(E-196) Polymeric delivery of epigallocatechin-3-gallate for inhibition of epithelial-to-mesenchymal transition in chronic kidney disease

Introduction:: Chronic kidney disease (CKD) is an incurable, deteriorating dysregulation of the kidney glomerulus, a network of blood vessels that filters circulating blood. CKD affects upwards of 700 million people globally and often progresses to end-stage renal disease, a debilitating terminal disease characterized by diminished glomerular function. A hallmark feature of CKD is the progression of renal fibrosis after initial glomerular injury, a process that is mediated by kidney tubular epithelial cells (TECs). During this process, TECs undergo epithelial-to-mesenchymal transition (EMT), through which they obtain pro-fibrotic and malignant properties like increased mobility and resistance to apoptosis. In recent years, epigallocatechin-3-gallate (EGCG) has been shown to be a promising candidate to inhibit EMT in kidney TECs. In this project, we conjugated EGCG to a low molecular weight polymer previously engineered at the Pun Lab for enhanced localization to the kidney TECs. By improving the localization of EGCG to the kidney TECs, we hope to increase the efficacy of EGCG in mitigating CKD progression.

Materials and Methods:: In vitro assays. Human kidney (HK-2) cells, cultured according to ATCC recommendations, were seeded onto a 96-well plate for 24 hours and then dosed with 0-320 nM unconjugated EGCG or poly-EGCG for 48 hours. Cell viability was determined by incubation with MTS reagent for 2 hours and measuring absorbance at 490 nm. To determine the effect of EGCG on EMT by RT-qPCR, 5 ng/mL TGF-β1 was added to HK-2 cells 1 hour after EGCG dosing (0-40 µM drug concentration). Cells were harvested 48 hours later and RNA was collected with TRIzol reagent. C_T values were measured and normalized to GAPDH. Protein concentrations were normalized through BCA assay and assessed by western blot analysis using mouse anti-GAPDH and rabbit anti-Vimentin, anti-ZO-1, and anti-Snail1 primary antibodies.

In vivo testing. To induce glomerular injury, anti-glomerular IgG was administered at a dose of 11 mg/20g at 8 weeks to C57bl/6 mice. Starting 24 hours after glomerular injury, mice were treated with either 20 mg/kg or 50 mg/kg of unconjugated EGCG three times a week for two weeks. Mice were sacrificed and tissues were collected 14 days after the start of EGCG treatment. Paraffin-embedded kidney samples were stained for Collagen IV according to standard protocols. Detection was completed using an Avidin Biotin Complex method, and DAB was used as the chromogen.


Results, Conclusions, and Discussions:: To evaluate EMT in cultured cells, we dosed HK-2 cells with varying concentrations of the growth factor TGF-β1 and confirmed a two-fold increase in Vimentin gene expression at 5 ng/mL. This concentration of TGF-β1 was therefore used in subsequent EMT studies. Cells treated with EGCG displayed a dose-dependent decrease in both gene expression and protein expression of mesenchymal markers Vimentin, ZO-1, and Snail1, with the most significant decreases seen in cells treated with 40 µM EGCG. This was also supported by morphological observations, with EGCG treatment rescuing healthy cell morphologies in a similar dose-dependent manner.

We then tested EGCG delivery using our polymeric carrier. In the same TGF-β1-induced EMT model, cells treated with poly-EGCG had a comparable decrease in mesenchymal markers like Snail1 and ZO-1, but not Vimentin, relative to cells treated with unconjugated EGCG. Both poly-EGCG and unconjugated EGCG were well tolerated across a broad range of concentrations spanning 0-320 nM.

Finally, we assessed the in vivo efficacy of unconjugated EGCG treatments in mice injured with anti-glomerular IgG. Mice receiving no EGCG after glomerular injury displayed a small increase in Collagen IV staining, which partially decreased at a 20 mg/kg dose of unconjugated EGCG. Conversely, we saw a drastic increase in Collagen IV staining at a 50 mg/kg dose of EGCG, suggesting that overly elevated doses of EGCG could be detrimental to renal scarring and fibrosis. Future in vivo studies will aim to optimize the dose and efficacy of poly-EGCG treatments relative to unconjugated EGCG treatments by assessing histology and urine samples for markers of kidney dysfunction.


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