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.