Assessment of metabolomic and proteomic biomarkers in detection and prognosis of progression of renal function in chronic kidney disease

Abstract

Chronic kidney disease (CKD) is part of a number of systemic and renal diseases and may reach epidemic proportions over the next decade. Efforts have been made to improve diagnosis and management of CKD. We hypothesised that combining metabolomic and proteomic approaches could generate a more systemic and complete view of the disease mechanisms. To test this approach, we examined samples from a cohort of 49 patients representing different stages of CKD. Urine samples were analysed for proteomic changes using capillary electrophoresis-mass spectrometry and urine and plasma samples for metabolomic changes using different mass spectrometry-based techniques. The training set included 20 CKD patients selected according to their estimated glomerular filtration rate (eGFR) at mild (59.9±16.5 mL/min/1.73 m2; n = 10) or advanced (8.9±4.5 mL/min/1.73 m2; n = 10) CKD and the remaining 29 patients left for the test set. We identified a panel of 76 statistically significant metabolites and peptides that correlated with CKD in the training set. We combined these biomarkers in different classifiers and then performed correlation analyses with eGFR at baseline and follow-up after 2.8±0.8 years in the test set. A solely plasma metabolite biomarker-based classifier significantly correlated with the loss of kidney function in the test set at baseline and follow-up (ρ = -0.8031; p<0.0001 and ρ = -0.6009; p = 0.0019, respectively). Similarly, a urinary metabolite biomarker-based classifier did reveal significant association to kidney function (ρ = -0.6557; p = 0.0001 and ρ = -0.6574; p = 0.0005). A classifier utilising 46 identified urinary peptide biomarkers performed statistically equivalent to the urinary and plasma metabolite classifier (ρ = -0.7752; p<0.0001 and ρ = -0.8400; p<0.0001). The combination of both urinary proteomic and urinary and plasma metabolic biomarkers did not improve the correlation with eGFR. In conclusion, we found excellent association of plasma and urinary metabolites and urinary peptides with kidney function, and disease progression, but no added value in combining the different biomarkers data.

Figure 1. Regulation of metabolites and peptides.

The fold changes of metabolites and peptides “mild CKD” vs. “advanced CKD”. A. Plasma metabolites. B. Urinary metabolites. C. Urinary peptides. C19∶0: Nonadecanoic acid. SM C26∶1: Sphingomyelin with acyl residue sum C26∶1. PC aa C42∶4: Phosphatidylcholine with acyl-alkyl residue sum C42∶4. C14∶2: Tetradecadienoylcarnitine. cis-C20∶1w9: cis-11-Eicosenoic acid. PC aa C42∶4: Phosphatidylcholine with acyl-alkyl residue sum C42∶4. C17∶0: Heptadecanoic acid. PC aa C42∶5: Phosphatidylcholine with acyl-alkyl residue sum C42∶5. C4: Nonanoylcarnitine. C5: Isovalerylcarnitine. ADMA: Asymmetric dimethylarginine. Total DMA: Total dimethylarginine. C9: Nonanoylcarnitine. C4∶1: Butenoylcarnitine. C5-DC(C6-OH): Acylcarnitine. C14∶1-OH: 3-Hydroxytetradecenoylcarnitine. dH: Deoxyhexose. HNAc(S2): (N-acetylhexosamine)-disulfate. C3∶1: Propenoylcarnitine. C7-DC: Pimelylcarnitine. H2-dH2: Dihexose-dideoxyhexose. Asn: Asparagine. Leu: Leucine. H1: Hexose. Pro: Proline. Cit: Citrulline.

Figure 2. Correlation analysis of metabolomic and proteomic based classifier scores with baseline eGFR.

The correlation analysis is performed by using the support vector machine classification scores obtained for the test set with baseline. A. Classifier MetaboP (plasma metabolites) ρ = −0.8031 and p<0.0001. B. Classifier MetaboU (urinary metabolites) ρ = −0.6557 and p = 0.0001. C. Classifier Pept (urinary peptides) ρ = −0.7752 and p<0.0001.

Figure 3. Correlation analysis of metabolomic and proteomic based classifier scores with follow-up eGFR.

The correlation analysis is performed by using the support vector machine classification scores obtained for the test set with follow-up eGFR. A. Classifier MetaboP (plasma metabolites) ρ = −0.6009 and p = 0.0019. B. Classifier MetaboU (urinary metabolites) ρ = −0.6574 and p = 0.0005. C. Classifier Pept (urinary peptides) ρ = −0.8400 and p<0.0001.

Figure 4. Correlation analysis of a combined proteomics and metabolomics based classifier with baseline or follow-up eGFR.

A. Classifier Pept_MetaboP (urinary peptides and plasma metabolites) with baseline eGFR ρ = −0.7833 and p<0.0001. B. Classifier Pept_MetaboP with follow-up eGFR ρ = −0.8061 and p<0.0001.