Ultra performance liquid chromatography-based metabonomic study of therapeutic effect of the surface layer of Poria cocos on adenine-induced chronic kidney disease provides new insight into anti-fibrosis mechanism

Abstract

The surface layer of Poria cocos (Fu-Ling-Pi, FLP) is commonly used in traditional Chinese medicine and its diuretic effect was confirmed in rat. Ultra performance liquid chromatography/quadrupole time-of-flight high-sensitivity mass spectrometry and a novel mass spectrometry(Elevated Energy) data collection technique was employed to investigate metabonomic characteristics of chronic kidney disease (CKD) induced from adenine excess and the protective effects of FLP. Multiple metabolites are detected in the CKD and are correlated with progressive renal injury. Among these biomarkers, lysoPC(18∶0), tetracosahexaenoic acid, lysoPC(18∶2), creatinine, lysoPC (16∶0) and lysoPE(22∶0/0∶0) in the FLP-treated group were completely reversed to levels in the control group which lacked CKD. Combined with biochemistry and histopathology results, the changes in serum metabolites indicate that the perturbations of phospholipids metabolism, energy metabolism and amino acid metabolism are related to adenine-induced CKD and to the interventions of FLP on all the three metabolic pathways. FLP may regulate the metabolism of these biomarkers, especially their efficient utilization within the context of CKD. Furthermore, these biomarkers might serve as characteristics to explain the mechanisms of FLP.

Figure 1. Physical parameter comparisons among the studied groups.

(A) Body weight; (B) urinary volume and (C) kidney weight index. The data were expressed as mean ± SD. *P<0.05, **P<0.01 compared to the control groups and ^# P<0.05, ^## P<0.01 compared to the CKD groups.

Figure 2. Basic serum biochemical parameter comparisons among the studied groups.

(A) BUN; (B) Scr; (C) cholesterol; (D) triglyceride. The data were expressed as mean ± SD. *P<0.05, **P<0.01 compared to the control groups and ^# P<0.05, ^## P<0.01 compared to the CKD groups.

Figure 3. Basic blood routine parameters of the studied groups.

(A) White blood cell count; (B) red blood cell; (C) hemoglobin and (D) hematocrit. The data were expressed as mean ± SD. *P<0.05, **P<0.01 compared to the control groups and ^# P<0.05, ^## P<0.01 compared to the CKD groups.

Figure 4. Histologic findings by HE-staining of transverse kidney sections.

(A) normal control rats, (B) adenine-induced CKD rats and (C) FLP-treated group with CKD rats. Large dilated tubule (asterisk); lymphocytic infiltrate (arrowheads); 2,8-dihydroxyadenine crystal (arrows). There was formation of foreign body granuloma in the renal tubules and interstitium and a marked fibrosis leading, in some extreme cases, to a contracted kidney. These results demonstrate that the rat model exhibited the typical pathological features associated with CKD.

Figure 5. The UPLC-MS base peak intensity (BPI) chromatogram.

(A) control group, (B) CKD group and (C) FLP-treated group.

Figure 6. PLS-DA scores plots (A) and Loading plots (B) derived from UPLC-MS data of serum samples.

(▴) control group, (•) CKD group and (♦) FLP-treated group. The variables marked (□) are the metabolites selected as potential biomarkers.

Figure 7. Correlation coefficient analysis between groups with corresponding markers in different groups.

Variables are presented in control, CKD and FLP groups. Values of correlations are shown in the vertical axis (upper for positive correlations and low for negative correlations) and corresponding markers represented to the right of the bars. Numbers are consistent with Table 1.

Figure 8. Differential expression levels (mean) of biomarkers in different groups.

The “asterisk” indicated the statistical significance of the biomarkers changes by Student’s t-test: *P<0.05, **P<0.01, ***P<0.001 significant difference compared with control group; ^# P<0.01, ^## P<0.01, ^### P<0.001 significant difference compared with CKD groups.