Introduction

All protein amino acids, except glycine, are chiral, with optical activity originating from the C-alpha atom. The L and D-amino acids that constitute these stereoisomers are enantiomers of one another. Chiral amino acids have identical chemical molecular formulas and differ only in their spatial configuration (Figure 1).

D-amino acids were once thought to be rare in higher organisms, but they are now recognized in many tissues and biological fluids. In bacteria, D-amino acids regulate cell wall remodeling and biofilm disintegration [1]. There is a strong connection between D-amino acids and various illnesses, including neurological disorders and cancer [2,3]. In particular, emerging research shows that D-amino acids are intricately linked to kidney health and disease. They have been detected in renal tissue and urine, and some D-amino acids show significant changes in concentration during kidney disease progression. D-amino acids therefore hold promise as novel biomarkers of kidney function and pathology [4,5].

Kidney diseases are a major global health concern, and current biomarkers (such as creatinine or proteinuria) have limitations in sensitivity and specificity. In this context, D-amino acids have attracted attention for their potential clinical significance in nephrology. For example, D-serine – a D-amino acid present in blood and urine – has been identified as a possible endogenous marker of glomerular filtration rate, showing a correlation with kidney function in patients. Understanding how D-amino acids originate, are metabolized, and function in the body could open new avenues for diagnosing and treating renal diseases [6–8].

The Introduction section of this article delineates the conceptual and empirical context of D-amino acids and frames the subsequent sections. We first synthesize current knowledge on their endogenous and exogenous sources in humans alongside principal metabolic and clearance pathways. We then survey analytical methodologies for enantiomeric separation and quantification, critically appraise evidence implicating D-amino acids in renal pathophysiology, and evaluate their performance and limitations as candidate biomarkers. The review concludes by identifying unresolved knowledge gaps and priorities for future investigation. Overall, the objective is to critically examine the sources and metabolism of D-amino acids and their roles as biomarkers in kidney disease.

The Origin and Metabolic Processes of Chiral Amino Acids Within the Human Body

Amino acids primarily originate from exogenous diets and metabolic processes within the cytoplasm [9]. Conversely, D-amino acids in the human body are derived from various sources, including exogenous dietary intake, production within the central nervous system, and the oral and gut microbiota [10]. Studies have shown that the human body consumes more than 100 mg of D-amino acids per day through dietary intake, mainly originating from starting materials, fermentation processes, racemization during food processing, or contamination [11]. Recent data indicate that serine racemase, the D-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that D-serine also has a neuronal origin [12].

Gut bacteria are a significant source of D-amino acids. Most of these amino acids are created by the intestinal flora and are both directly eliminated in feces and absorbed into the bloodstream via the intestines [13]. Only a small number of transporter proteins, including LAT1, ASCT1, ASCT2, ATB(0,+), and EAAT, can transport D-amino acids. These proteins are selective for the transport of D-Leu, D-Ser, D-Met, D-Phe, and D-Asp, among other amino acid types [14–16]. Renal excretion selectively removes unbound D-amino acids from the serum [17] (Figure 2).

The primary elements of peptidoglycan on the bacterial cell wall are D-Ala and D-Glu [11,18]. Peptidoglycan is a crucial barrier that protects bacteria by helping to control bacterial osmotic pressure and preserve cell shape [1]. Bacteria primarily manufacture D-amino acids through various internal racemase and epimerase enzymes and release them to control biofilm breakdown and bacterial spore development [18]. Bacteria utilize 2 strategies to produce D-amino acids: stereospecific amination of the corresponding α-ketoacid or inverting the stereochemistry of the corresponding L-amino acids. Racemase or epimerase catalyzes the former, a reversible reaction resulting in the direct interconversion of L- and D-stereoisomers. D-amino acid aminotransferase enzymes catalyze the latter, which is also reversible and requires an amino acid-containing co-substrate. Epimerase and racemase include Ala racemase, Serine racemase, Lys racemase, Asp racemase, Pro racemase, Glu racemase, and Broad-Spectrum Epimerase [19]. Tian et al used isotopically labeled L-/D-Ala and germ-free mice to track their biodistribution and racemization in vivo [20]. When the diet was changed, Konno et al discovered that the amount of D-Met in the urine decreased proportionally, but the amount of D-Ala did not. However, the amount of D-Ala was substantially lower when sterile mice were used, proving that most of the D-Ala in urine came from the gut microbiota [20,21].

