4. DISCUSSION
In this study, untargeted metabolomics was employed in a mouse model of
cisplatin-induced AKI to investigate early metabolic changes following
cisplatin administration. Previous studies have utilized untargeted
metabolomics to study rodent models of cisplatin-induced AKI , but have
typically focused on studying one or two biological matrices.
Furthermore, though some of these studies have looked at the metabolic
alterations induced by cisplatin-induced AKI over multiple timepoints,
others have only studied a single timepoint after establishment of AKI.
To our knowledge, our study is the first to comprehensively assess
temporal metabolic alterations in all three of plasma, urine, and kidney
samples throughout AKI progression. Additionally, our study used two
separate strains of mice, allowing for the corroboration of metabolic
changes observed in either strain. Metabolites that exhibited early
alterations in plasma, urine, and kidney samples were identified as
potential early biomarkers for cisplatin-induced nephrotoxicity; in
total, 26 such metabolites were identified.
The extent of renal injury throughout the day 1-4 timepoints was
assessed by plasma creatinine and histological assessment. Similar to
cisplatin nephrotoxicity in humans, AKI only manifested in the later
timepoints, and kidney injury was not apparent in all cisplatin treated
mice. Previous studies have reported that FVB/N mice are more sensitive
to cisplatin-induced AKI compared to C57BL/6 mice , but surprisingly,
there was no difference between the two strains in this study. This lack
of difference in cisplatin sensitivity may be due to the relatively mild
injury observed. This is likely a result of using a cisplatin dose on
the lower end of the established 10-30 mg kg-1 range
for mouse models .
Mitochondrial dysfunction is a key component of the pathogenesis of AKI,
with the kidneys being an organ with very high energy demand . Many
metabolites identified as early biomarkers were related to mitochondrial
energy metabolism, including metabolites related to fatty acid
β-oxidation (FAO) and the tricarboxylic acid (TCA) cycle. Cisplatin has
previously been shown to inhibit FAO by deactivating PPAR-α, a nuclear
receptor that plays a crucial role in the regulation of FAO .
L-carnitine plays a vital role in FAO, allowing for the transport of
long chain fatty acids across the mitochondrial inner membrane . Plasma
accumulation of acylcarnitines, formed by the conjugation of L-carnitine
and fatty acids, are indicative of disorders FAO . Both L-carnitine and
L-acetylcarnitine were altered by cisplatin treatment, with early
elevations observed for urinary L-carnitine in both mouse strains and
early increases in plasma L-acetylcarnitine levels in C57BL/6 mice.
These cisplatin-induced alterations in urinary L-carnitine and plasma
L-acetylcarnitine were sustained throughout the duration of the study.
L-carnitine and acylcarnitines have previously been shown to be altered
in other rodent models of cisplatin-induced nephrotoxicity and human
patients undergoing cisplatin chemotherapy . In patients receiving
cisplatin, urinary excretion of L-carnitine was significantly increased
even on the first day of cisplatin treatment, with increased urine
levels being maintained for the next two days. Though studies have shown
accumulation of acylcarnitines in the blood as early as 24 hours
following cisplatin administration, our study is the first metabolomics
study to show an early elevation of plasma L-acetylcarnitine,
specifically, following cisplatin administration. In addition to markers
of dysfunctional FAO, 15urine levels of TCA cycle intermediates citric
acid and succinate showed significant decreases as early as two days
after cisplatin treatment in C57BL/6 mice, in accordance with previous
studies linking cisplatin and early alterations of TCA cycle
intermediates in urine . Moreover, creatine, which plays a crucial role
in the creatine kinase-phosphocreatine circuit, was consistently
increased in the plasma, urine, and kidneys of both strains of mice
throughout progression of kidney damage, though alterations were more
pronounced in FVB/N mice. The creatine kinase system is responsible for
coupling cellular sites of ATP production to sites of ATP consumption
and is particularly sensitive to oxidative stress . An overall
accumulation of creatine suggests an impairment in the generation of
high energy phosphate molecules.
