Abstract: PD12-09
Sources of Funding: NIH RO1 GM 057242; Department of Veterans Affairs

Introduction

Chronic kidney disease (CKD) is a widely prevalent and often silent condition. Effective treatment to halt renal fibrosis progression to CKD is largely lacking. Connective tissue growth factor (CTGF) renal upregulation is evident in various nephropathies including obstructive uropathy and is linked to fibrosis progression. Precise mechanisms of CTGF contribution to the maladaptive phenotype (e.g., epithelial dedifferentiation, growth arrest, fibrotic factor induction) however, are largely unknown.

Methods

Human kidney tubular epithelial cells (HK-2) were stably transduced with either control or CTGF expression via lentiviral transduction to mimic CTGF induction in kidney injury. Western analysis was used to confirm CTGF overexpression and to investigate the effects of CTGF overexpression on various fibrotic markers. Epithelial cell-cell communication studies involved transfer of conditioned media from control or CTGF stably expressing epithelial cells to similarly seeded normal HK-2 cultures. Microscopy was used to evaluate cell morphologic changes. Cell death is confirmed by Annexin staining.

Results

Prolonged epithelial CTGF overexpression resulted in upregulation of pro-fibrotic factors including fibronectin and plasminogen activator inhibitor 1 (PAI-1), induction of plasticity maker, vimentin, and downregulation of expression of epithelial cell adhesion molecule (E-cadherin), compared to vector transduced HK-2 controls. Epithelial cells with CTGF overexpression assume a mesenchymal morphology and undergo growth inhibition. Cell-cell communication experiments reveal that paracrine factors secreted by CTGF expressing HK-2 cells trigger growth inhibition and a fibrotic response in normal HK-2 cells. CTGF expression in older epithelial cultures also induces markers of DNA damage and cell death compared to similarly aged control vector transduced cultures.

Conclusions

Epithelial CTGF induction promotes upregulation of fibrotic factors, induction of epithelial dedifferentiation, suppression of cell growth and orchestrates (via paracrine mechanisms) dysfunction in normal renal epithelia. CTGF, therefore, could be an attractive drug target against renal fibrosis and CKD progression.

Funding

NIH RO1 GM 057242; Department of Veterans Affairs