Contribution of Dietary Oxalate and Oxalate Precursors to Urinary Oxalate Excretion

Kidney stone disease is increasing in prevalence, and the most common stone composition is calcium oxalate. Dietary oxalate intake and endogenous production of oxalate are important in the pathophysiology of calcium oxalate stone disease. The impact of dietary oxalate intake on urinary oxalate excretion and kidney stone disease risk has been assessed through large cohort studies as well as smaller studies with dietary control. Net gastrointestinal oxalate absorption influences urinary oxalate excretion. Oxalate-degrading bacteria in the gut microbiome, especially Oxalobacter formigenes, may mitigate stone risk through reducing net oxalate absorption. Ascorbic acid (vitamin C) is the main dietary precursor for endogenous production of oxalate with several other compounds playing a lesser role. Renal handling of oxalate and, potentially, renal synthesis of oxalate may contribute to stone formation. In this review, we discuss dietary oxalate and precursors of oxalate, their pertinent physiology in humans, and what is known about their role in kidney stone disease.


Introduction
Kidney stone disease affects approximately 10% of the population [1] and calcium oxalate (CaOx) is the most common stone composition [2]. Supersaturation of urine with CaOx markedly increases the risk of stone formation [3], and supersaturation is the driving force behind CaOx crystal precipitation [4]. The contribution of urinary oxalate to supersaturation of CaOx is significant. For example, in one study, the supersaturation of CaOx was up to 23 times more sensitive to changes in urinary oxalate compared to changes in urinary calcium [5]. In a subsequent study, urinary calcium and urinary oxalate were found to contribute equally to supersaturation of CaOx [6]. Urinary oxalate is derived from dietary oxalate intake and endogenous oxalate synthesis [7]. The diet-derived portion depends primarily on three factors: the amount of oxalate consumed, the amount of calcium and other divalent cations consumed, and oxalate handling by the intestine. This is further complicated by the degradation of some of the ingested oxalate by intestinal bacteria [8] and differences in oxalate absorption in different regions of the intestinal tract [9]. Endogenous oxalate production occurs primarily in the liver and is influenced by dietary intake of precursors, notably ascorbic acid (AA) [10]. Subsequent renal handling [11] and, perhaps, endogenous synthesis by the kidney are final determinants of the urinary oxalate pool. In this paper, we review sources of dietary oxalate and oxalate precursors, their impact on the urinary oxalate pool, and their influence on kidney stone risk.

Quantifying Oxalate Consumption
An estimate of the amount of oxalate consumed in large populations has been performed primarily by the use of FFQs. This tool was crucial in identifying the critical role of dietary calcium in CaOx stone formation [32]. The main mechanism for this effect was proposed to be the binding of calcium to oxalate limiting intestinal oxalate absorption [33]. Through a similar approach, however, the relationship between dietary oxalate and kidney stone disease was less pronounced [12]. Furthermore, the difference in urinary oxalate between the highest and lowest quartiles of oxalate consumption based on an FFQ was small, 1.7 mg/day [13]. There are several reasons for these inconsistencies: (1) The daily intake of oxalate on a molar basis is much less than calcium, approximately one-tenth (2.5 mmol vs. 25 mmol); (2) The amount of bound oxalate ingested is higher than bound calcium; (3) The amount of oxalate ingested can be difficult to determine due to the variability of oxalate in plants and plant-based foods. These differences may be due to growth conditions, genetic divergence and analytical variability in oxalate analyses [34]; (4) FFQs are subject to errors and when targeting a single nutrient, they should be validated by comparison with another technique such as a weighed 3-4-day food record [35].
Due to the plurality of these influencing factors, it would appear that validating the accuracy of FFQs in determining oxalate consumption could be a fruitless, if not impossible task. Studies utilizing controlled dietary conditions overcome many of the limitations of FFQs with respect to oxalate intake.

