Panoply™ Human LPAR1 Knockdown Stable Cell Line

Epithelial-mesenchymal transition (EMT) is a differentiation process associated with fibrosis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA), a naturally occurring small glycerophospholipid, has been implicated in the pathogenesis of DN. Here, researchers observed decreased E-cadherin expression and increased vimentin expression in the renal tubules of diabetic db/db mice, and treatment with ki16425 (an LPAR1/3 inhibitor) suppressed the expression of these EMT markers. Ki16425 treatment also reduced the expression of the profibrotic factors fibronectin and α-smooth muscle actin (α-SMA) in db/db mice. Similarly, researchers found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, a phenomenon that could be reversed by treatment with ki16425 or AM095 (an LPAR1 inhibitor). Furthermore, LPA increased the expression levels of fibronectin and α-smooth muscle actin (α-SMA), an effect that could be reversed by treatment with ki16425 and AM095 or knockdown of LPAR1. Furthermore, LPA induced the expression of the transcription factor Krüppel-like factor 5 (KLF5), which was reduced by AM095 treatment or knockdown of LPAR1. In HK-2 cells, knockdown of KLF5 reduced the expression levels of LPA-induced EMT markers and fibrotic factors. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways reduced LPA-induced expression of KLF5 and EMT markers. Together, these data suggest that LPA promotes the pathogenesis of diabetic nephropathy by regulating KLF5 through LPAR1, thereby inducing EMT and renal tubular fibrosis.

To confirm that LPAR1 mediates LPA-induced expression of EMT markers and fibrotic factors, HK-2 cells were transfected with LPAR1 siRNA and the expression levels of EMT markers and fibrotic factors were measured after LPA treatment. LPAR1 expression in LPAR1-knockdown cells was significantly lower than that in control cells (Figure 1A). Compared with LPA-treated control siRNA-transfected cells, LPAR1 knockdown significantly increased E-cadherin expression and significantly decreased vimentin expression (Figure 1B). Furthermore, LPAR1 knockdown significantly reduced LPA-induced expression of fibrotic factors (α-SMA and fibronectin) (Figure 1C). These results indicate that LPA/LPAR1 signaling is involved in the EMT and fibrotic responses of HK-2 cells.

Figure 1. LPAR1 knockdown inhibited LPA-induced changes in the expression levels of EMT markers and fibrotic factors in HK-2 cells. (Lee G H, et al., 2022)