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.