In addition to serving as the building blocks of proteins and polypeptides, L-amino acids serve as the precursors to significant biomolecules, including bioactive amines [22]. They play a key role in metabolic pathways and physiological processes involved in health, growth, development, and reproduction. L-amino acids regulate the synthesis and metabolism of nutrients, endocrine homeostasis, neurotransmission, and immunity [23]. They are essential for antibodies and energy metabolism and are integral to all proteins, whereas D-amino acids lack these physiological activities and functions [21]. However, previous research has shown that D-amino acids are present in various tissues and organs, including the brain, skin, bone, and biological fluids, indicating that they may serve multiple physiological roles [24,25].

The activity and stability of peptide antibiotics are improved by D-amino acids, which are typically found in natural antibiotics extracted from bacteria, invertebrates, and amphibians [26]. For example, ranalexin, a powerful antimicrobial peptide, is produced by the skin of American bullfrogs. Despite ranalexin’s potent antimicrobial activity against gram-positive bacteria, it has disadvantages, such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting solely of D-amino acids, named danalexin, was developed. Danalexin showed greater biodistribution and a longer retention period in Wistar rats compared to ranalexin [27]. Antibiotics work by preventing the metabolism of proteolytic enzymes and interfering with or eliminating biofilms via the hydrogen peroxide formed by the DAO metabolic pathway [1,13]. As a result, D-amino acids are frequently used as the structural foundation for creating pharmaceuticals and antibiotics, such as the D-Val residue in antibacterial cyclic hexapeptides [28]. In mammals, D-amino acids can regulate the immune system and aid in coordinating host-microbe interactions [29–31]. The oxidative deamination of D-amino acids by DAO results in hydrogen peroxide, which has an antibacterial effect, leading some researchers to hypothesize that the expression of DAO in leukocytes may be related to the bactericidal activity of these cells. DAO also modifies the microbiota composition and is associated with microbial induction of intestinal sIgA [32]. In neutrophils, GPR109B mRNA is widely expressed. Human neutrophils treated with D-Phe and D-Trp had a brief rise in intracellular Ca2+ and a fall in cAMP. Additionally, D-amino acids-induced reduction of cellular cAMP levels in neutrophils was prevented by knocking down GPR109B by RNA interference [33].

Furthermore, D-Trp, D-Phe, and D-Leu are involved in immune regulation in different parts of the respiratory tract, and a high intracellular D-Ser concentration in urethral pathogenic bacteria influences the expression of virulence factors, reducing the bacteria’s virulence. D-Ser has an antibacterial action in the urinary system [30]. Feeding D-Trp to asthmatic mice increased the number of regulatory T cells in their lungs and colons and increased gut microbial diversity, which was decreased by allergic airway inflammation [31].

Notably, D-amino acids lack the widespread roles of L-amino acids (which are building blocks of proteins and crucial for metabolism). However, the presence of D-amino acids in skin, bone, brain, and other tissues indicates they may have specialized functions yet to be fully elucidated.

In summary, the body manages D-amino acids through a balance of microbial production, limited human synthesis, and active degradation/excretion mechanisms. This balance determines circulating and urinary D-amino acid levels, which – as discussed in later sections – can change markedly in kidney disease.

Detection of Chiral Amino Acids

Separating and measuring L- and D-amino acid enantiomers is analytically challenging due to their identical chemical properties. This section describes how researchers detect chiral amino acids in biological samples.

General Principles: Separating and measuring the L and D isomers of chiral amino acids is crucial for determining their concentration in living organisms. Chiral amino acid analysis relies on converting enantiomers into distinguishable forms or using chiral environments to separate them. Two primary approaches are employed:

Indirect Method (Derivatization): This method separates chiral molecules by producing diastereoisomers with the aid of a chiral derivatizing agent. However, this approach has drawbacks. The derivatization outcome directly influences the lower limit of quantification (LLOQ), and various amino acids compete for the derivatizing agent during the process. Amino acids may be lost due to cyclization at high temperatures or in extremely acidic or alkaline conditions [34,35].