Relationship between Dietary Oxalate and Urinary Oxalate
Several studies have been performed in healthy human subjects that have addressed the relationship between dietary oxalate to its urinary excretion using diets tightly controlled in their nutrient content. In one study, the mean contribution of dietary oxalate to urinary oxalate ranged from 25% (10 mg dietary oxalate per day) to 42% (250 mg dietary oxalate per day); when the calcium content of the 250 mg oxalate/day diet was decreased from 1002 mg to 391 mg, the mean contribution increased to 53% [7]. Further analysis demonstrated a linear increase in urinary oxalate excretion with respect to dietary oxalate intake over a range of 50-750 mg daily intake under defined dietary conditions including calcium intake of 1000 mg/day [36]. It is important to note that dietary oxalate intake on self-selected diets can vary by more than 200 mg/day [37], producing substantial variation in oxalate excretion without dietary control. We have previously advocated that reducing or eliminating oxalate-rich foods in the diet, when appropriate, should be beneficial in reducing the contribution of diet to urinary oxalate excretion [38].

Dietary Oxalate and the Gut
Gastrointestinal oxalate absorption is influenced by absorptive and secretory fluxes of this dicarboxylic acid. The influence of oxalate-degrading organisms in the fecal microbiome including Oxalobacter formigenes on these processes is an area of active research.

Gut Absorption of Dietary Oxalate
Oxalate is absorbed along the intestinal tract via transcellular anion transporters, notably the solute-linked carrier (SLC)-26 family, as well as paracellular fluxes through tight junctions [9,39]. The amount of ingested oxalate that is absorbed by the gut appears to be between 5 and 15%, depending on intake of calcium, magnesium, and fiber [15]. Hyperabsorption of oxalate has been associated with surgical resection of intestinal segments, including bypass surgery for weight loss, and malabsorptive intestinal diseases. This secondary hyperoxaluria, known as enteric hyperoxaluria (reviewed in [40]), has been attributed to the increased amount of soluble oxalate available for absorption as a consequence of free fat malabsorption resulting in sequestration and saponification of calcium, as well as the presence of increased amounts of bile acids, again due to malabsorption, which is thought to augment gastrointestinal oxalate uptake.

Gut Microbial Oxalate Degradation
The mammalian body does not possess enzymes capable of metabolizing oxalate. However, multiple bacterial species in the gastrointestinal tract have the ability to degrade oxalate, and thus it is hypothesized these gut microbes play an important role in reducing the risk of CaOx stone disease. There are two major groups of oxalate-degrading bacteria in the gastrointestinal tract. There are the "generalist oxalotrophs", which do not depend entirely on oxalate as an energy source, and the "specialist oxalotrophs", which use oxalate as their sole or major carbon and energy source [41]. To date, only one specialist oxalotroph has been discovered in the mammalian gut, the obligate anaerobic bacterium O. formigenes [42,43]. Most of the research has centered on this bacterium [44][45][46][47][48][49], with only a few studies addressing the role of the general oxalotrophs on stone disease [50,51].
A few studies have examined the impact of probiotic preparations designed to degrade gastrointestinal oxalate with the intent of attenuating urinary oxalate excretion, including those containing O. formigenes, Lactobacillus, and/or Bifidobacterium spp. [23,[52][53][54]. Although these trials did not show a significant effect on urinary oxalate excretion, it is not clear whether the ingested bacteria in these trials maintained viability. Enzymatic degradation of oxalate throughout the gastrointestinal tract using an oral preparation of oxalate decarboxylase is a more recent potential treatment strategy [55]. A phase 2, openlabel trial found that dosing with oxalate decarboxylase significantly reduced 24-h urinary oxalate excretion in participants with idiopathic (−10.2 mg) and enteric hyperoxaluria (−22.0 mg) [56]. The relative role of generalist oxalotrophs and O. formigenes on gut oxalate degradation has only been examined in one study with healthy non-stone formers [25]. This study utilized diets controlled in nutrients, including oxalate, and demonstrated that the oxalate degrading capacity of the microbiome of individuals not colonized with O. formigenes is negligible at low dietary oxalate intake and increases with ingestion of higher levels of dietary oxalate. This is consistent with early dietary oxalate feeding studies in ruminants [57]. This study also found that individuals colonized with O. formigenes excreted significantly lower levels of fecal oxalate compared with those individuals not colonized with O. formigenes, highlighting the importance of a specialist oxalotroph on Similar controlled dietary studies need to be performed in stone formers to determine the relative role of generalist oxalotrophs and O. formigenes on gut oxalate degradation, and how colonization of the gut with these microbes impacts urinary oxalate excretion and risk of stone formation. Future studies utilizing state of the art sequencing technologies [58] could lead to the identification of networks of microbes that play an important role in enhancing oxalate degradation in the human gastrointestinal tract. Furthermore, as oral antibiotic use and antibiotic exposure at a young age have been associated with an increased risk for stone formation [59], future research should also focus on determining the mechanisms by which antibiotic exposure increases the risk of stone disease, including the impact of antibiotic treatment on microbial oxalate degradation.

