Free-catch, fasted urine samples were collected from cats clinically diagnosed with CKD from urinalysis, GFR and serum biochemistry assessments and centrifuged immediately (3500 rpm at 4 °C for 15 min). Healthy subjects were recruited from the local institutional cat population and had no clinically diagnosed underlying health issues. All subjects were administered similar diets. The method of urine collection was identical to case subjects above. A minimum of 0.5 ml of urine is required for analysis. The supernatants were then stored at − 80 °C prior to analysis. NMR spectra were obtained on an Oxford Instruments X-Pulse 60 MHz benchtop NMR spectrometer, operating at + 40 °C. Samples were defrosted, and diluted by addition of 20% (by volume) deuterium oxide, D2O. One-dimensional proton NMR spectra (with and without solvent suppression), and proton-proton gradient-selective COSY spectra were obtained for each sample. One-dimensional 1H spectra were collected with 64 scans, 6 s acquisition time and 5 s relaxation delay; one-dimensional solvent suppressed 1H spectra [using a WET (Water suppression Enhanced through T1 effects) sequence] were collected with 128 scans, and the same acquisition time, and relaxation delay; COSY spectra were obtained with 8 scans of 256 slices. All spectra were internally referenced to H2O/HOD at δH + 4.66 ppm. The Kyoto Encyclopedia of Gene and Genomics (KEGG) was used to ascribe significant biomolecular modifications and describe linkages between metabolic cycles.
High-field metabolomics has already demonstrated translational capability for diagnostic and therapeutic aims in humans with renal conditions . Herein, we use CKD as a case study for the application of this technology and present pilot data from four subjects consisting of two control (Subjects S2,S4) and two with CKD IRIS stage 2 (S1, S3). Subjects S2 and S4 were clinically assessed by a veterinarian for confounding conditions and assessment as control participants for this pilot study as healthy controls with no renal conditions and serum creatinine concentrations of < 145 μmol/L. Further details on participants availbale within data protection regulations is available in the Additional file 1.
Subjects S1 and S3 were diagnosed with azotaemic CKD at Stage 2 according to IRIS guidelines from their serum creatinine concentrations of 193 and 188 μmol/L, respectively. Additionally, these CKD subjects show stronger resonances in the aromatic region (signal 17) ascribed to hippurate and phenylacetylglycine aromatic protons. Increases in urinary creatinine have previously been confirmed in cats with CKD . Creatine is essential for energy transfer to skeletal muscle through the formation of ATP. Renal dysfunction can lead to an increase in creatinine in urine; therefore, the level of creatinine in urine is a principal indicator of CKD.
Furthermore, the relatively weak acetate signal compared to healthy controls is indicative of decreased excretion in individuals with CKD, as displayed in Fig. 1 (S1, S3). Indeed, an inverse correlation between urinary excretion of acetate and renal function has been established in comparative physiological studies . It has been observed elsewhere that the level of acetate was lower in humans with diabetes mellitus and CKD than those with CKD alone . Reduced excretion of acetate in urine indicates further metabolism to acetyl coenzyme A which has a central role in fatty acid metabolism. Acetyl coenzyme A is involved in the central carbon metabolism that subsequently generates ATP through catabolism of the acetyl moiety in the tricarboxylic acid cycle . Indeed, a reduction in urinary excretion of TCA cycle metabolites and renal expression of the genes which regulate these metabolites has been demonstrated in human cases of CKD, linking to mitochondrial dysfunction and CKD progression. Moreover, recent genomic and metabolomic assessments of human patients with non-diabetic CKD identified reduced TCA cycle activity in cases when compared to a control group. This reduction in urinary excretion of TCA cycle metabolites was linked to a reduction in overall mitochondrial biogenesis in kidney tissues from CKD patients likely caused by reduced expression of genes such as isocitrate dehydrogenase 3 in the tubointerstitial compartment of the kidney .
Metabolic pathway analysis (MPA) from these pilot data identified that glycine, serine and threonine metabolism was associated with classification between CKD and control subjects. An impact score from pathway topological analysis was 0.3, whilst a p value adjusted by the Holm-Bonferroni correction was 0.00426. This suggests that glycine, serine and threonine metabolism are modified in CKD subjects compared to the controls. Concentration decreases in metabolites excreted through both serine and threonine metabolism were detected elsewhere [10, 28]. Such a significance of serine metabolism between groups could be an indicator of its biological role in renal dysfunction. Serine acts as a mediator for methylation and the lowering of blood pressure in renal mechanisms. Serine excretion is correlated to glomerular filtration rate (GFR) which in-turn is used to define reductions in renal function. Therefore, if GFR is reduced, D-serine begins to accumulate in tissue [10, 15].
Furthermore, changes in glycine concentration in biofluids over the course of CKD has identified perturbations in amino acid metabolism in both rat and human models . Additionally, hippuric acid metabolite signals in CKD subjects are linked to glycine conjugation with benzoic acid in hepatic, intestinal and renal activity . Taken together, these glycine and glycine-conjugated metabolites are linked to oxidative stress and inflammation through both the IκBα/NF-κB and Keap1/Nrf2 pathways . Moreover, glycine forms a central node in glutathione metabolism, which with tocopherol act as baseline markers of oxidation and concentrations of downstream metabolites are highly mediated by CKD stage. Cellular processes determining immunity, but particularly inflammation can be assessed by sphingolipid metabolites where sphingosines act as signalling molecules. These metabolites, also detected as N-acetyl functions (signal 5) have been demonstrated to be highly sensitive in their concentration to dietary interventions for the treatment of CKD in cats. Indeed, successful fibre supplementation aligned with positive clinical outcomes to changes in diet were ascribed to increased sphingolipid metabolite concentrations in plasma of cats with CKD .
Regarding other metabolites, the reduction of urine citrate concentration has also been associated with CKD where urine citrate can prevent the formation of calcium-based kidney stones . Furthermore, the dysfunction of taurine was connected to CKD in other studies . Taurine is involved in osmoregulation, calcium ion kinetics and regulation of the membrane potential in skeletal muscle. Moreover, taurine can be considered as anti-inflammatory and an antioxidant agent . Kidneys have a crucial role in maintaining the level of taurine. However, the levels of taurine can be dramatically decreased in patients with CKD. Therefore, taurine can also feature one of the main regulatory metabolites for the detection of CKD .
Renal function can be assessed by the measurement of glomerular filtration rate and is often referred to as the gold standard, however this can involve clinically and technically challenging measurements. Indeed, CKD can be diagnosed in small animals through a combinatorial approach involving creatinine concentration and urine specific gravity, and whilst these may be widely used, they remain insensitive as prognostic and monitoring markers. Our preliminary assessment of a pilot feline cohort identifies (a) the ability of low field NMR spectroscopy to detect > 15 metabolites in feline urine and (b) the potential of the technology when applied to large cohorts and informed by machine learning, to provide fast biofluid analysis to support clinical decision making.
Benchtop NMR metabolic profiling offers an opportunity to leverage the selectivity of NMR spectroscopy in a portable format capable of being more widely applied, whilst taking advantage of chemometric methods to deconvolute spectra and offer the facility as a technique with diagnostic potential. Benchtop NMR is therefore a potential tool for the early detection of diseases and the evaluation of health conditions that can provide necessary, timely treatments [29, 30].
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.