Mechanism of Chronic Kidney Disease Progression and Novel Biomarkers: A Metabolomic Analysis of Experimental Glomerulonephritis

Abstract

While a complex network of cellular and molecular events is known to be involved in the pathophysiological mechanism of chronic kidney disease (CKD), the divergence point between reversal and progression and the event that triggers CKD progression are still unknown. To understand the different mechanisms between reversible and irreversible kidney disease and to search for urinary biomarkers that can predict prognosis, a metabolomic analysis was applied to compare acute and chronic experimental glomerulonephritis (GN) models. Four metabolites, namely, epoxyoctadecenoic acid (EpOME), epoxyeicosatetraenoic acid (EpETE), α-linolenic acid (ALA), and hydroxyretinoic acid, were identified as predictive markers after comparing the chronic nephritis model with acute nephritis and control groups (false discovery rate adjusted p-value (q-value) < 0.05). Renal mRNA expression of cytochrome P450 and epoxide hydrolase was also identified as being involved in the production of epoxide metabolites from these polyunsaturated fatty acids (p < 0.05). These results suggested that the progression of chronic kidney disease is associated with abnormally activated epoxide hydrolase, leading to an increase in EpOME and EpETE as pro-inflammatory eicosanoids.

Keywords: chronic glomerulonephritis; chronic kidney disease; experimental; untargeted metabolomics.

Figure 1

Schematic illustration of the definition of acute and chronic nephritis.

Figure 2

Experimental design of the nephritis rat model. The AN animals were produced via the injection of mouse anti-Thy1.1 antibody. The CN group was induced through the administration of the mouse anti-Thy1.1 antibody to unilaterally nephrectomized rats. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis.

Figure 3

The 24-h urine protein-to-creatinine ratio in experimental glomerulonephritis rat groups. There was a marked increase in proteinuria in the CN groups compared to the AN groups from Week 4 to 12. * p < 0.05 vs. AN; # p < 0.05 vs. AN-C/CN-C. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis.

Figure 4

The glomerular matrix and interstitial fibrosis (IF) scores among the four groups at both the 2W and 12W time points. (a) glomerular matrix score, (b) interstitial fibrosis score. There were significant differences in both the glomerular matrix and IF scores among the four groups at both the 2W and 12W time points. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis. * p < 0.05 vs. CN, # p < 0.05 vs. AN.

Figure 5

Scheme of the untargeted metabolomics strategy to determine urinary biomarkers predictive of the progression of nephritis. The predictive criterion were as follows: (1) q-value < 0.05 when the CN group was compared to the AN animal at Week 1 or 2, representing early-stage nephritis, even if there was no significant difference in proteinuria between the AN and CN groups; and (2) q-value < 0.05 when the CN group was compared to the CN-C animal between Week 2 and 12. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis.

Figure 6

Relative intensity of the five identified urinary biomarkers predictive of the progression of nephritis. (a) EpOME, (b) EpETE, (c) α-linolenic acid, (d) hydroxyretinoic acid, and (e) DiHOME. Data are shown as mean values ± SEM. * q  < 0.05 vs. AN; # q  < 0.05 vs. CN-C. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis; DiHOME, dihydroxyoctadecenoic acid; EpETE, epoxyeicosatetraenoic acid; EpOME, epoxyoctadecenoic acid.

Figure 7

Renal mRNA expression of (a) CYP2J4, (b) CYP2C23, (c) CYP2E1, (d) Ephx2, and (e) Ephx3 at the Week 2 and 12 time points. CYP2C23 and CYP2E1 were lower in the CN group than in AN or CN-C animals. Ephx3 expression was higher with CN than in AN or CN-C groups. The Mann–Whitney U test was used to calculate statistical significance. * p < 0.05 vs. AN; # p < 0.05 vs. CN-C. AN, acute nephritis; AN-C, control group for acute nephritis; CN, chronic nephritis; CN-C, control group for chronic nephritis; Ephx2, epoxide hydrolase 2; Ephx3, epoxide hydrolase 3.

Figure 8

Potential mechanism of cytochrome P450 (CYP450)-dependent metabolism of polyunsaturated omega-6 and omega-3 fatty acids involved in CKD progression. (a) omega-6 PUFA pathway, (b) omega-3 PUFA pathway. Decreased mRNA expression of CYP2C23 and CYP2E1 prevent the production of EETs, as a vasodilator, from arachidonic acid. Increased mRNA expression of CYP2J4 induces the generation of EpOME and the corresponding DiHOME as a protoxin via the soluble epoxide hydrolase, leading to the progression of CKD. Metabolites in the red colored squares are increased. mRNA is increased in the red line circle and decreased in the blue line circle. CKD, chronic kidney disease; COX, cyclooxygenase; DHET, dihydroxyeicosatrienoic acid; DiHETE, dihydroxyeicosatetraenoic acid; DiHOME, dihydroxyoctadecenoic acid; EETs, epoxyeicosatrienoic acids; EPA, eicosapentaenoic acid; Ephx2, epoxide hydrolase 2; Ephx3, epoxide hydrolase 3; EpOME, epoxyoctadecenoic acid; EpETE, epoxyeicosatetraenoic acid; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; PUFA, polyunsaturated fatty acid.