Naringin attenuates renal interstitial fibrosis by regulating the TGF-β/Smad signaling pathway and inflammation

Abstract

Interstitial fibrosis is a typical feature of all progressive renal diseases. The process of fibrosis is frequently coupled with the presence of pro-fibrotic factors and inflammation. Naringin is a dihydroflavone compound that has been previously reported to exhibit anti-fibrotic effects in the liver, where it prevents oxidative damage. In the present study, a rat model of renal interstitial fibrosis and fibrosis cell model were established to evaluate the effects of naringin on inflammatory proteins and fibrosis markers in kidney of rats and NRK-52E cells, and to elucidate the role of the TGF-β/Smad signaling pathway in this mechanism. Compared with those in fibrotic NRK-52E cells that were stimulated by transforming growth factor-β (TGF-β), gene expression levels of α-smooth muscle actin (α-SMA), collagen 1 (COL1A1), collagen 3 (COL3A1), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) were all found to be significantly decreased in fibrotic NRK-52E cells following treatment with naringin (50, 100 and 200 ng/ml). Results from the histopathological studies showed that naringin treatment preserved the renal tissue structure and reduced the degree of fibrosis in the kidney tissues of rats that underwent unilateral ureteral obstruction (UUO). In addition, naringin administration reduced the expression of α-SMA, COL1A1, COL3A1, IL-1β, IL-6 and TNF-α in the kidneys of rats following UUO. The current study, using western blot analysis, indicated that naringin also downregulated the activation of Smad2/3 and the expression of Smad4, high-mobility group protein B1, activator protein-1, NF-κB and cyclooxygenase-2 whilst upregulating the expression of Smad7 in fibrotic NRK-52E cells and rats in the UUO group. In conclusion, naringin could antagonize renal interstitial fibrosis by regulating the TGF-β/Smad pathway and the expression of inflammatory factors.

Keywords: anti-inflammatory; naringin; renal interstitial fibrosis; transforming growth factor-β/Smad.

Figure 1

Effect of naringin on the fibrosis of NRK-52E cells induced by TGF-β. (A) Toxicity of naringin on NRK-52E cells. (B) The effect of naringin on NRK-52E cell viability following treatment with TGF-β. (C) The effect of naringin on α-SMA expression in NRK-52E cells following treatment with TGF-β as examined by immunofluorescence. Magnification, x400. Data is presented as the mean ± SD (n=6). ^*P<0.05, ^**P<0.01 vs. Control; ^#P<0.05 and ^##P<0.01 vs. TGF-β. Nar, naringin; TGF-β, transforming growth factor-β.

Figure 2

Effect of naringin on renal interstitial fibrosis induced by UUO in renal cortex of rats. Effect of naringin treatment on (A) BUN and (B) Scr levels. (C) Effect of 20, 40 and 80 mg/kg naringin on renal sections as assessed using H&E staining. Magnification, x400. Data is presented as mean ± SD (n=6). ^**P<0.01 vs. Sham; ^#P<0.05, ^##P<0.01 vs. UUO. Nar, naringin; UUO, unilateral ureteral obstruction; BUN, blood urea nitrogen; Scr, serum creatinine.

Figure 3

Effect of naringin on renal interstitial fibrosis induced by UUO in rats. Collagen and amyloid deposition in renal tissues following naringin administration was assessed using Masson’s trichrome and Sirius Red staining. Magnification, x400. The length of the scale bars is 50 μm. Nar, naringin; UUO, unilateral ureteral obstruction.

Figure 4

Effect of naringin on α-SMA expression in renal tissues following UUO in rats as examined using immunofluorescence. Magnification, x400. Nar, naringin; UUO, unilateral ureteral obstruction.

Figure 5

Effect of naringin on the mRNA expression of fibrotic markers and inflammatory factors. (A) Effect of naringin on the gene expression of α-SMA, COL1A1 and COL3A1 in NRK-52E cells. (B) Effect of naringin on the gene expression of TNF-α, IL-1 and IL-6 in NRK-52E cells. (C) Effect of naringin on gene expression of α-SMA, COL1A1 and COL3A1 in rat kidney tissues. (D) Effect of naringin on the gene expression of TNF-α, IL-1 and IL-6 in rat kidney tissues. Data is presented as the mean ± SD (n=6). ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. UUO or TGF-β. Nar, naringin; UUO, unilateral ureteral obstruction; COL, collagen; TNF-α, tumor necrosis factor-α; α-SMA, α-smooth muscle actin; IL, interleukin.

Figure 6

Effect of naringin on the protein expression of fibrotic markers in NRK-52E cells and rat kidneys. (A) Effect of naringin on TGF-β, α-SMA and COL1A1 protein expression in NRK-52E cells. (B) Quantitative analysis of the protein expression of TGF-β, α-SMA and COL1A1 in NRK-52E cells. (C) Effect of naringin on TGF-β, α-SMA and COL1A1 in rat kidneys. (D) Quantitative analysis of the protein expression of TGF-β, α-SMA and COL1A1 in rat kidneys. Data is presented as mean ± SD (n=6). ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. UUO or TGF-β. Nar, naringin; UUO, unilateral ureteral obstruction; COL, collagen; TNF-α, tumor necrosis factor-α; α-SMA, α-smooth muscle actin; IL, interleukin; TGF-β, transforming growth factor-β.

Figure 7

Effect of naringin on the expression of key components of the TGF-β/Smad pathway. (A) Effect of naringin on the protein levels of Smad2, Smad3, p-Smad2/3, Smad7 and Smad4 in NRK-52E cells. (B) Quantitative analysis of the protein levels of Smad2, Smad3, p-Smad2/3, Smad7 and Smad4 in NRK-52E cells. (C) Effect of naringin on the protein levels of Smad2, Smad3, p-Smad2/3, Smad7 and Smad4 in rat kidneys. (D) Quantitative analysis of the protein levels of Smad2, Smad3, p-Smad2/3, Smad7 and Smad4 in rat kidneys. Data is presented as mean ± SD (n=6). ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. UUO or TGF-β. Nar, naringin; UUO, unilateral ureteral obstruction; COL, collagen; TNF-α, tumor necrosis factor-α; α-SMA, α-smooth muscle actin; IL, interleukin; TGF-β, transforming growth factor-β.

Figure 8

Effects of naringin on the expression of HMGB1, AP-1, NF-κB and COX-2 in NRK-52E cells and rat kidneys. (A) Effect of naringin on HMGB1, AP-1, NF-κB and COX-2 in NRK-52E cells. (B) Quantitative analysis of the protein expression of HMGB1, AP-1, NF-κB and COX-2 in NRK-52E cells. (C) Effect of naringin on HMGB1, AP-1, NF-κB and COX-2 in rat kidneys. (D) Quantitative analysis of the protein expression of HMGB1, AP-1, NF-κB and COX-2 in rat kidneys. Data is presented as mean ± SD (n=6). ^***P<0.001 vs. Control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. UUO or TGF-β. Nar, naringin; UUO, unilateral ureteral obstruction; HMGB-1, high-mobility group protein B1; COX-2, cyclooxygenase-2; AP-1, activator protein-1; TGF-β, transforming growth factor-β.