Direct Method (Chiral Chromatography): This method employs commercially available chiral stationary phases or chiral mobile phase additives. It separates chiral amino acids using a specialized column without derivatization, minimizing amino acid loss and reducing detection time. However, due to the distinct spatial structures of various amino acids, this approach cannot separate all types of amino acids and is highly dependent on the performance of the stationary phase [36,37].

Analytical Advances: Recent years have seen significant improvements in chiral amino acid detection techniques. Trace amounts of amino acids are detected by 4 main assays: capillary electrochromatography (CEC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS).

For example, Han et al successfully determined the content of chiral amino acids in the serum of patients with hepatocellular carcinoma and healthy individuals using a CROWNPAKCR-I(+) chiral column. They discovered that D-Glu and D-Gln were significantly down-regulated serum markers in hepatocellular carcinoma and separated 19 pairs of proteogenic amino acid enantiomers and achiral glycines in human serum in 13 minutes [2]. Using the CEC method, Rizvi et al used natural α-cyclodextrin and β-cyclodextrin synergistic chiral selective agents to separate L-Kyn and D-Kyn in human serum and urine [38]. Studies on the separation and analysis of multiple enantiomers using CEC have been published [39].

By combining 2 different types of chiral columns (a crown ether based on a binaphthyl group and an amphoteric stationary phase derived from cinchona alkaloids), Nakano et al developed an LC-MS/MS high-throughput method to analyze 115 chiral and achiral metabolites [40]. Kimura et al examined the blood levels of 305 women using 2 columns, QN-AX and ZWIX(+), in series [41]. A novel chiral selective agent called Taniaphos, created by Wenjie Xiao and colleagues, effectively extracts D-amino acids [42].

In terms of accuracy and practicality, liquid chromatography (LC), gas chromatography combined with mass spectrometry (GC-MS/MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are still the most commonly used methods for analyzing chiral amino acids. Accurate quantification of biomarkers is crucial to study outcomes, and each method has benefits and drawbacks. Future research should focus on developing more user-friendly tools for quantitative analysis.

Applications in Renal Research: Robust analytical methods have paved the way for examining D-amino acids in kidney disease contexts:

Researchers have profiled plasma and urine D-amino acid levels in patients with various renal diseases. For instance, Kimura et al (2016) used a two-dimensional HPLC system to analyze plasma from 108 chronic kidney disease (CKD) patients, finding that certain D-amino acids (like D-serine, D-asparagine, D-proline) were correlated with CKD progression [43]. In rapidly progressive glomerulonephritis (RPGN), serial D/L-serine measurements by HPLC-MS provided insight into disease dynamics, as discussed later [44]. D-Serine has been measured by LC-MS in kidney transplant donors and recipients to evaluate it as an endogenous GFR marker, with promising results [45].

In summary, state-of-the-art detection techniques allow clinicians and researchers to accurately quantify D-amino acids in biological samples. This capability is fundamental to investigating D-amino acids as biomarkers or mediators in kidney disease.

Roles of D-Amino Acids in Renal Disease Development

NEPHROTOXICITY OF D-AMINO ACIDS:

High concentrations of D-amino acids can damage the liver and kidneys, inhibit growth, and disrupt the production of critical neurotransmitters. Studies have shown that high D-Ser administered orally or intravenously to rats causes proteinuria, glycosuria, and severe acute necrosis of the proximal tubules of the kidneys. Acute kidney injury (AKI) is characterized by rapid cellular atrophy, cytoplasmic loss, and nuclear content lysis, leaving only the basement membrane as a barrier between the interstitial fluid and luminal fluid [42,46,47].

IMMUNE REGULATION AND IGA NEPHROPATHY:

Changes in chiral amino acid levels are strongly correlated with the development of IgA nephropathy (IgAN). The intestine, abundant in lymphoid tissues and immune cells, is crucial for maintaining mucosal immunity. Abnormal intestinal mucosal immunity is a major cause of IgAN. Recent research indicates that the composition of gut flora significantly influences mucosal immunity. The intestinal microecology enhances the tight junctions of intestinal epithelial cells and regulates lymphocyte differentiation, thereby improving the mucosal barrier function and controlling the immune system response. The interaction between the host and intestinal flora influences the chiral ratio of amino acids, B-cell survival and activation, and Gd-IgA1 synthesis [32,46,48].

