The role of EDA⁺ fibronectin in tubulo-interstitial injury in lupus nephritis

Abstract

Lupus nephritis (LN) is a severe manifestation of SLE and tubulo-interstitial injury is associated with poor renal prognosis. Proximal tubular epithelial cells (PTEC) are the most predominant cell type in the tubulo-interstitium and play a central role in tubulo-interstitial injury and fibrosis. Anti-dsDNA antibody production is a hallmark of SLE, and they may bind to PTEC to induce downstream inflammatory and fibrotic processes. Fibronectin (FN) is a large glycoprotein that is present in the extracellular matrix of all tissues. During tubulo-interstitial injury, increased deposition of FN serves as a scaffold for other matrix proteins to be deposited in the kidney. In the normal adult kidney, EDA+ spliced variant of fibronectin (EDA+ FN) is weakly expressed, and its presence portends the onset of tissue fibrosis. The expression of EDA+ FN, its functional role and mechanism of induction in the pathogenesis of LN have not been explored. The aims of this study are to investigate tubulo-interstitial EDA+ FN expression in patients and mice with LN, the effect of anti-dsDNA antibodies on EDA+ FN expression in cultured PTEC and its role in the pathogenesis of LN. In renal specimens from patients with biopsy-proven Class III/IV±V LN, EDA+ FN expression was markedly increased in the tubulo-interstitium, especially in proximal tubules showing atrophy, when compared to normal kidney specimens. In NZB/W F1 mice, EDA+ FN expression was present in the glomerular compartment in proliferative LN and in the tubulo-interstitium when tubulo-interstitial injury, that is, influx of infiltrating immune cells and tubular atrophy, were apparent. A proportion of infiltrating immune cells in the tubulo-interstitium of NZB/W F1 mice also expressed EDA+ FN. Tubulo-interstitial EDA+ FN expression increased with progressive renal failure and it correlated with the extent of kidney fibrosis. Anti-dsDNA antibodies did not correlate with tubulo-interstitial EDA+ FN expression. Stimulation of cultured PTEC with human polyclonal anti-dsDNA antibodies induced EDA+ FN expression that was predominantly localized to the ECM. This was accompanied by increased FN and laminin expression and induction of collagen I, mediated in part through PI3K and mTOR activation, and increased TGF-1, IL-1 and TNF-α secretion. To investigate the functional role of EDA+ FN in PTEC, we generated EDA+ and EDA- FN peptides using molecular cloning. Stimulation of PTEC with EDA+ FN, but not EDA- FN peptide, increased ZEB2, SNAIL, FN, collagen I and laminin expression, accompanied by decreased E-cadherin expression compared to unstimulated cells. The addition of EDA+ FN peptide to PTEC also increased IL-6, IL-8 and MCP-1 secretion. In summary, we demonstrated that tubulo-interstitial EDA+ FN expression is increased in patients and mice with chronic kidney damage. Induction of EDA+ FN expression in the kidney may be attributed to anti-dsDNA antibody stimulation of resident renal cells, in particular PTEC, mediated in part, through increased TGF-1, IL-1 and TNF-α secretion, and activation of PI3K and mTOR signaling pathways. EDA+ FN could also induce mediators of EMT, inflammation and fibrosis, suggesting its pathogenic importance in tubulo-interstitial injury and fibrosis in the setting of LN.