Reduced kidney levels of lysophosphatidic acids in rats after chronic administration of aristolochic acid: Its possible protective role in renal fibrosis

Abstract

Aristolochic acid (AA) is considered to be a causative agent for progressive interstitial renal fibrosis, leading to AA nephropathy. Lysophosphatidic acid (LPA) is a mediator in the onset of renal fibrosis. In this study, we analyzed the molecular species of LPA and its precursor lysophospholipids in kidney tissue from rats exposed to AA. Daily intraperitoneal injections of AA for 35 days to rats gave rise to fibrosis in kidney, decreased the kidney levels of LPA, lysophosphatidylserine and lysophosphatidylinositol. In rat renal cell lines (NRK52E and NRK49F), AA-induced cytotoxicity was potentiated by Ki16425, LPA_1,3 receptor antagonist. The level of mRNA encording α-smooth muscle actin was significantly increased by AA-treatment only in NRK52E cells, while the mRNA level of collagen III was decreased in both NRK52E and NRK49F cells. These results suggest that endogenous LPA in rat kidney prevents AA-induced renal fibrosis.

Keywords: 18S, ribosomal protein S18; AA, aristolochic acid; AZ, azan Mallory; Aristolochic acid; Chronic kidney disease; Fibrosis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HE, hematoxylin/eosin; LC–MS/MS, liquid chromatography–tandem mass spectrometry; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatidylinositol; LPL, lysophospholipid; LPS, lysophosphatidylserine; Lysophosphatidic acid; Lysophospholipid; Nephrotoxicity; PLA1, phospholipase A1; PLA2, phospholipase A2; lysoPLD, lysophospholipase D; α-SMA, α-smooth muscle actin.

Fig. 1

Photomicrographs of renal cortices of rats. Rats were exposed to AA (10 mg/kg/day) or vehicle by daily intraperitoneal injection for 35 days. The kidneys were excised and stained with HE (A and B) or AZ (C and D). Data are representatives of each group with 4 or 6 test and sham rats. Scale bar = 100 μm.

Fig. 2

Effects of AA on mRNA expression of collagen I, collagen III, collagen IV and α-SMA. RNAs were extracted from kidneys of rat that received AA (10 mg/kg/day) for 35 days. mRNAs encoding collagen I, collagen III, collagen IV and α-SMA were quantified by real-time RT-PCR. Results are means ± S.E. (n = 6, 4). *p < 0.05 compared to the sham.

Fig. 3

Effects of AA on total amount and molecular species of five LPL in kidney tissue of rats. Rats were treated with AA (10 mg/kg/day) for 35 days. Extracted lipids were analyzed by LC–MS/MS. *p < 0.05 compared to the sham.

Fig. 4

Effects of AA and Ki16425 on cell viability. FCS-starved NRK52E and NRK49F cells were incubated with various concentrations (5, 10, 20 μM) of AA and Ki16425 for 24, 48 and 65 h. Cell viability was determined as described in Section 2. Results are means ± S.E. (n = 3). *p < 0.05 compared to the cells treated with vehicle.

Fig. 5

Effects of AA on mRNA expressions of collagen I, collagen III, collagen IV and α-SMA in rat kidney-derived cell lines. RNAs were extracted from NRK52E or NRK49F cells treated with 5 μM AA and/or 20 μM Ki16425 for 65 h. mRNAs encoding collagen I, collagen III, collagen IV and α-SMA were quantified by real-time RT-PCR. Results are means ± S.E. (NRK52E; n = 6, NRK49F; n = 4). *p < 0.05 compared to the cells treated with vehicle.