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. 2021 Feb 16:12:636154.
doi: 10.3389/fphar.2021.636154. eCollection 2021.

Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats

Chongxiang Xiong  1 , Jin Deng  1 , Xin Wang  1 , Xiaofei Shao  1 , Qin Zhou  1 , Hequn Zou  1 , Shougang Zhuang  2   3
Affiliations

Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats

Chongxiang Xiong et al. Front Pharmacol. .

Abstract

Hyperuricemia is an independent risk factor for renal damage and promotes the progression of chronic kidney disease. In this study, we investigated the effect of I-BET151, a small-molecule inhibitor targeting the bromodomain and extraterminal (BET) proteins, on the development of hyperuricemic nephropathy (HN), and the mechanisms involved. Expression levels of bromodomain-containing protein 2 and 4, but not 3 were increased in the kidney of rats with HN; administration of I-BET151 effectively prevented renal dysfunction, decreased urine microalbumin, and attenuated renal fibrosis as indicated by reduced activation of renal interstitial fibroblasts and expression of fibronectin and collagen I in HN rats. Mechanistic studies show that I-BET151 treatment inhibited transition of renal epithelial cells to a mesenchymal cell type as evidenced by preservation of E-cadherin and reduction of vimentin expression. This was coincident with reduced expression of TGF-β1 and dephosphorylation of Smad3 and ERK1/2. I-BET151 was also effective in inhibiting phosphorylation of NF-κB, expression of multiple cytokines and chemokines, and infiltration of macrophages to the injured kidney. Although there were increased serum levels of uric acid and xanthine oxidase, an enzyme that catalyzes production of uric acid, and decreased expression of renal organic anion transporter 1 and 3 that promote urate excretion in the model of HN, and reduced expression levels of urine uric acid, I-BET151 treatment did not affect these responses. Collectively, our results indicate that I-BET151 alleviates HN by inhibiting epithelial to mesenchymal transition and inflammation in association with blockade of TGF-β, ERK1/2 and NF-κB signaling.

Keywords: I-BET151; bromodomain and extra-terminal proteins; epithelial-to-mesenchymal transition; hyperuricemic nephropathy; inflammation; renal fibrosis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
I-BET151 reduces Brd4 levels in the kidney of HN rats. (A) A rat model of HN was established and treated with I-BET151 as indicated in “Material and Methods.” After 3 weeks, kidneys were taken for immunoblot analysis for Brd2, Brd3, Brd4 or GAPDH. Expression levels of Brd2 (B), Brd3 (C), Brd4 (D) were quantified by densitometry analysis and then normalized with GAPDH. (E) Photomicrographs (original magnification, ×400) illustrate immunohistochemical Brd4 staining of kidney tissues. (F) Brd4 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 2
FIGURE 2
I-BET151 improves renal function and alleviates proteinuria and renal pathology in HN rats. HN rats were treated with I-BET151 for 3 weeks and then blood, urine and kidneys were collected. Levels of (A) serum creatinine, (B) blood urea nitrogen (BUN), and (C) urine microalbumin were examined using biochemical assays. (D) Photomicrographs illustrating hematoxylin–eosin (HE) and Masson’s trichrome staining of kidney tissue from the different groups (original magnification ×200). (E) Tubular changes were scored as described in “Materials and Methods.” (F) The graph shows the percentage of Masson-stained tubulointerstitial area (blue) relative to the whole area from 10 random cortical fields (original magnification×400). Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 3
FIGURE 3
I-BET151 treatment reduces expression of fibronectin, collagen I and α-SMA in kidneys of HN rats. (A) Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against α-SMA, fibronectin, collagen I, or GAPDH. Expression levels of fibronectin (B), collagen I (C), α-SMA (D) were quantified by densitometry and normalized with GAPDH. (D) Photomicrographs (original magnification ×400) illustrate immunohistochemical fibronectin staining of kidney tissues. (E) Photomicrographs illustrating Fibronectin and α-SMA immunochemical staining of kidney tissue. Fibronectin (F) and α-SMA (G) positive areas relative to the whole area was quantified. Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 4
FIGURE 4
I-BET151 inhibits renal tubular epithelial-to-mesenchymal transition in the kidney of HN rats. (A) Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against E-cadherin, vimentin or GAPDH. (B) Expression levels of E-cadherin (B) or vimentin (C) were quantified by densitometry and normalized to GAPDH. (D) Photomicrographs (original magnification, ×400) illustrate immunohistochemical staining of E-cadherin and vimentin in in kidney tissues. Graphic presentation of quantitative data for the staining of E-cadherin (E) and vimentin (F). Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 5
FIGURE 5
I-BET151 abrogates the TGF-β/Smad3 signaling pathway in kidneys of HN rats. (A) Protein was extracted from kidneys and subjected to measurement of TGF-β1 by ELISA as indicated. (B) Expression levels of p-Smad3 were quantified by densitometry and normalized to Smad3. (C) The kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against p-Smad3, Smad3, p- ERK1/2, ERK1/2, or GAPDH. (D) Expression levels of P-ERK1/2 were quantified by densitometry and normalized to ERK1/2. (E) Photomicrographs (original magnification, ×400) illustrate immunohistochemical staining for p-ERK1/2 in kidney tissues. (F) p-ERK1/2 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. *p < 0.05; **p < 0.01.
FIGURE 6
FIGURE 6
I-I-BET151 suppresses the activation of NF-κB and expression of proinflammatory cytokines/chemokines and infiltration of mononuclear cells in the kidney of HN rats (A) Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against p-NF-κB (p65), NF-κB (p65), or GAPDH. (B) Expression levels of p-NF-κB (p65) were quantified by densitometry and normalized to NF-κB (p65). Protein levels of (C) MCP-1, (D) RANTES, (E) TNF-α, and (F) IL-1β were measured by the ELISA. (G) Photomicrographs (original magnification, ×400) illustrate immunohistochemical staining of CD68 in kidney tissues. (H) CD68 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. *p < 0.05 vs. sham group and sham + I-BET151 group. **p < 0.05 vs. HN group.
FIGURE 7
FIGURE 7
I-BET151 reduces serum uric acid and XOD activity and promotes uric acid excretion in HN rats. Levels of serum uric acid (A) and urine uric acid (C) were examined using an automatic biochemical assay. (B) Serum XOD activity was examined using a XOD kit. (D) OAT1 and (E) OAT3 mRNA expression in renal tissue from the different groups were determined by RT-PCR and normalized to GAPDH). Kidney tissue lysates were subjected to immunoblot analysis with specific antibodies against OAT1, OAT3 and GAPDH (F). Expression levels of OAT1 (G) and OAT3 (H) were quantified by densitometry and normalized to GAPDH. Data are represented as the mean ± SEM. **p < 0.01.
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