Precursors of Endogenous Oxalate Production
The initial insights into endogenous oxalate synthesis were generated over 60 years ago by the study of the rare hereditary kidney stone/nephrocalcinosis generating diseases, primary hyperoxalurias (PH). Studies using 14 C-and 13 C-labeled compounds in rats and in healthy volunteers and patients with PH were instrumental in determining the importance of the liver in oxalate production and in identifying precursors to oxalate synthesis ( Figure 1). The central role of the liver in oxalate synthesis was demonstrated by studies performed in isolated rat liver and hepatectomized rats [60,61]. The contribution of other tissues, such as the kidney, have yet to be fully elucidated. In the liver, glyoxylate has been identified as a direct precursor to oxalate, and lactate dehydrogenase as the key enzyme in oxidizing glyoxylate to oxalate [62][63][64][65]. Further supporting the central role of the liver in glyoxylate to oxalate metabolism, the enzyme deficiencies in PH are involved in glyoxylate metabolism and are highly or even entirely expressed in the liver [66][67][68][69][70]. Thus, the search for precursors to oxalate synthesis has been driven by pathways involving glyoxylate metabolism [71,72]. A notable exception is AA, which has long been known to be a non-enzymatic source of oxalate synthesis [73,74].

Amino Acids and Proteins
Among the oxalate precursors that have been studied, a number of amino acids have been proposed (glycine, serine, tyrosine, tryptophan, phenylalanine, and hydroxyproline), but only a few have been evaluated and validated in humans, fewer still with dietary control [60,[75][76][77][78]. The role of glycine in oxalate synthesis was initially thought to be a major one with contribution to oxalate synthesis estimated as high as 40% in older studies [79][80][81]. However, a study of 6 healthy volunteers using improved analytical method including dietary control, and primed, constant infusions of 13 C-glycine demonstrated that glycine metabolism contributed <5% to urinary oxalate [26]. Hydroxyproline, a product of collagen breakdown, when orally ingested in the form of gelatin, showed conversion to oxalate in healthy volunteers [19]. This study prompted a more quantitative examination of hydroxyproline catabolism to oxalate using primed, constant infusions of 15 N, 13 C 5 -hydroxyproline in healthy volunteers and subjects with PH, on controlled diets [30]. This study showed that the metabolism of hydroxyproline contributed to at least 15% of endogenous oxalate synthesis in healthy subjects and played a greater role in patients with PH, notably PH type 2 and type 3.
has been identified as a direct precursor to oxalate, and lactate dehydrogenase as the key enzyme in oxidizing glyoxylate to oxalate [62][63][64][65]. Further supporting the central role of the liver in glyoxylate to oxalate metabolism, the enzyme deficiencies in PH are involved in glyoxylate metabolism and are highly or even entirely expressed in the liver [66][67][68][69][70]. Thus, the search for precursors to oxalate synthesis has been driven by pathways involving glyoxylate metabolism [71,72]. A notable exception is AA, which has long been known to be a non-enzymatic source of oxalate synthesis [73,74].

