissue of BTBR ob/ob mice starting from 8 weeks of age, together with an increase in Mecp2 levels in 16-week-old mice. Besides, there was a positive correlation between renal expression of Grem1 and Mecp2 in 16-week-old mice. Also, we observed that renal expression of Grem1 was significantly increased in Mecp2-null mice. Bioinformatic analysis identified a~ 2 kb CpG island including the proximal promoter, the first exon, and part of Grem1 single intron. Next, by ChIP we found that in the kidney of wild- type mice, Mecp2 binds to several regions of the CpG island identified in Grem1 coding gene. CONCLUSIONS: Our results show that the expression level of both Grem1 and Mecp2 are associated with diabetic nephropathy progression in a murine model of the disease. Furthermore, our results strongly suggest that Mecp2 represses Grem1 expression by binding to a CpG island identified in the promoter and coding region of Grem1. Altogether, these results allow us to propose that an epigenetic mechanism underlies the deregulation of Grem1 gene expression associated with the development of diabetic nephropathy. Funding Acknowledgements: PFB CECs 01/2007, FONDECYT 1160465, FONDECYT 1181574, Beca Doctorado Nacional CONICYT 21160495. FP074 EMPLOYING MACROPHAGE-DERIVED MICROVESICLE FOR KIDNEY-TARGETED DELIVERY OF DEXAMETHASONE: AN EFFICIENT THERAPEUTIC STRATEGY AGAINST RENAL INFLAMMATION AND FIBROSIS Tang Tao-Tao 1 , Lv Lin-Li 1 , Cao Jing-Yuan 1 , Zuolin Li 1 , Ye Feng 1 , Bi-Cheng Liu 1 1 Southeast University, Nanjing, China INTRODUCTION: Although glucocorticoids are the mainstays in the treatment of renal diseasesfor decades, the dose dependent side effects have largely restricted their clinical use. Microvesicles (MVs) are small lipid-based membrane-bound particles generated by virtually all cells. Here, we investigated whether macrophage-derived MVs can function as efficient carriers of dexamethasone (DEX), thereby, leading to better therapeutic efficacy. METHODS: MVs were prepared from the supernatants of RAW 264.7 cells treated with DEX by centrifugation. NTA, TEM and HPLC were used to characterize the MVs. MVs distribution were analyzed by flow cytometer and Maestro in vivo imaging system. In vivo, the therapeutic potential of DEX-packaging MVs (MVs-DEX) was assessed in LPS and Adriamycin (ADR) nephropathy model. In vitro, the therapeutic efficacy was assessed in glomerular endothelial cells (GECs). RESULTS: Firstly, we found that RAW 264.7 cells incubated with DEX can package drugs into MVs, which can be collected and deliver DEX into inflamed endothelial cells. The drug-packaging MVs were characterized from several aspects. The mean diameter of MVs-DEX was 140.764.8 nm and the average drug content was 6.2lg/ 1Â1010MVs. Compared with GECs without LPS stimulation, more MVs were accumulated in the inflamed GECs. Consistently, the higher renal radiance signal of DID-labelled MVs was observed in LPS and ADR model. The extent of average radiance was positive correlated with renal TNF-a or IL-6 protein levels. Proteomic analysis revealed distinct integrin expression patterns on the MVs-DEX surface, in which the integrin aLb2 (LFA-1) and a4b1(VAL-4) enabled them to adhere to the inflamed kidney.In vitro, we found that the mRNA levels of proinflammatory cytokines, protein expression of NF-jB p65 and p-p65, and levels of TNF-a and IL-6 in the supernatants were significantly inhibited by MVs-DEX treatment. MVs-DEX with about one-fifth of the doses of bare DEX achieved significant anti-inflammatory efficacy by inhibiting NF-jB activity.In LPS model, treatment with MVs-DEX increased survival rate. In ADR model, albuminuria, glomerulosclerosis and foot process effacement were dramatically ameliorated with MVs-DEX therapy. In both models, the mRNA levels of proinflammatory cytokines, protein expression of p65 and p-p65, and infiltration of inflammatory cells were inhibited after MVs-DEX treatment. Of note, MVs-DEX also showed better therapeutic efficacy than bare DEX. Mechanistically, MVs-DEX could package and deliver glucocorticoid receptors to renal cells, thereby, increasing cellular levels of the receptor and improving cell doi:10.1093/ndt/gfz106 | i67 Nephrology Dialysis Transplantation Abstracts Downloaded from https://academic.oup.com/ndt/article-abstract/34/Supplement_1/gfz106.FP072/5515935 by guest on 08 June 2020