Metabolomics and Gene Expression Analysis Reveal Down-regulation of the Citric Acid (TCA) Cycle in Non-diabetic CKD Patients

Abstract

Chronic kidney disease (CKD) is a public health problem with very high prevalence and mortality. Yet, there is a paucity of effective treatment options, partly due to insufficient knowledge of underlying pathophysiology. We combined metabolomics (GCMS) with kidney gene expression studies to identify metabolic pathways that are altered in adults with non-diabetic stage 3-4 CKD versus healthy adults. Urinary excretion rate of 27 metabolites and plasma concentration of 33 metabolites differed significantly in CKD patients versus controls (estimate range-68% to +113%). Pathway analysis revealed that the citric acid cycle was the most significantly affected, with urinary excretion of citrate, cis-aconitate, isocitrate, 2-oxoglutarate and succinate reduced by 40-68%. Reduction of the citric acid cycle metabolites in urine was replicated in an independent cohort. Expression of genes regulating aconitate, isocitrate, 2-oxoglutarate and succinate were significantly reduced in kidney biopsies. We observed increased urine citrate excretion (+74%, p=0.00009) and plasma 2-oxoglutarate concentrations (+12%, p=0.002) in CKD patients during treatment with a vitamin-D receptor agonist in a randomized trial. In conclusion, urinary excretion of citric acid cycle metabolites and renal expression of genes regulating these metabolites were reduced in non-diabetic CKD. This supports the emerging view of CKD as a state of mitochondrial dysfunction.

Keywords: Chronic kidney disease; Citric acid cycle; Gene expression; Metabolomics; Mitochondria.

Fig. 1

The urinary excretion of citric acid (TCA) cycle metabolites and the renal expression of genes that regulate these metabolites were significantly reduced among participants with versus without non-diabetic CKD. Urine excretion of citric acid cycle metabolites in the proximal part of the pathway were reduced in samples from patients with non-diabetic CKD, as was the mRNA expression of the enzymes catalyzing the proximal steps of the citric acid cycle in the tubulointerstitial compartment in biopsies of patients with non-diabetic CKD.

Fig. 2

Expression of mRNA regulating mitochondrial biogenesis was significantly different comparing participants with versus without non-diabetic CKD. A. Increased AMPK activity induces activation of PGC-1α, a major regulator of mitochondrial biogenesis. Activated PGC-1α sets in motion several transcriptional programs to stimulate replication of mitochondrial DNA, as well as expression of mitochondrial enzymes such as those involved in fatty acid oxidation, citric acid cycle or the electron transport chain. B. Compared with healthy controls, subjects with non-diabetic CKD showed reduced mRNA levels for two subunits of the AMPK protein (PRKAB1, PRKAB2), as well as PGC-1α, while PPARγ mRNA was increased. PRKAA1 to PRKAG2: AMPK subunits; PARGC1A: PPARγ coactivator 1α (PGC-1α); AMPK: AMP-dependent kinase; PPARγ: Peroxisome proliferator-activated receptor γ; ERR α: Estrogen related receptor α; NRF: Nuclear respiratory factor; TFAM: Transcription factor A, mitochondrial.

Fig. 3

Urine and blood concentrations of citrate and 2-oxoglutarate in CKD were increased by paricalcitol in non-diabetic CKD. Urine excretion of citrate is reduced in CKD, compared with healthy controls, and is increased significantly after paricalcitol treatment (panel A). Plasma citrate shows a similar trend, however did not reach significance (panel B). Whisker plots depict maximum, upper quartile, median, lower quartile and the minimum values of each metabolite.