Gut-derived metabolites of tryptophan indole-3-carboxaldehyde and
indole-3-carboxylic acid were found to be significantly decreased as
early as day 1 and day 2, respectively, in the urine of
cisplatin-treated mice. Both indole-3-carboxaldehyde and
indole-3-carboxylic acid have shown the ability to activate aryl
hydrocarbon receptor (AhR) , which has been shown to induce renal
fibrosis, podocyte injury, glomerular damage, inflammation, and is
correlated with exacerbation of chronic kidney disease in both animal
models and human patients . Phenylalanine and downstream gut-derived
metabolites of phenylalanine also exhibited early alterations following
cisplatin treatment in our study. Phenylalanine was elevated in plasma,
urine, and kidney samples following cisplatin exposure, with plasma
levels being significantly elevated as early as day 1 in C57BL/6 mice
and day 2 in FVB/N mice. Phenylalanine is metabolized by intestinal
bacteria to p-cresol , which is subsequently metabolized to p-cresol
sulfate or p-cresol glucuronide in the liver . Both p-cresol conjugates
were elevated in plasma, urine, and kidneys with cisplatin treatment,
where the most striking and consistent differences were observed in
FVB/N mice. P-cresol derivatives have predominantly been studied in the
setting of chronic kidney disease, where they have been associated with
exacerbation of renal injury and cardiovascular disease . Additionally,
urinary excretion of acylglycines phenylpropionylglycine and
phenylacetylglycine, gut-derived down-stream metabolites of
phenylalanine , were found to be altered in cisplatin-treated mice.
Urinary excretion of phenylpropionylglycine was consistently decreased
in both mouse strains throughout cisplatin treatment, whereas
phenylacetylglycine was shown to accumulate in the kidneys, altogether
suggesting tissue accumulation of acylglycine compounds. Acylglycines
have historically been used as markers of disorder of metabolism and
FAO, where a defect in β-oxidation is typically characterized by an
increased urinary excretion of acylglycines . An accumulation of
intracellular acyl-CoA due to disorders of FAO is associated with
toxicity and detrimental to mitochondrial function , and conjugation of
glycine with these acyl-CoA compounds to form acylglycines has shown to
have a detoxification effect .
Metabolomics analysis also revealed dietary metabolites to be affected
by cisplatin treatment. In both strains of mice, urinary excretion of
dietary phenolic metabolites homovanillic acid sulfate, pyrocatechol
sulfate, and tyrosol 4-sulfate were significantly decreased two days
after cisplatin treatment and remained decreased throughout the study.
Metabolomics studies in CKD rats and CKD patients have also reported the
accumulation of plasma pyrocatechol sulfate in the setting of renal
decline . Polyphenolic compounds found in olive oil, including
unconjugated precursors of homovanillic acid sulfate and tyrosol
4-sulfate (homovanillic acid and tyrosol, respectively) have routinely
been reported to possess a multitude of health benefits in a variety of
pathophysiological conditions . Trigonelline is a dietary plant alkaloid
that has been reported to possess hypoglycemic, anti-diabetic,
antioxidant and overall renoprotective properties. Trigonelline was
consistently reduced in the urine and kidneys of cisplatin treated mice.
Studies have shown trigonelline to have beneficial effects against
diabetic nephropathy , and in attenuating epithelial-to-mesenchymal
transition and ROS generation in an oxalate-induced in vitromodel of renal fibrosis . Our study adds to previous findings from a
metabolomics study in a mouse model of cisplatin-induced AKI which
reported an early decrease in urinary trigonelline , by showing
decreased levels of trigonelline in the plasma and kidneys of
cisplatin-treated mice in addition to urine.
Taurine is a β-amino acid that has a protective role against oxidative
stress, reducing mitochondrial production of oxidants . Additionally,
taurine plays a crucial role in post-translation modification of
mitochondrial tRNAs, which are important in leucine and lysine
synthesis, and by extension mitochondrial protein translation of
electron transport chain complexes . In our study, taurine was
significantly higher in the plasma of day 2 cisplatin treated C57BL/6
mice, though a significant elevation was not observed until day 3 in the
plasma of FVB/N mice. Taurine enters tubular cells via uptake by taurine
transporter (TauT). TauT is found on both apical and basolateral
membranes of renal tubular cells, with expression varying based on
tubular cell type . Cisplatin exposure to proximal tubule cells for 24
hours in vitro has been shown to downregulate gene and protein
expression of TauT and reduce the function of TauT, potentially through
stimulation of p53 . Alterations in taurine transport may help to
explain the plasma accumulation of taurine observed in our study.
As previously discussed, the main strength of our study was the
comprehensive metabolomic analysis of three biological matrices sampled
at multiple timepoints throughout the progression of kidney injury.
Moreover, two separate strains of mice were investigated, demonstrating
that cisplatin-induced metabolic alterations could be reproduced in two
different strains of mice. There were also some limitations to this
study. Firstly, only male mice were studied, and sex differences could
not be assessed. Additionally, diet is known to affect metabolomics;
mice had free access to chow ad libitum , and cisplatin-treated
mice were observed to eat less compared to saline controls.