Amino Acids and Proteins
Among the oxalate precursors that have been studied, a number of amino acids have been proposed (glycine, serine, tyrosine, tryptophan, phenylalanine, and hydroxyproline), but only a few have been evaluated and validated in humans, fewer still with dietary control [60,[75][76][77][78]. The role of glycine in oxalate synthesis was initially thought to be a major one with contribution to oxalate synthesis estimated as high as 40% in older studies [79][80][81]. However, a study of 6 healthy volunteers using improved analytical method including dietary control, and primed, constant infusions of 13 C-glycine demonstrated that glycine metabolism contributed <5% to urinary oxalate [26]. Hydroxyproline, a product of collagen breakdown, when orally ingested in the form of gelatin, showed conversion  Figure 1. Pathways of endogenous oxalate synthesis. Synthesis of oxalate occurs via enzymatic (names in italics) and non-enzymatic reactions taking place in different subcellular compartments (cytosol, mitochondria, and peroxisome). The molecular formula is given in brackets underneath each compound. Glycolaldehyde is a product of fructose and xylitol metabolism. 1P5C: 1-pyrroline-3-hydroxy-5-carboxylic acid, LDH: lactate dehydrogenase, GO: glycolate oxidase, GR: glyoxylate reductase, AGT: alanine:glyoxylate aminotransferase, DAO: D-amino oxidase, ALDH: aldehyde dehydrogenase, GLO: glyoxalase, HOGA: 4-hydroxy-2-oxoglutarate aldolase, AspAT: aspartate aminotransferase, 1P5CDH: 1P5C dehydrogenase, HYPDH: 4-hydroxyproline dehydrogenase.

Other Sources of Glyoxylate
Glycolate, a precursor to glyoxylate, is a major source of oxalate synthesis in PH type 1. Its metabolism to glyoxylate has been established by numerous studies in humans and rodents for over 60 years [60,61,63,71,82]. Inhibition of glycolate oxidation to glyoxylate by RNA interference or CRISPR/Cas9 targeting of glycolate oxidase (GO) has shown effectiveness in reducing urinary oxalate excretion in mice deficient in alanine:glyoxylate aminotransferase (AGT), a model of PH type 1 [83][84][85]. The central role of GO to enhanced oxalate synthesis in PH type 1 stems from the deficiency in AGT; however, in healthy volunteers with normal AGT activity, the role of glycolate as an oxalate precursor is unknown. Glyoxal, a 2-carbon reactive dialdehyde, is produced through a number of oxidative and metabolic reactions [86] and can be enzymatically converted to glyoxylate [87] and glycolate [88]. In a study utilizing human erythrocytes, glyoxal was shown to be preferably converted to glycolate with 1% ultimately converted to oxalate via glyoxylate [89]. The formation of oxalate was increased when intracellular glutathione was depleted, highlighting a potential role of oxidative stress in endogenous oxalate synthesis through this pathway. Glyoxal is found in a vast number of food items and beverages, such as bread, cookies, yogurt, sardine oil, coffee, tea, beer and wine, and is a food processing contaminant [90][91][92][93][94][95]; however, nothing is known about the intestinal absorption of these dietary sources of glyoxal. The reactivity of glyoxal in vivo also makes it a difficult target to study and its role in endogenous oxalate synthesis remains unclear.

Fructose, Glucose and Pentose Sugars
Epidemiological evidence of the association between fructose intake and kidney stone formation [96,97] supports the hypothesis that fructose consumption could lead to oxalate synthesis, directly or indirectly through serine metabolism [77]. However, a controlled diet study in healthy volunteers fed with increasing fructose (4-21% of calories) failed to demonstrate a change in urinary oxalate excretion [22]. Furthermore, in vitro experiments in liver cells did not show any conversion of 13 C-labeled sugars (fructose and glucose) to oxalate [22]. The reported association of fructose intake and kidney stone disease may be explained by confounding factors such as obesity and insulin resistance [98,99]. The pentose pathway and xylulose has been proposed as a source of oxalate synthesis, via glycoladehyde and glycolate synthesis [77]. Although there have been case reports of oxalosis following xylitol intake, in vitro experiments [100] do not support xylitol as an important source of oxalate, and thus it is still unclear whether the intake of xylitol is a risk factor for increased urinary oxalate excretion.

