Comprehensive Lipidome Profiling of the Kidney in Early-Stage Diabetic Nephropathy

Figures

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  Figure 1. Confirmation of the early-stage diabetic nephropathy (DN) rat model. After diabetic nephropathy was induced by feeding high-fat diet (HFD) combined with injection of low-dose streptozotocin (STZ) for 8 weeks, (A) fasting blood glucose; (B) fructosamine; serum level of (C) TG and (D) TCHO; (E) kidney coefficient; serum level of (F) creatinine and (G) BUN; (H) 24-h microurinary albumin; (I) creatinine clearance; and (J) N-acyl-β-glucosidase (NAG) activity were determined. (K) Periodic acid-Schiff (PAS) staining and (L) periodic acid-silver methenamine (PASM) staining were conducted to examine renal structural changes. Data are presented as means ± SEM, n = 6 per group; ***P < 0.001.

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  Figure 2. General analysis of lipidomic profiles in the kidney cortex of diabetic nephropathy (DN) and normal rats. (A) Principal component analysis (PCA) of whole lipidome of the two groups. (B) Volcano plot of all lipid species detected in the two groups. Data are shown as ratios of DN/NC, false discovery rate (FDR) < 0.05, and fold change > 1.5 is highlighted. Heatmap analysis of (C) main lipid class, (D) overall enrichment of chain length for lipid species, and (E) overall quantification of unsaturated bonds for lipid species; n = 6 per group.

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  Figure 3. Differences in glycerides, fatty acids, and sterol lipids between normal and diabetic nephropathy (DN) rats. (A) Changes in the numbers of total carbons were analyzed for free fatty acid (FFA), diacylglycerol (DAG), and triacylglycerol (TAG) total concentrations. (B) Analysis of the changes in the number of unsaturated bonds in FFA, DAG, and TAG lipid species. (C) Bubble map showing the differences in TAG species content between the two groups, with the sizes of circles representing the level of significance calculated by –log 2 (P), colored by the number of unsaturated side chains. The content of DAG species (D), FFA species (E), and CE species (F) was analyzed. Data are presented as means ± SEM, n = 6 per group; *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 4. Phospholipid remodeling in the kidney cortex of diabetic nephropathy (DN) and normal rats. (A) Total content of each phospholipid class. Ratio heat plot analysis of (B) phosphatidylethanolamine (PE), (C) phosphatidylinositol (PI), (D) phosphatidic acid (PA), (E) phosphatidylcholine (PC), (F) phosphatidylserine (PS), and (G) phosphatidylglycerol (PG) lipid species. The color bars represent the ratio of DN/NC in each lipid species, with the relative abundance of the normal group fixed as a value of 1. Only statistically significant changes are shown. Data are presented as means ± SEM, n = 6 per group; *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 5. Diabetic nephropathy differentially affected the abundance of lyso-phospholipids. After diabetic nephropathy (DN) was induced by feeding high-fat diet (HFD) in combination with injection of low-dose streptozotocin (STZ) for 8 weeks, (A) the content of specific species of lyso-bisphosphatidic acid (LBPA) within the kidney cortex was analyzed; n = 6 per group. (B) Immunohistochemical staining of CD68, at 400× magnification, and quantitative analysis of positivity in kidney glomeruli are shown. Five glomeruli were assessed for each slide; n = 3 per group. Content of specific species of (C) lyso-phosphatidylcholine (LPC), (D) lyso-phosphatidylethanolamine (LPE), (E) (LPI), (F) LPS, and (G) LPA in the kidney cortex was analyzed; n = 6 per group. Data are presented as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6. Sphingolipids were significantly altered in the diabetic kidney cortex. Total content of sphingolipid classes (A) and specific species of Gb3 (B), glucosylceramide (GluCer) (C), galactosylceramide (GalCer) (D), and monosialo-dihexosyl gangliosides (GM3) (E) were analyzed. Data are presented as means ± SEM, n = 6 per group; *P < 0.05, **P < 0.01, ***P < 0.001.