Reciprocal Relationship Between HDAC2 and P-Glycoprotein/MRP-1 and Their Role in Steroid Resistance in Childhood Nephrotic Syndrome

Abstract

Background: Reduced HDACs levels have been reported in steroid resistant chronic obstructive pulmonary disease and bronchial asthma patients. P-glycoprotein (P-gp) over expression in peripheral blood mononuclear cells (PBMCs) has been reported in patients with steroid resistant nephrotic syndrome (NS). Whether and how HDACs and P-gp are linked with each other is not clear, especially in NS patients. Aim: To evaluate mRNA expression of P-gp/MRP-1 and HDAC2 in PBMCs of steroid sensitive (SSNS) and steroid resistant nephrotic syndrome (SRNS) patients, and determine the relationship between expression of HDAC2 and P-gp/ MRP-1in NS patients. Methods: Twenty subjects (10 in each group), SSNS (mean age 7.54 ± 3.5 years), and SRNS (mean age 8.43 ± 3.8 years) were recruited. mRNA expression of HDAC2 and P-gp/MRP-1 was studied by quantitative real time PCR. PBMCs were treated with Theophylline, 1 μM, and Trichostatin A, 0.8 μM, for 48 h for induction and suppression of HDAC2, respectively. Results: At baseline, expression of P-gp (4.79 ± 0.10 vs. 2.13 ± 0.12, p < 0.0001) and MRP-1 (3.99 ± 0.08 vs. 1.99 ±0.11, p < 0.0001) on PBMCs were increased whereas, HDAC2 mRNA levels (2.97 ± 0.15 vs. 6.02 ± 0.13, p < 0.0001) were significantly decreased in SRNS as compared to that of SSNS patients. Compared to baseline, theophylline reduced mRNA expression of P-gp and MRP-1 (fold change 2.65 and 2.21, ^* p < 0.0001 in SRNS) (fold change 1.25, 1.24, ^* p < 0.0001 in SSNS), respectively. However, it increased the expression of HDAC2 (fold change 5.67, ^* p < 0.0001 in SRNS) (fold change 6.93, ^* p < 0.0001 in SSNS). Compared to baseline, TSA treatment increased mRNA levels of P-gp and MRP-1 (fold change 7.51, 7.31, ^* p < 0.0001 in SRNS) and (fold change 3.49, 3.35, ^* p < 0.0001 in SSNS), respectively. It significantly decreased the level of HDAC2 (fold change 1.50, ^* p < 0.0001 in SRNS) (fold change 2.53, ^* p < 0.0001 in SSNS) patients. Conclusion: Reduced HDAC2 and increased P-gp/MRP-1 activity may play a role in response to steroids in childhood NS. HDAC2 and P-gp/MRP-1 are in reciprocal relationship with each other.

Keywords: HDAC inhibitor and HDAC stimulator; P-glycoprotein (P-gp); histone deacetylase2 (HDAC2); multidrug resistance-associated protein 1 (MRP-1); steroid resistance.

Figure 1

Expression of P-gp, MRP-1 and HDAC2 on unstimulated PBMCs of SRNS and SSNS patients. Peripheral Blood Mononuclear Cells were isolated from SRNS and SSNS patients and the mRNA levels of P-gp, MRP-1 and HDAC2 were quantified by real-time PCR technique (A–C). The experiments are representative of three independent series. Pooled data of all the experiments are represented as mean ± SEM. Significant differences compared to control were indicated by p < 0.05.

Figure 2

Effect of HDAC2 stimulator Theophylline on expression levels of P-gp, MRP-1 and HDAC2 in SRNS and SSNS patients (A,B). Quantitative real-time PCR of P-gp, MRP-1 (A) and HDAC2 (B) in peripheral blood mononuclear cells of SRNS and SSNS patients treated with different concentrations of Theophylline (0, 0.01, 0.1, 1.0 μM) for 48 h. The experiments are representative of three independent series. Pooled data of all the experiments are represented as mean±SEM. Significant differences compared to control are indicated by *p < 0.05.

Figure 3

Effect of HDAC2 inhibitor Trichostatin A expression levels of P-gp, MRP-1 and HDAC2 in SRNS and SSNS patients (A,B). Quantitative real-time PCR of P-gp, MRP-1, and HDAC2 in peripheral blood mononuclear cells of SRNS and SSNS patients treated with different concentrations of Trichostatin A (0, 0.2, 0.4, 0.8 μM) for 48 h. The experiments are representative of three independent series. Pooled data of all the experiments are represented as mean ± SEM. Significant differences compared to control were indicated by *p < 0.05.

Figure 4

Relationship between corticosteroids and HDAC2. Glucocorticoids (GCs) on entering into the cell binds with Glucocorticoid receptor (GCR) present in the cytosol. Thereafter, GC-GCR complex enters inside the nucleus and binds to Glucocorticoid Receptor Elements (GREs) present on the promotor regions of various genes. Depending upon its binding to either positive GREs or negative GREs, transactivation or trans-repression of genes is resulted. In another trans-repression mechanism, GC-GCR complex induces the recruitment of HDAC2, which binds to GREs of certain genes and results in deacetylation of MDR-1 gene, other pro-inflammatory genes. Thus, inhibitors of HDAC2 (Trichostatin A) may suppress the steroid response whereas stimulators of HDAC2 (Theophylline) may increase the steroid response. HAT: Histone Acetylases, HDAC2: Histone Deacetylases 2.