GUT–KIDNEY AXIS AND RENAL PROTECTION:

Two enzymes, D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO), catabolize and catalyze the creation of hydrogen peroxide from D-amino acids, which is toxic to pathogens. DAO catalyzes the oxidative deamination of neutral and basic D-amino acids, while DDO reacts with acidic D-amino acids to create 2-oxo acids, hydrogen peroxide, and ammonia. These enzymes are primarily generated in intestinal epithelial cells and neutrophils. Reduced DAO enzyme activity results in increased IgA synthesis, dysbiosis, and altered IgA coating on bacteria due to the absence of D-amino acid catabolism. DAO-mediated control of the small-intestine microbiota influences its level and composition [32,46].

DAO is linked to the microbial stimulation of intestinal sIgA and also modifies the microbiota’s makeup. In conclusion, D-amino acids, as metabolites of the gut microbiota, affect the production of IgA, potentially leading to the development of IgAN. D-amino acids could also be protective against acute renal damage. In a mouse model of renal ischemia/reperfusion, it has been demonstrated that chiral amino acids metabolized by the intestinal microbiota reduce tubular injury. AKI-induced disruption of intestinal ecology impacts D-amino acid metabolism. DAO activity decreases, and serine-abrogating enzyme activity increases in damaged kidneys. Oral administration of D-serine to rats lessens kidney damage, prevents hypoxia-induced tubular damage, and encourages post-hypoxic tubular cell growth. Blood levels of D-serine in AKI patients are significantly correlated with renal function, indicating a nephroprotective role of intestinal-derived D-serine in AKI [49].

In summary, chiral amino acids can have diverse effects on renal disease development, and these insights underscore the complex role of the microbiome and metabolism in renal pathology.

D-Amino Acids as Biomarkers in Kidney Disease

Currently, there are no disease-specific biomarkers other than blood creatinine, urine protein, and glomerular filtration rate to accurately and dynamically monitor renal function in kidney disease. Existing studies on kidney disease and chiral amino acid changes are shown in Figure 3. Patients with kidney disease have very small quantities of chiral amino acids in their blood or urine.

Plasma chiral amino acid dynamics react to disease development in rapidly progressing glomerulonephritis (RPGN). Plasma levels of D-serine are abnormally high during its eruptive phase, and its urine fractional excretion (FE) is much higher than those of L-serine. Elevated plasma amino acid levels gradually return to normal after treatment. Blood creatinine levels decline concurrently with D-serine levels, suggesting a gradual restoration of glomerular filtration rate. Prior to patient recovery, there is a brief rise in the amount of D-serine in the urine, indicating renal facilitation allowing free D-serine levels in the blood to revert to normal after kidney function restoration. Chiral amino acids have the potential to serve as RPGN biomarkers [44].

A study on kidney transplantation clarified the connection between D-amino acids and glomerular filtration rate (eGFR). Using a linear regression model, the relationship between plasma D-serine clearance and eGFR was confirmed. Two endogenous substances, D-serine and creatinine, were used to assess eGFR and renal function with good accuracy, suggesting these substances could serve as markers for clinical assessment of renal function indicators after kidney transplantation [45].

Chiral amino acids in the blood can also be utilized as biomarkers to detect kidney disease in chronic kidney disease (CKD) patients. Kimura et al analyzed 16 chiral amino acids in the plasma of 108 CKD patients using micro-two-dimensional high-performance liquid chromatography (2D-HPLC) and found serine, proline, and asparagine were most strongly connected with chiral amino acids and CKD progression [43].

In diabetic nephropathy (DN), the FE of D-serine rises as eGFR declines due to tubulin damage affecting proximal renal tubule reabsorption. The excretion of D-serine increases, showing a relatively minor positive association with urine β2-microglobulin (a measure of decreased reabsorption). This phenomenon is not observed in patients with IgAN and MCD who still have residual renal units. Thus, D-serine could act as a special biomarker for DN [50].

