The kidney plays a fundamental role in the body, as it allows the elimination of waste and/or harmful substances for the body. The kidney concentrates these substances in the urine for elimination. Therefore, urine is a fluid that collects the end point of many metabolic processes in the body (Duarte et al. 2014). It offers us invaluable information on the situation of the organism at a given moment or the organism's response to a certain situation, such as SAR-CoV-2 infection (Alvarez-Belon, Sarnowski and Forni 2020). The kidney is also a very important organ in intermediate metabolism due to its participation in fundamental metabolic pathways for the organism. Considering both roles of the kidney, that of a filtering agent and that of an important metabolic center, together with the liver, allows us to understand the metabolic alterations caused by SARS-CoV-2 infection. In order to understand the metabolic changes caused by COVID-19, we have to evaluate each of the metabolites altered by the disease. Figure 2.C shows the importance of the different signals to obtain a good classification and separation of the samples of COVID-19 patients and healthy people. Signals that are below zero indicate that they are dominant in urine samples from COVID-19 patients, while signals that are positive indicate that they are higher in healthy people. In 1H NMR, the intensity of the signal in the spectrum indicates the number of protons in a given molecule that contribute to the signal. That is, the more signal intensity, the greater number of protons of that particular molecule. Therefore, the PLS-LDA multivariate analysis method is generating a tpLoadings profile that indicates the importance of the 1H NMR signal to generate the mathematical model. The mathematical model is what allows us to identify a sample as being from a COVID-19 patient or from a healthy person. The signals corresponding to ketone bodies (acetoacetate, 2-hydroxybutyrate and acetone), TMAO, glucose and the amino acids phenylalanine and tyrosine are more intense in COVID-19 patients than in healthy people. This increase in ketone bodies would be in line with the increase in these substances found in the blood (Bruzzone et al. 2020). Ketosis is generated in the liver because there has been a metabolic shift to a state of gluconeogenesis. When this ketogenic metabolic pathway is activated in the liver and kidney, all available precursors are used for de novo glucose synthesis (gluconeogenesis). One of the most important precursors is oxaloacetate (Nelson and Michael 2014). This compound is the one that initially forms citrate when condensing with acetyl-CoA that comes from the oxidative decarboxylation of pyruvate or from the β-oxidation of fatty acids, initiating the Krebs cycle. The lack of oxaloacetate causes the concentration of acetyl-CoA to increase, which is directed towards the formation of ketone bodies. In addition, these ketone bodies will compensate for the lack of glucose in different tissues, such as the central nervous system and muscle (Nelson and Michael 2014). We must also take into account that the amino acids phenylalanine and tyrosine are ketogenic amino acids, which would be mobilized to serve as a source of acetyl-CoA. However, the presence of glucose in the urine makes us consider another more serious metabolic situation, since a situation of ketosis is relatively normal in metabolism (Nelson and Michael 2014) and should not produce an increase in ketone bodies or glucose in the urine. urine. The presence of glucose in the urine, together with the increase in ketone bodies, would indicate a situation of type II diabetes. Glucose transporters in the nephritic tubes are unable to recover glucose from urine because there is excess glucose that saturates the transporters (Mather and Pollock 2011, Schetz et al. 2010, Wen et al. 2021). It is also important to point out that the presence of ketone bodies in urine could be due to the role that these compounds play as molecular signals and protection against kidney damage. The presence of β-hydroxybutyrate has an anti-inflammatory effect and suppresses oxidative stress (Rojas-Morales, Pedraza-Chaverri and Tapia 2020, Rojas-Morales, Pedraza-Chaverri and Tapia 2021).
In different studies carried out with analyzes of COVID-19 patients, it could be deduced that the metabolism was changing to this situation of ketosis and type II diabetes or a certain resistance to insulin (Bruzzone et al. 2020). This metabolism alteration would be supported by the increase in the concentration of TMAO (trimethylamine N-oxide). TMA (trimethylamine) is produced by the bacterial flora from dietary metabolites such as choline and L-carnitine present in animal products in the diet (Hoyles et al. 2018). TMA is transported to the liver, where it is oxidized to TMAO by hepatic flavin monooxygenases (FMO1 and FMO3). The increased concentration of TMAO in the body has been associated with health problems such as inflammatory processes, risk of arteriosclerosis, neurological disorders or kidney damage, among other diseases. However, the mechanism associated with these damages is still unknown (Chhibber-Goel et al. 2017, Coras et al. 2019, Dove et al. 2012, Farmer et al. 2021, Gatarek and Kaluzna-Czaplinska 2021, Janeiro et al. 2018, Barrea et al. 2018, Koeth et al. 2013, Brial et al. 2018). One of the characteristics of COVID-19 is the appearance of inflammatory processes, which could be leading to increased levels of TMAO in the urine of COVID-19 patients, as occurs with other inflammatory processes (Tang et al. 2015, Yang et al. 2019). Kidney damage is also associated with an increased concentration of TMAO (Tang et al. 2015).
