Inhibition of histone deacetylase activity attenuates renal fibroblast activation and interstitial fibrosis in obstructive nephropathy

Abstract

Activation of renal interstitial fibroblasts is critically involved in the development of tubulointerstitial fibrosis in chronic kidney diseases. In this study, we investigated the effect of trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, on the activation of renal interstitial fibroblasts in a rat renal interstitial fibroblast line (NRK-49F) and the development of renal fibrosis in a murine model of unilateral ureteral obstruction (UUO). alpha-Smooth muscle actin (alpha-SMA) and fibronectin, two hallmarks of fibroblast activation, were highly expressed in cultured NRK-49F cells, and their expression was inhibited in the presence of TSA. Similarly, administration of TSA suppressed the expression of alpha-SMA and fibronectin and attenuated the accumulation of renal interstitial fibroblasts in the kidney after the obstructive injury. Activation of renal interstitial fibroblasts was accompanied by phosphorylation of signal transducer and activator of transcription 3 (STAT3), and TSA treatment also abolished these responses. Furthermore, inhibition of the STAT3 pathway with AG490 inhibited expression of alpha-SMA and fibronectin in NRK-49F cells. Finally, TSA treatment inhibited tubular cell apoptosis and caspase-3 activation in the obstructive kidney. Collectively, we suggest that pharmacological HDAC inhibition may induce antifibrotic activity by inactivation of renal interstitial fibroblasts and inhibition of renal tubular cell death. STAT3 may mediate those actions of HDACs.

Fig. 1.

Effect of trichostatin A (TSA) and AG490 on expression of α-smooth muscle actin (SMA) and fibronectin. NRK-49F cells were incubated with 100 nM TSA (A) or 40 μM AG490 (C) alone for the indicated time or together for 24 h (E) and harvested for immunoblot analysis with antibodies against α-SMA, fibronectin, or GADPH. Representative immunoblots from 3 experiments are shown. Expression levels of α-SMA or fibronectin were quantified by densitometry and are expressed as fold-induction over controls (B, D, and F). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.05).

Fig. 2.

Effect of TSA on serum-induced phosphorylation and acetylation of signal transducer and activator of transcription 3 (STAT3). Serum-starved NRK-49F cells were exposed to 5% FBS for 0–60 min (A) or pretreated with TSA for 1 h at the indicated concentrations and then exposed to 5% FBS for an additional 1 h (C). Cell lysates were subjected to immunoblot analysis with antibodies to phospho-STAT3 (Tyr705), acetyl-STAT3 (K685), STAT3, or actin. Representative immunoblots from 3 experiments are shown. Expression levels of phospho-STAT3 or acetyl-STAT3 were quantified by densitometry and are expressed as fold-induction over controls (B and D). Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.05).

Fig. 3.

Effect of TSA on serum-induced translocation of STAT3. NRK-49F cells were serum-starved for 24 h and then treated with 5% FBS in the presence or absence of 100 nM TSA for 1 h (A) or 30 min (B). A: cells were stained with anti-STAT3 antibody and then subjected to microscopic analysis (×600). B: cytosolic and nuclear proteins were separated and subjected to immunoblot analysis of STAT3, GADPH, or lamin B. C: expression levels of cytosolic and nuclear STAT3 were quantified by densitometry and normalized to GADPH or lamin B levels, respectively. Bars with different letters (a–c) are significantly different from one another (P < 0.05).

Fig. 4.

Effect of TSA on expression of α-SMA and phosphorylation of STAT3 in the kidney after unilateral ureteral obstruction (UUO). Kidney tissue lysates prepared from the obstructed kidneys were subjected to immunoblot analysis with specific antibodies against α-SMA, phospho-STAT3, GAPDH, acetyl-H3, or acetyl-H4 (A and D). Expression levels of the indicated proteins in different groups were quantified by densitometry and are expressed as fold-induction over sham controls (value = 1.0) after normalization with GADPH (B, C, and E). Values are means ± SD (n = 4). Bars with different letters (a–c) are significantly different from one another (P < 0.05).

Fig. 5.

Effect of TSA on expression of fibroblast-specific protein-1 (FSP-1) in the kidney after UUO. A: immunofluorescence staining of kidney sections exhibited FSP-1-positive cells localizing in the peritubular areas. B: FSP-1-positive cells/high-power field (×600) were counted. Values are means ± SD of 4 animals/group. Bars with different letters (a–c) are significantly different from one another (P < 0.05).

Fig. 6.

Effect of TSA on expression of fibronectin in the kidney after UUO. A: representative micrographs demonstrating the expression and localization of fibronectin in the mouse kidney as indicated. B: graph showing the percentage of fibronectin-positive area (arrows) relative to the whole area from 10 random cortical fields (×200). Values are means ± SD. Bars with different letters (a–c) are significantly different from one another (P < 0.05).

Fig. 7.

Effect of TSA on renal fibrosis after UUO. A: photomicrographs illustrating sirius red staining of sham, UUO-injured kidneys with/without TSA administration. B: graph showing the percentage of sirius red-positive tubulointerstitial area (red) relative to the whole area from 10 random cortical fields (×200). Values are means ± SD. Bars with different letters (a–c) are significantly different from one another (P < 0.05).

Fig. 8.

Effect of TSA on renal tubular cell apoptosis after UUO injury. A: representative micrographs demonstrating the expression and localization of apoptosis in different groups as indicated (×400). B: the number of apoptotic tubular cells was counted and expressed as means ± SD of 4 animals/group. Bars with different letters (a–c) are significantly different from one another (P < 0.05). C: whole kidney tissue lysates were subjected to immunoblot analysis with antibodies against caspase-3 or GAPDH. D: expression levels of active caspase-3 were quantified by densitometry and are expressed as fold-induction over controls. Values are means ± SD of 3 independent experiments. Bars with different letters (a–d) are significantly different from one another (P < 0.05).