Integrative phosphatidylcholine metabolism through phospholipase A2 in rats with chronic kidney disease

Abstract

Dysregulation in lipid metabolism is the leading cause of chronic kidney disease (CKD) and also the important risk factors for high morbidity and mortality. Although lipid abnormalities were identified in CKD, integral metabolic pathways for specific individual lipid species remain to be clarified. We conducted ultra-high-performance liquid chromatography-high-definition mass spectrometry-based lipidomics and identified plasma lipid species and therapeutic effects of Rheum officinale in CKD rats. Adenine-induced CKD rats were administered Rheum officinale. Urine, blood and kidney tissues were collected for analyses. We showed that exogenous adenine consumption led to declining kidney function in rats. Compared with control rats, a panel of differential plasma lipid species in CKD rats was identified in both positive and negative ion modes. Among the 50 lipid species, phosphatidylcholine (PC), lysophosphatidylcholine (LysoPC) and lysophosphatidic acid (LysoPA) accounted for the largest number of identified metabolites. We revealed that six PCs had integral metabolic pathways, in which PC was hydrolysed into LysoPC, and then converted to LysoPA, which was associated with increased cytosolic phospholipase A₂ protein expression in CKD rats. The lower levels of six PCs and their corresponding metabolites could discriminate CKD rats from control rats. Receiver operating characteristic curves showed that each individual lipid species had high values of area under curve, sensitivity and specificity. Administration of Rheum officinale significantly improved impaired kidney function and aberrant PC metabolism in CKD rats. Taken together, this study demonstrates that CKD leads to PC metabolism disorders and that the dysregulation of PC metabolism is involved in CKD pathology.

Keywords: Rheum officinale; chronic kidney disease; lipidomics; phosphatidylcholine metabolism; phospholipase A2.

Fig. 1. Adenine leads to renal function decline and altered lipid profiles in CKD rats.

a Creatinine clearance rate in control and CKD rats induced by adenine. b The geometric mean ratio of each variable in CKD rats versus control rats is presented in positive and negative ion modes. c OPLS-DA of 710 variables with P < 0.05 by using two-tailed unpaired Student’s t test between two groups in positive electrospray ionization mode. d OPLS-DA of 89 variables with P < 0.05 by using two-tailed unpaired Student’s t test between two groups in negative electrospray ionization mode. e Dendrogram analysis based on 50 lipid species identified in positive and negative ion modes. f The levels of total lipids and intraclass comparison of the distribution of 50 lipid species. *P < 0.05, **P < 0.01 compared with CKD rats.

Fig. 2. Aberrant levels of lipid species are involved in CKD.

a Heatmap clustering analysis based on 50 lipid species between the two groups. b Dendrogram of hierarchical clustering analysis of 50 lipid species between the two groups. c PCA of 50 lipid species between the two groups. d OPLS-DA of 50 lipid species between the two groups. e Summary of ingenuity pathway analysis with lipidomics pathway analysis. f Lipid species set enrichment overview. The size and colour of each circle are based on the enrichment ratio and P values, respectively. The enrichment ratio was calculated by hits/expected, where hits = observed hits and expected = expected hits.

Fig. 3. Aberrant PC metabolism is involved in CKD.

a PC metabolic pathway with phospholipase A₂ enzymes and LPLD. Phospholipase A₂ enzymes hydrolyse the sn-2 acyl bond of glycerophospholipids [PC(42:8)] to release fatty acids and lysophospholipids [LysoPC(20:3)]. Lysophospholipids [LysoPC(20:3)] are converted into lysophosphatidic acids [LysoPA(20:3)] by LPLD. b The levels of PC and its metabolites, including LysoPC and LysoPA, between the two groups by PC metabolism analysis. c Intrarenal PLA₂ protein in adenine-induced CKD and control rats. d Quantitative analysis of intrarenal PLA₂ protein expression levels in adenine-induced CKD and control rats. **P < 0.01 compared with control rats.

Fig. 4. The aberrant levels of PC metabolites could distinguish CKD rats from CTL rats.

a PCA of 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, between the two groups. b Dendrogram of hierarchical clustering analysis of 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, between the two groups. c Analysis of PLS-DA-based ROC curves for distinguishing CKD rats from control rats with the combination of the 18 lipid species. d Analysis of PLS-DA-based ROC curves for distinguishing CKD rats from control rats with the combination of the 6 PCs, 6 LysoPCs or 6 LysoPAs. e Analysis of PLS-DA-based ROC curves for distinguishing CKD rats from control rats with PCs, LysoPCs or LysoPAs individually.

Fig. 5. Treatment with EAA improves impaired kidney function in CKD rats induced by adenine.

a The levels of serum creatinine, urea, uric acid, total cholesterol, and triglycerides in control, CKD and CKD+EAA rats. b Immunohistochemical analyses with anti-α-SMA, collagen I and fibronectin antibodies in rat kidney tissues in control, CKD and CKD+EAA (200 mg/kg) rats. Magnification, ×400. **P < 0.01 compared with control rats; ^#P < 0.05, ^##P < 0.01 compared with CKD rats.

Fig. 6. Treatment with EAA improves PC metabolism in CKD rats induced by adenine.

a PCA of 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, in control, CKD and CKD+EAA (200 mg/kg) rats. b Heatmap clustering analysis based on 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, in control, CKD and CKD+EAA (200 mg/kg) rats. c Dendrogram of hierarchical clustering analysis based on 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, in control, CKD and CKD+EAA (200 mg/kg) rats. d The levels of 18 lipid species, including 6 PCs, 6 LysoPCs and 6 LysoPAs, in the different rats. *P < 0.05, **P < 0.01 compared with control rats; ^#P < 0.05, ^##P < 0.01 compared with CKD rats