Another compound that also has high values in COVID-19 patients is formate. Formate is synthesized from precursors such as choline or methanol, but it is mainly obtained from the amino acid serine (Pietzke, Meiser and Vazquez 2020). In some COVID-19 patients, we have found very high levels of formate. Formate is used as a witness for exposure to environmental contaminants in workplaces, such as methanol. However, COVID-19 patients have not been exposed to these environments, so the elevated formate levels could be due to massive production of amino acids during sarcopenia caused by the infection or to kidney damage that affects osmoregulation. (Pietzke et al. 2020, Gil et al. 2018, Liesivuori, Laitinen and Savolainen 1992). In addition, formate induces changes in energy metabolism, increasing AICAR and increasing AMPK activity (Oizel et al. 2020), which would cause an increase in intracellular glucose generation via gluconeogenesis and glycogenolysis. However, there are still many unknowns about the role of formate in the body. Sarcopenia is one of the most serious problems associated with COVID-19 (Anker et al. 2021, Morley, Kalantar-Zadeh and Anker 2020, Pleguezuelos et al. 2021, Wang, Li and Wang 2020), which may be causing the huge decrease in creatine/creatinine seen in COVID-19 patients vs. healthy people. Losses of muscle mass and strength pose a serious health risk (Isoyama et al. 2014, Newman et al. 2006). The loss of muscle mass would be caused by the breakdown of muscle proteins to use amino acids as an energy source, as well as glucose precursors in hepatic and renal gluconeogenesis. The situation of type II diabetes caused by the infection would be aggravating this process of sarcopenia as the necessary amount of glucose does not enter the muscle cells. The difference in creatine/creatinine values between COVID-19 patients and healthy people is key in the PLS-LDA model, indicating the severity of muscle loss during the disease. Creatine is generated in the muscle as a reserve of phosphate bonds, by the synthesis of phosphocreatine. Creatinine is a derivative of creatine that is formed by a non-enzymatic reaction (Nelson and Michael 2014).
In healthy people we find differences with respect to COVID-19 patients, in other metabolites such as acetic acid, citric acid and hippuric acid. Acetic acid is part of the short-chain fatty acids (SCFA) that are synthesized in the gastrointestinal tract by the microbiota, particularly in the colon. They are produced by fermentation of dietary fibre (Canani et al. 2011). Propionate and butyrate are used as an energy source by colonocytes, while acetate is excreted in the urine (Pomare, Branch and Cummings 1985, Boets et al. 2017). In COVID-19 patients, there is a decrease in this metabolite in urine samples. The change in this metabolite is probably due to changes in the diet of COVID-19 patients, due to their hospital admission, although we cannot rule out negative effects of SARS-CoV-2 infection on the intestinal flora. These SCFAs also have anti-inflammatory effects (Boets et al. 2017), so if their concentration decreases, it may also be contributing to the state of inflammation caused by the infection.
Citric acid virtually disappears in COVID-19 patients. This metabolite is produced by condensation of oxaloacetate with acetyl-CoA to initiate the Krebs cycle (Nelson and Michael 2014). Its metabolic function is to be the beginning of the Krebs cycle to oxidize acetyl-CoA and obtain energy. It is also the precursor for fatty acid synthesis in a metabolic situation of excess sugars (Nelson and Michael 2014). However, the metabolic situation in COVID-19 patients is totally different, since the body is in a gluconeogenic situation. Using all possible precursors for glucose synthesis is a priority for cells. As we have discussed, oxaloacetate is one of those glucose precursors (Nelson and Michael 2014). The kidney, along with the liver, are the gluconeogenic organs, which would explain the lack of citric acid in the urine. Citric acid in urine has the function of binding Ca2+ (Moe and Preisig 2006) to prevent the formation of kidney stones, since it prevents the formation of calcium oxalate, which would form the stones (Tiselius, Fornander and Nilsson 1993). Multivariate analysis of 1H NMR spectra indicates that this metabolite is the most important for identifying COVID-19 patients (Fig. 2 and Figure S3). All healthy people have citric acid, while COVID-19 patients show an absence of this metabolite. In other words, we would not only be observing a metabolic change due to this absence of citric acid, but it could also be a fast and non-invasive method to detect the infection (Seker et al. 2009).
Glycine is also a glucogenic amino acid, so its significant decrease in samples from COVID-19 patients compared to healthy people would also support the hypothesis of this gluconeogenic situation in the body due to type II diabetes caused by the infection. COVID-19 patients suffer an enormous loss of muscle mass due to the breakdown of muscle proteins. Amino acids are used in the muscle as a source of energy to compensate for the lack of glucose (Nelson and Michael 2014).
Hippuric acid is produced in the liver as a metabolite from the detoxification of benzoic acid (Irwin et al. 2016). Therefore, the decrease in this metabolite could be indicating that this detoxification process is not working properly, which would cause liver damage. In other words, the infection itself could be affecting the liver's detoxifying capacity, which would explain the liver damage observed in COVID-19 patients (Bruzzone et al. 2020). These results should be confirmed by further investigations focused on hepatic metabolism. On the other hand, hippuric acid also plays a role in the kidney, preventing the formation of stones (Atanassova and Gutzow 2013), so its decrease in urine could be associated with the decrease in citrate.