Pirfenidone is renoprotective in diabetic kidney disease - PubMed (original) (raw)

Pirfenidone is renoprotective in diabetic kidney disease

Satish P RamachandraRao et al. J Am Soc Nephrol. 2009 Aug.

Abstract

Although several interventions slow the progression of diabetic nephropathy, current therapies do not halt progression completely. Recent preclinical studies suggested that pirfenidone (PFD) prevents fibrosis in various diseases, but the mechanisms underlying its antifibrotic action are incompletely understood. Here, we evaluated the role of PFD in regulation of the extracellular matrix. In mouse mesangial cells, PFD decreased TGF-beta promoter activity, reduced TGF-beta protein secretion, and inhibited TGF-beta-induced Smad2-phosphorylation, 3TP-lux promoter activity, and generation of reactive oxygen species. To explore the therapeutic potential of PFD, we administered PFD to 17-wk-old db/db mice for 4 wk. PFD treatment significantly reduced mesangial matrix expansion and expression of renal matrix genes but did not affect albuminuria. Using liquid chromatography with subsequent electrospray ionization tandem mass spectrometry, we identified 21 proteins unique to PFD-treated diabetic kidneys. Analysis of gene ontology and protein-protein interactions of these proteins suggested that PFD may regulate RNA processing. Immunoblotting demonstrated that PFD promotes dosage-dependent dephosphorylation of eukaryotic initiation factor, potentially inhibiting translation of mRNA. In conclusion, PFD is renoprotective in diabetic kidney disease and may exert its antifibrotic effects, in part, via inhibiting RNA processing.

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Figures

Figure 1.

(A) PFD effects on glucose-induced TGF-β1 promoter A 835-luciferase activity. PFD decreases the glucose-induced activity of TGF-β1 promoter A835-luciferase in MMCs at 100 μg/ml, that becomes significant at 500 μg/ml. *P < 0.05 PFD 500 μg/ml versus control (n = 4). (B) PFD reduces TGF-β1 protein levels. To determine whether PFD reduces matrix by lowering TGF-β production, we measured TGF-β1 levels by ELISA in the conditioned media. As compared with control, PFD decreased secreted TGF-β1 significantly at all dosages tested.*P < 0.05, PFD treatment versus control (n = 3).

Figure 2.

(A) Effect of PFD on phospho-Smad2 protein upregulation by TGF-β. As shown in this Western blot, low-dosage PFD had no effect on Smad2 phosphorylation, but, at higher dosages, it partially reduced Smad2 phosphorylation. (B) PFD effects on 3TP-lux activity and TGF-β1 promoter activity. TGF-β1 increased 3TP-LUX promoter activity by six-fold in HEK293 cells. PFD reduced TGF-β1 promoter activity only at dosages 600 μg/ml and significantly blocked 50% of the increase at 1000 μg/ml dosage. *P < 0.05, TGF-β treatment versus control (n = 5); **P < 0.05, PFD + TGF-β treatment versus TGF-β treatment alone (n = 5).

Figure 3.

ROS quantification in MMCs treated with TGF-β and PFD. MMCs grown in 96-well plates were serum-deprived and treated with TGF-β in the presence of varying amounts of PFD as described in the Concise Methods section. ROS generated in MMCs subsequent to treatment with these reagents was quantified using Amplex Red Assay kit. *P < 0.05, TGF-β treatment versus control (n = 6); **P < 0.05, PFD+TGF-β treatment versus TGF-β treatment alone (n = 6).

Figure 4.

MMCs were treated with TGF-β alone, PFD alone, or PFD and TGF-β (30 min before TGF-β treatment) and left in culture for 24 h before harvesting. Total RNA was isolated from the MMCs using the RNeasy Mini Kit (Qiagen, Valencia, CA) according the manufacturer's instructions. (A and B) Real-time PCR was performed using the primers for α1-type I collagen (A) and α1-type IV collagen (B) and normalized against 18S as listed in supplemental section. *P < 0.05, TGF-β treatment versus control.

Figure 5.

(A) PFD reduces the mesangial matrix expansion in db/db mice. Representative micrographs taken from kidney in db/m, untreated db/db and PFD-treated db/db mice. See Table 2 for semi-quantitative scoring of the glomerular matrix. (B) PFD inhibits renal collagen and fibronectin expression in db/db mice. Quantitative real time PCR was performed with kidney cortex from all 3 groups (type I collagen, type IV collagen, and fibronectin), each normalized against 18S. *P < 0.05 db/db versus db/m.

Figure 6.

(A) Venn diagram depicting the distribution of proteins in normal db/m mouse kidney. (B) PFD-treated db/db diabetic mouse kidney. (C) db/db diabetic mouse kidney (n = 4).

Figure 7.

Protein–protein interaction network of PFD-unique db/db kidney proteome. The figure depicts the human protein–protein interaction network centered on 11 human proteins orthologous to the mouse proteins identified only in PFD-treated db/db kidneys. The network comprises 518 proteins and 655 interactions. The PFD-unique interacting proteins are grouped in the meta-nodes depicted by rectangular structures. The enriched gene ontology categories are reported for each meta-node with shades based on the enrichment P value. The total number of proteins in each meta-node is also featured.

Figure 8.

(A) Effect of PFD dosage on phosphorylation of eIF4E protein. As shown in this Western blot, PFD decreased eIF4E phosphorylation in a dosage-dependent manner. (B) Quantification of P-eIF4E/total eIF4E from immunoblots in A (n = 4). Data are means ± SEM. *P < 0.05 PFD treatment versus control.

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Pirfenidone is renoprotective in diabetic kidney disease - PubMed (original) (raw)