Ascorbic Acid
The relationship between AA and oxalate synthesis has been known since the mid-20th century [73]. The formation of oxalate is the result of the non-enzymatic breakdown of AA into 2,3-diketogulonic acid (DKG) (Figure 1). DKG can be split into threosone or erythrulose, accompanied by oxalate in both cases [10,74]. The conversion is pH and temperature sensitive, a factor that may impact oxalate measurement in some biological samples rich in AA. Studies by Atkins et al. and Baker et al. indicate that AA may be the largest contributor to endogenous oxalate synthesis [81,101], contributing approximately 40% to urinary oxalate excretion. The methods used at that time had limitations and thus studies readdressing the role of AA in endogenous oxalate synthesis are needed. The association between AA intake and the risk of kidney stone risk has been shown in several large cohort studies, although lack of dietary control, short duration of the studies and potential sample handling issues may have influenced the results [13,102]. AA intake has also been reported to promote oxalate excretion to a greater degree in kidney stone formers [13]. Though one study performed under conditions of dietary control found that both kidney stone formers and healthy controls had increased urinary oxalate attributed to oral AA supplementation [17]. Several case reports have also indicated that individuals with compromised renal function can develop oxalate nephropathy following large oral doses or intravenous infusions of AA [103][104][105]. Despite the importance of AA to oxalate synthesis in both healthy subjects and kidney stone patients, there remain gaps in our knowledge of the mechanisms involved. Mitochondria have been regarded as a major site of intracellular AA recycling and act as a major component of endogenous antioxidant defense [106]. It is possible that as AA acts as an antioxidant in the mitochondria in which oxalate is produced. Investigating the importance of this pathway in oxalate synthesis is warranted and may identify novel approaches to reducing oxalate synthesis.

Oxalate and the Kidney
Oxalate is an end product of metabolism in humans. In healthy subjects, approximately 95% of the oxalate absorbed by the gut and produced endogenously is excreted in urine [107]. Oxalate is delivered to the nephron through glomerular filtration and secretion [108,109]. While oxalate reabsorption has been demonstrated in the rat proximal tubule [110], this has not been characterized in humans. Oral loading of a single dose of sodium oxalate in healthy volunteers on controlled diets showed a rapid absorption and net renal secretion, with transient elevations in both urinary and plasma oxalate [11]. A later study evaluating oxalate excretion following oral oxalate loads demonstrated no differences in urinary oxalate excretion or the ratio of oxalate to creatinine clearance between normal Nutrients 2021, 13, 62 9 of 13 subjects and kidney stone formers [20]. However, Bergsland et al. found that patients with idiopathic hypercalciuria or stone formers who have had bariatric surgery had significantly more rapid increases in urinary oxalate compared to normal controls while on controlled diets, and that tubular secretion of oxalate was a key mediator, potentially to maintain tight regulation of plasma oxalate [24]. In addition to its role in excreting oxalate, there is some evidence that the kidney might play a role in oxalate synthesis. Both glyoxylate reductase (GR) and 4-hydroxyproline dehydrogenase (HYPDH) enzymes are highly expressed in the kidney. In rats, the proximal tubule has been shown to be the segment of the nephron where these enzymes are highly expressed [111][112][113][114]. Experiments by Farinelli and Richardson in the hepatectomized rat have suggested that some metabolism of glycolate to oxalate might occur in the kidney via unknown mechanisms [61]. A study in the GR-deficient mouse, a model of PH2, has shown that dietary hydroxyproline feeding rapidly induces CaOx nephrocalcinosis due to increased renal oxalate synthesis [115]. In light of these findings, future studies should examine the role of renal oxalate synthesis and renal oxalate handling in urinary oxalate excretion.

Conclusions
Intake of dietary oxalate and dietary oxalate precursors are key determinants of urinary oxalate excretion and CaOx stone disease. Quantification of dietary oxalate intake in large populations via FFQs has significant limitations which can be overcome through studies with strict dietary control. Through such studies, it has been determined that dietary oxalate intake on average accounts for~50% of urinary oxalate excretion in normal healthy subjects. The role of oxalate-degrading gut microbes in CaOx kidney stone disease is still unclear and requires further investigation using controlled diets. AA has been identified as the major dietary oxalate precursor while other putative precursors seem to have a lesser role. However, a limitation of the majority of these studies is they have only been conducted in healthy volunteers. Controlled dietary studies in patients with stone disease to examine the role of dietary oxalate, oxalate precursors, and both renal and gastrointestinal handling are needed. Institutional Review Board Statement: Ethical review and approval were waived for this study, due to this is a review article.