SP8
The effect of 5-aza-2’-deoxycytidine (DAC) on Nephrotoxic Nephritis and
DNA cytosine methylation in macrophages
Thomas Oates1,2, Stephen McAdoo3, Zelpha D’Souza2, Charles Pusey3, Terry
Cook4, Tim Aitman2 & Enrico Petretto1
1
IGM & 2PGM Groups, MRC Clinical Sciences Centre; 3 Vascular and Renal
Inflammation Section, Division of Medicine and 4CCIR, Imperial College London
INTRODUCTION: Nephrotoxic nephritis (NTN) is a model of crescentic
glomerulonephritis (CRGN) in which the Wistar-Kyoto (WKY) rat shows a marked
susceptibility to disease, whilst the Lewis (Lew) rat is resistant. Previous work has
demonstrated the importance of macrophages in NTN and defined some of the genetic
determinants underlying disease susceptibility. Epigenetic modifications, such as DNA
cytosine methylation, have been linked to macrophage activation and autoimmune
disease. As a result, we hypothesised that variation in DNA methylation could
determine dysregulation of macrophage activity and contribute to the
glomerulonephritis susceptibility of the WKY rat. We investigated this hypothesis by
examining the effect of the inhibition of DNA methylation using 5-aza-2’-deoxycytidine
(DAC) on NTN.
METHODS: NTN was induced in 8-10 week old male WKY rats. Two groups of six rats
were used; one group was treated with 5mg/kg intra peritoneal DAC and the other with
phosphate buffered saline vehicle. Treatment was given every three days prior to
sacrifice at 10 days. The effect of DAC on NTN phenotypes was examined. In addition,
multiplexed PCR sequencing of bisulfite converted DNA extracted from nephritic
glomeruli was used to assess the effect of DAC on DNA cytosine methylation.
RESULTS: DAC treated animals showed fewer glomerular crescents and less
proteinuria and glomerular macrophage infiltration (assessed by ED1 staining) without
significant bone marrow suppression (Table 1).
Table 1 DAC ameliorates NTN. Values are mean (standard deviation) and P-Values by Mann-Whitney
test
Variable (Unit)
DAC group
Vehicle group
P-Value
Crescents (%)
92 (1.7)
56 (6.8)
4.8x10-3
Proteinuria
43 (21)
87 (15)
4.3x10-3
(mg/24hr)
Glomerular area
11 (3.0)
16 (2.7)
0.015
ED1 staining (%)
Hb (g/dL)
12.1 (0.64)
12.7 (0.50)
NS
WBC (X109/L)
3.14 (0.62)
7.54 (1.99)
0.036
In addition, the distribution of cytosine methylation across 200 sequenced cytosine
bases showed a small but significant decrease in methylation in the DAC treated
samples (P-Value = 0.049, Wilcoxon rank sum test). This overall decrease was driven
by 39 out of 200 cytosines which showed demethylation in DAC treated animals. These
39 cytosine bases were associated with genes that are relevant to the pathogenesis of
CRGN, such as P2rx7 and Ccl22.
CONCLUSION: These experiments show that DAC treatment of WKY rats in which
NTN had been induced, had a protective effect on NTN phenotypes with a
demonstrable reduction in DNA cytosine methylation. These results suggest that DNA
cytosine methylation is involved in the pathogenesis of CRGN and could represent a
target for intervention in this disease.