4. DISCUSSION
Untargeted metabolomics was employed in urine and serum samples of adult head and neck cancer patients receiving cisplatin for the identification of predictive or early biomarkers of cisplatin-induced AKI. This allowed for the identification of important metabolites that are early or predictive biomarkers of cisplatin AKI. Future metabolomics studies in larger adult and pediatric cohorts recruited as part of the ACCENT study will employ fully quantitative metabolite analysis. Metabolomics has been used to investigate cisplatin-induced acute kidney injury in the past but has predominantly been utilized in rodent models. To our knowledge, this study is the first metabolomic investigation of cisplatin-induced AKI in human patients, providing insight into the metabolic differences present between patients who present with clinical AKI upon cisplatin infusion and those who do not, in addition to highlighting the early metabolic alterations induced by cisplatin.
Four urinary biomarkers were identified as predictive markers of clinical AKI: glycine, 3-hydroxydecanedioc acid, hippuric acid sulfate, and suberate. All four metabolites were significantly different between the no AKI and AKI groups at the pre timepoint with fold changes of -2.2-fold, 3.62-fold, 8.85-fold, and 1.91-fold, respectively, in AKI patients relative to no AKI patients (Table 2 ).
Glycine is an amino acid component of the potent antioxidant molecule glutathione and has been associated with beneficial effects in reducing oxidative stress. Alterations in glycine levels have been observed previously in a mouse model of ischemia-reperfusion AKI, where glycine levels were decreased in kidney and heart tissues following ischemic AKI. A metabolomic investigation of urine samples from combat casualties also revealed that lower levels of glycine were associated with need for renal replacement therapy, and glycine levels were higher in patients with moderate to severe AKI compared to mild AKI. In both cases, it was suggested that decreases in glycine levels were associated with upregulation of glutathione production under oxidative stress. Furthermore, glycine was shown to protect against cisplatin nephrotoxicity and ischemia reperfusion renal injury in vivo when administered to rats before cisplatin treatment or ischemic insult. Cisplatin is well documented to cause mitochondrial dysfunction and oxidative stress, and availability of glycine may be an important factor in antioxidant defense against cisplatin-induced oxidative stress.
3-hydroxydecanedioc acid and suberate are dicarboxylic acids that have been associated with fatty acid β-oxidation disorders. Increased urinary excretion of 3-hydroxydecanedioc acid and suberate have been used to diagnose medium-chain acyl-CoA dehydrogenase deficiency (MCAD) and indicates a block in fatty acid oxidation. Dysfunctional mitochondrial fatty acid oxidation is believed to be a crucial mechanism in cisplatin-induced AKI. Cisplatin has previously been shown to inhibit mitochondrial fatty acid β-oxidation by deactivating PPAR-α, a crucial nuclear receptor in the regulation β-oxidation. An accumulation of intracellular acyl-CoAs due to disorders of fatty acid β-oxidation is associated with lipotoxicity and detrimental to mitochondrial function. Additionally, serum levels of acylcarnitines octanoylcarnitine and octenoylcarnitine were significantly higher at the post timepoint in AKI patients relative to the no AKI group and showed increased trends in the pre and 24-48h timepoints, though the differences were not significant (Figure 5D, 5E ). Elevation of serum acylcarnitines is also a marker of dysfunction in fatty acid β-oxidation. Taken together, the elevation of urinary 3-hydroxydecanedioc acid, urinary suberate, and serum acylcarnitines in AKI patients suggest a lower capacity for fatty acid oxidation in patients who develop clinical AKI following cisplatin therapy. An underlying diminished capability for fatty acid oxidation may leave these patients more susceptible towards cisplatin-induced mitochondrial dysfunction and accumulation of toxic lipid compounds.
Hippuric acid sulfate was identified to potentially be both a predictive and early diagnostic marker of cisplatin-induced AKI. Hippuric acid sulfate is not well studied, and very few articles have been published regarding this metabolite. Hippuric acid sulfate is a sulfated derivative of hippuric acid, a uremic toxin that accumulates in chronic kidney disease (CKD). Hippuric acid is derived from the conversion of dietary polyphenols into benzoic acid by the gut microbiome, followed by conjugation with glycine by hepatic or renal glycine-N-acyltransferase. Though hippuric acid has been implicated in both CKD and AKI, hippuric acid sulfate has yet to be implicated with kidney disease.
Cisplatin is well known to be nephrotoxic, manifesting as AKI in approximately one third of patients. It is likely that patients who don’t develop AKI are able to withstand the nephrotoxic insult mediated by cisplatin. To evaluate the metabolic response to cisplatin in patients that don’t progress to AKI, we evaluated metabolic alterations in no AKI patients over the three timepoints of this study. This analysis revealed cisplatin induces early metabolic changes in both the urine and serum even in patients who don’t progress to AKI (Figure 6 ). Many of the metabolites found to be altered at the 24-48h were intermediates of the citric acid cycle or associated with fatty acid oxidation, further emphasizing the central role of mitochondrial dysfunction in cisplatin-induced nephrotoxicity. Of special interest was TMAP, a dipeptide biomarker of reduced kidney function in CKD, which was elevated in both no AKI and AKI patients at the 24-48h timepoint but only remained elevated at the post timepoint in patients with clinical AKI (Figure 5G ). These findings are in accordance with the concept of subclinical AKI induced by cisplatin whereby there is an increase in AKI biomarkers without presentation of clinical AKI. In other words, subclinical AKI is kidney damage without substantial loss of function. Though there has been some work highlighting potential prognostic benefits of using markers of subclinical AKI, the clinical relevance of subclinical AKI is unclear. Further investigation of these early subclinical markers of AKI may provide further insight into the mechanisms of cisplatin nephrotoxicity.
One strength of our study was the high degree of similarity in baseline patient demographics such as age, BMI, ethnicity, and baseline SCr/eGFR between the AKI and no AKI groups, minimizing interindividual variability that could potentially confound metabolic profiling (Table 1 ). Furthermore, the collection of three separate timepoints allowed for comprehensive metabolic profiling of patients prior to and shortly after cisplatin infusion, as well as upon establishment of clinical AKI (or lack thereof).
There were some limitations to this study. Firstly, the sample size for our study was relatively small, ranging from 11 in the AKI group and 20 in the no AKI group. Despite this small sample size, a number of key metabolic alterations were characterized. Future metabolomics studies with larger discovery cohorts may help extract more distinct and robust differences between AKI and no AKI patients. Furthermore, the incidence of head and neck cancer is 2-4-fold higher in men compared to women, and this disparity was reflected in our cohort. As only 2 out of 31 patients were female, sex differences could not be investigated in our analysis. Similarly, all 31 patients in this study were Caucasian which limits the generalizability of our findings to other ethnicities.
Though serum creatinine remains the principal biomarker in AKI diagnosis, cisplatin nephrotoxicity occurs prior to the detection of elevated serum creatinine. Accordingly, there is a need for biomarkers capable of early diagnosis of AKI or prediction of AKI onset prior to cisplatin therapy. In this study, we identified glycine, 3-hydroxydecanedioc acid, hippuric acid sulfate, and suberate as potential predictive markers of clinical cisplatin-induced AKI. Additionally, we provided insight into early metabolic alterations following cisplatin infusion. Further investigations are necessary to validate the applicability and clinical utility of these proposed biomarkers. Future metabolomics studies are planned in large discovery and validation cohorts to further investigate the metabolic effects of cisplatin and elucidate the underlying metabolic differences between patients who present with clinical AKI and patients who do not.