Chiral amino acids exhibit specificity in serum versus urine in some acute kidney injuries. This was demonstrated by serine enantiomers found in the serum and urine of mice following renal ischemia-reperfusion injury (IRI). Blood levels of D-serine increase with creatinine levels in mice with renal IRI, while L-serine levels rapidly drop. Only the proximal tubules displayed DAO activity, but IRI rendered DAO inactive, increasing serum levels. The L/D-amino acid ratios in blood and urine offer a more sensitive response to kidney injury than the kidney injury biomarkers KIM-1, NGAL, creatinine, cystatin C, or urea ratios [51].

D-methionine, another compound with potential as a marker, is affected by renal insufficiency. Studies in rats revealed that the clearance of D-methionine in the renal insufficiency group was only one-sixth that of the sham-operated group [51].

Future Directions

EXPANDING BEYOND D-SERINE:

Thus far, D-serine has received the most attention, but other D-amino acids may be relevant to renal pathology. For example, D-aspartate and D-alanine are abundant microbial D-amino acids that could influence host metabolism or immune signaling. Preliminary metabolomic data suggest D-proline and D-asparagine levels change in CKD patients; these findings warrant deeper investigation. Future work should systematically screen a broad panel of D-amino acids in various kidney diseases to identify novel biomarkers or effectors. There may be condition-specific patterns (a “D-amino acid signature”) that we have yet to discover.

MECHANISTIC STUDIES:

We need a better mechanistic understanding of how D-amino acids interact with the kidneys. For instance, how exactly does D-serine cause tubular toxicity? Does it induce oxidative stress, or does it trigger apoptosis via a specific receptor? Conversely, how do D-amino acids confer protection in AKI? Unraveling these mechanisms could reveal new therapeutic targets (eg, modulating DAO activity or blocking D-serine uptake in certain contexts). Animal models, such as mice with genetic alterations in D-amino acid metabolizing enzymes (DAO knockouts, serine racemase knockouts), will be valuable tools to study outcomes in models of CKD or AKI.

In summary, the “D-amino acid nephrology” field is ripe for exploration. By addressing these questions, we will clarify whether D-amino acids are merely correlated with kidney disease or actively contributing, and how we can harness this knowledge. Given the early promising results, future research holds tremendous potential for deeper pathophysiological insight and clinical innovation.

CLINICAL TRANSLATION AND BIOMARKER VALIDATION:

Before D-amino acids can be used in practice, large-scale clinical studies are needed. Longitudinal cohort studies should evaluate whether baseline or changing D-amino acid levels predict outcomes in CKD (eg, time to dialysis) or AKI (eg, need for RRT, recovery). It will be important to establish reference ranges for plasma and urine D-amino acids in healthy populations and various disease states. Additionally, we must consider confounding factors: diet, for instance, can acutely alter D-amino acid levels (eg, after a high-dairy meal), and gut microbiome variability between individuals is large.

Conclusions

Research into the origins, metabolism, and detection of chiral amino acids is gradually elucidating their physiological roles and connections to kidney diseases. Significant progress has been made in understanding the interplay between D-amino acids, the gut microbiome, and host metabolism, revealing unique functions and pathways that distinguish D-amino acids from their L-counterparts. There is growing evidence that D-amino acids and kidney health are closely intertwined. In particular, D-serine has emerged as a potential predictor and marker of renal function and injury. Early clinical studies suggest that measuring D-amino acids could improve the detection and monitoring of conditions like AKI, CKD progression, and glomerular diseases, offering information beyond traditional biomarkers.

Nevertheless, this field is still nascent. Many D-amino acids remain underexplored in the renal context, and we are just beginning to grasp how modulation of D-amino acid levels might affect disease outcomes. The tantalizing question is whether D-amino acids could be not only indicators but also effectors in kidney disease processes; for example, influencing immune responses in IgA nephropathy or aiding recovery in AKI. Additionally, the specific contributions of different gut bacteria to the host D-amino acid pool and subsequent renal impacts represent a new frontier in gut-kidney axis research.

In conclusion, chiral amino acids, once a biochemical curiosity, are now recognized as important molecules in nephrology. They hold promise for advancing our understanding of renal pathophysiology and improving patient care. Future work will determine how we can translate these insights into clinical practice, whether through novel diagnostics (D-amino acid panels) or even therapeutic modulation of D-amino acid pathways. Embracing this new perspective may provide significant benefits in our fight