P1817DOES EPIGENETIC FACTORS LIKE HDAC2 PLAYS ROLE IN STEROID RESISTANCE VIA REGULATION OF P-GP AND MRP-1 IN CHILDHOOD IDIOPATHIC NEPHROTIC SYNDROME?

Abstract

Abstract Background and Aims The action of glucocorticoids is to switch off activated inflammatory genes. The activated glucocorticoid receptors (GR) interact with co-repressor molecules to impair NFκB-associated co-activator activity, reducing histone acetylation, chromatin remodelling. Reduction in histone acetylation occurs via recruitment of histone deacetylase (HDAC) 2 to the activated inflammatory gene complex by activated GR, resulting in efficacious suppression of activated inflammatory genes within the nucleus. To evaluate the effect HDAC 2 on P-gp and MRP-1 expression and function. Method Total of 78 subjects were considered in the study out of which 50 were steroid sensitive nephrotic syndrome (SSNS), and 28 were steroid resistant nephrotic syndrome (SRNS) patients. mRNA expression was analyzed on peripheral blood mononuclear cells (PBMCs) in SRNS patients (mean age 8.43±3.8 years), SSNS patients (mean age 7.54±3.5 years). PBMCs were treated with 1µM of Theophylline (HDAC2 stimulator) and 0.8µM of Trichostatin A (HDAC2 inhibitor) for a period of 48 hours. Quantitative PCR was performed using light cycler LC480 using SYBR green PCR technology with SYBR premix relative gene expression levels were calculated and normalized to the corresponding levels of the housekeeping gene (GAPDH). Results Expression of P-gp (4.79±0.970 v/s 2.13±0.72, p<0.0001) and MRP-1 (3.99 ±0.8 v/s 1.99 ±0.91, p<0.0001) on PBMCs was increased in SRNS as compared to that of SSNS. HDAC2 mRNA levels were significantly decreased in SRNS patients as compared to that of SSNS patients (2.97 ± 0.95 v/s 6.02 ± 1.13, p<0.0001). Theophylline(HDAC stimulator) for a period of 48 hours decreased mRNA levels of P-gp and MRP-1 in PBMCs of SRNS with maximal induction at 1µM (fold change 2.65 and 2.21, *p<0.0001) However HDAC2 mRNA expression increased significantly (fold change5.67, *p<0.0001). In SSNS patients P-gp and MRP-1 mRNA expression decreased at1µM (fold change 1.25, 1.24, *p<0.0001) while the mRNA expression was increased (fold change 6.93, *p<0.0001). TSA(HDAC inhibitor) for a period of 48 hours increased mRNA levels of P-gp and MRP-1 in PBMCs of SRNS with maximal induction at 0.8µM (fold change 7.51, 7.31, *p<0.0001) and significantly decreased the level of HDAC2 (fold change1.50, *p<0.0001) similarly in SSNS patients P-gp and MRP-1 mRNA expression increased at 0.8µM (fold change 3.49, 3.35, *p<0.0001) and HDAC2 decreased (fold change2.53, *p<0.0001) at 0.8µM. The functional activity of P-gp and MRP-1 was significantly higher in SRNS group as compared to SSNS group (p<0.001), whereas enzymatic activity of HDAC2 was increased in SSNS group as compared to SRNS group (p<0.001) Conclusion As we observed that HDAC2 regulates P-gp and MRP-1 efflux pumps, Inducer of HDAC2 may be a probable treatment stratergy for patients of Idiopathic Nephrotic Syndrome.