Novel Therapeutics Identification for Fibrosis in Renal Allograft Using Integrative Informatics Approach

Abstract

Chronic allograft damage, defined by interstitial fibrosis and tubular atrophy (IF/TA), is a leading cause of allograft failure. Few effective therapeutic options are available to prevent the progression of IF/TA. We applied a meta-analysis approach on IF/TA molecular datasets in Gene Expression Omnibus to identify a robust 85-gene signature, which was used for computational drug repurposing analysis. Among the top ranked compounds predicted to be therapeutic for IF/TA were azathioprine, a drug to prevent acute rejection in renal transplantation, and kaempferol and esculetin, two drugs not previously described to have efficacy for IF/TA. We experimentally validated the anti-fibrosis effects of kaempferol and esculetin using renal tubular cells in vitro and in vivo in a mouse Unilateral Ureteric Obstruction (UUO) model. Kaempferol significantly attenuated TGF-β1-mediated profibrotic pathways in vitro and in vivo, while esculetin significantly inhibited Wnt/β-catenin pathway in vitro and in vivo. Histology confirmed significantly abrogated fibrosis by kaempferol and esculetin in vivo. We developed an integrative computational framework to identify kaempferol and esculetin as putatively novel therapies for IF/TA and provided experimental evidence for their therapeutic activities in vitro and in vivo using preclinical models. The findings suggest that both drugs might serve as therapeutic options for IF/TA.

Figure 1. Work Flow of Identifying the Drug Targets through Integrative Informatics Approach.

Figure 2. IPA regulatory network using 75 of the 85 genes specific to IF/TA.

We used 85 significantly expressed genes by two meta-analysis methods as input to IPA to create a gene-gene interaction network. We chose the direct relationship option in the IPA to create the interaction networks, resulting in 75 genes. The gradient of the red and green represent the positive and negative meta-effect size respectively. We highlighted the IF/TA biological relevant molecules which were significantly associated with communication between innate and adaptive immune cells in green rectangle, Fcγ receptor-mediated phagocytosis in macrophages and monocytes in blue rectangle, B cell development in red rectangle, T cell receptor signaling in dark red rectangle, dendritic cell maturation in orange rectangle, and natural killer cell signaling in yellow rectangle.

Figure 3. 8 genes associated with kidney failure and tubular toxicity.

Forest plots are presented as in 3(A–H) in 6 independent studies consisting of 275 kidney transplant samples. Meta effect size was in black diamond, and biopsy and peripheral blood samples were in red and blue respectively.

Figure 4. Validation of kaempferol and esculetin through TGF-β and Wnt/β-catenin pathways respectively in HK2 cells.

(A) SNAI1 and CDH1 gene expression determined by RT-PCR (normalized to GAPDH) for cells treated with kaempferol (1–15 μM) for 12 hours followed by 12 hours of kaempferol in the presence of TGF-β1 (5 ng/ml). (B) Western blots and associated densitometries for P-P65, total P65, P-SMAD3, total SMAD3 and β-Actin for HK2 cells treated with kaempferol (1–15 μM) for 16–24 hours followed by 20 minutes of kaempferol in the presence of TGF-β1 (5 ng/ml). Quantifications were shown next to western blots. (C) Western blot and associated densitometry for CCND1 and β-Actin for HK2 cells treated with esculetin (10–80 μM) for 16 hours followed by 36 hours of esculetin in the presence of Wnt-agonist (1.25 μM). Quantifications were next to western blot. (D) CCND1 and MYC gene expression determined by Q-PCR (normalized to GAPDH) for cells treated with esculetin (10–80 μM) for 16 hours followed by 8 hours of esculetin in the presence of Wnt-agonist (1.25 μM). N = 3 in each arm. Western blot experiments on (B) and (C) were run under the same experimental conditions. Data were represented in mean and standard error of the mean.

Figure 5. Kaempferol and esculetin inhibit pro-fibrotic mediators in 7 days UUO model.

(A) Snai1 gene expression of kaempferol treated mice (N = 5) compared with PBS/DMSO-treated mice (N = 6) in UUO model and controls. (B) Western blots in UUO model for P-Smad3, total Smad3, P-p65, total p65, and GAPDH between kaempferol treated mice (N = 3) and PBS/DMSO treated mice (N = 2). Quantifications were shown next to western blots. (C) Cyclin D1 gene expression of esculetin treated mice (N = 5) compared with PBS/DMSO treated mice (N = 6) in UUO model and controls. (D) Western blots in UUO model for Cyclin d1 and GAPDH between esculetin treated mice (N = 5) and PBS/DMSO treated mice (N = 6). Western blot experiments on (B) and (D) were run under the same experimental conditions. Quantifications were shown next to western blots. Data were represented in mean and standard error of the mean.

Figure 6. Kaempferol and esculetin reduce renal fibrosis in 7 days UUO model.

(A) Representative images of picrosirius red stain (collagen 1 and collagen 3) and collagen 1 IHC of renal cortex. (B) Quantification of collagen 1 IHC of renal cortex between UUO kidney and control kidney with PBS/DMSO (N = 6) or kaempferol treatment (N = 5). (C) Quantification of Picrosirius red (collagen 1 and collagen 3) stained renal cortex between UUO kidney and control kidney with PBS/DMSO (N = 6) or esculetin treatment (N = 5). Note: 10–15 random hpfs/animal, original magnification, x40. Data were represented in mean and standard error of the mean.