1 Introduction

Chronic kidney disease (CKD) is a serious condition characterized by the gradual decline of kidney function, eventually requiring dialysis or kidney transplantation to sustain life. The number of patients is increasing worldwide, and according to data from the World Health Organization (WHO), CKD is estimated to affect more than 800 million people globally, making it a significant public health issue [1]. In Japan, the number of CKD patients is estimated to be approximately 13 million, with the number of patients undergoing dialysis therapy due to CKD progression expected to reach around 350 000 by 2023 [2]. Once CKD reaches an advanced stage, it becomes essentially irreversible, making early diagnosis and appropriate therapeutic interventions critical to preventing the progression of chronic kidney failure. Accurate assessment of individual renal function is essential for early diagnosis and effective therapeutic interventions. However, current renal function assessment methods used in clinical practice face challenges in terms of accuracy and simplicity. Meanwhile, our recent studies have increasingly demonstrated that the properties of d-amino acids may serve as ideal biomarkers for assessing renal function. Therefore, we aimed to develop a novel method for evaluating renal function using d-amino acids.

2 What Is Renal Function?

Glomerular filtration rate (GFR) is a representative index used to evaluate renal function. The kidneys perform various functions, including regulating fluid volume, maintaining electrolyte balance, regulating acid–base equilibrium, controlling blood pressure, and promoting red blood cell production. Among these, the most critical function is filtering blood and eliminating waste products from the body as urine.

The kidneys contain hundreds of thousands to millions of functional units called nephrons. Nephrons are broadly divided into three main components: the glomeruli, Bowman's sacs, and tubules, each playing a specific role. The glomerulus filters blood, collecting waste products and excess fluid as “primary urine” in Bowman's sac. This primary urine is subsequently reabsorbed and secreted within the renal tubules, ultimately being discharged as urine.

Renal function, or the filtration capacity of the kidneys, is expressed as the volume of blood filtered by the glomeruli per minute, which is defined as GFR. GFR reflects the efficiency of blood filtration in the glomeruli and serves as an important indicator of kidney health [3] (Figure 1).

In healthy adults, GFR is generally considered normal within the range of 90–120 mL/min/1.73 m². A decrease in GFR below this range suggests a potential decline in renal function [4].

3 Necessity of Renal Function Assessment

As mentioned, the measurement of GFR is used to diagnose CKD and monitor its progression. This enables the early detection of declining renal function and the evaluation of disease progression.

In addition, the adjustment of dosages for renally excreted drugs and drugs with nephrotoxic potential is also based on GFR. Furthermore, GFR is a critical factor in the selection of donors in renal transplantation. Accurate assessment of renal function is therefore essential for the safe and effective treatment of patients.

4 Methods and Issues in Renal Function Assessment

As the direct measurement of GFR is highly challenging, clinical practice typically relies on the estimation of GFR using biomarkers of renal function. An ideal renal function biomarker should meet the following criteria: (i) free filtration in the glomerulus, (ii) no reabsorption or secretion in the tubules, and (iii) stable dynamics independent of GFR [5, 6]. Furthermore, from the perspective of minimizing patient invasiveness, the use of endogenous substances for estimation is preferred.

In routine clinical practice, GFR is estimated and evaluated using the following methods.

5 d-Amino Acids as a Biomarker for Kidney Disease

Proteins, which play a major role in biological functions, are composed of 20 amino acids, their smallest units. Although Krebs et al. discovered the presence of d-amino acid oxidase (DAO) in the kidney in 1935 [8], the physiological role of d-amino acids in the human body remains unclear. However, recent advancements in measurement technology have revealed that d-amino acids are present in trace amounts in mammalian bodies and exhibit a variety of physiological functions.

d-Amino acids, especially d-serine and d-asparagine, were reported to be elevated in kidney and plasma in patients with worse kidney function [9]. d-Serine and d-aspartate delivered to the kidneys are excreted almost 100% in urine. However, when kidney blood flow decreases with the decline of kidney function, the amount of d-amino acids reaching the kidneys also decreases, resulting in the accumulation of d-amino acids in the blood. This reflects kidney function [10]. On the other hand, in Dao-deficient mice, the metabolism of d-serine is impaired, leading to an increase in its concentration in the blood [11, 12]. Dao is primarily expressed in the kidneys [13], but as d-serine is almost entirely excreted in urine, the amount of d-serine metabolized by the kidneys may not be very high. The effects of Dao are based on studies conducted with completely gene-deficient animals. However, the impact of partial DAO deficiency, similar to what has been studied in relation to kidney function in humans, remains unclear.

Regarding the excretion of d-amino acids, the decrease in kidney blood flow is likely an upstream mechanism, making it the primary reason for the increase in blood concentration in worse kidney function.

Given the close relationship between the kidney and d-amino acids, we have been investigating the potential of d-amino acids as biomarkers for kidney disease. Our findings indicate that patients with CKD who exhibit high levels of d-amino acids, particularly d-serine and d-asparagine, in their blood tend to experience earlier onset of end-stage renal failure and dialysis compared to those with intermediate or low levels of d-amino acids [9, 14, 15].

Furthermore, differences in d-amino acid dynamics among the underlying diseases causing CKD suggest that these dynamics may aid in diagnosing specific diseases [16, 17].

The primary diseases of CKD vary, and each disease has different treatments and prognoses. The dynamics of d-amino acids in each disease are expected to reflect differences in tubular function, and these differences can be used to diagnose the primary disease of CKD. On the other hand, the dynamics of d-amino acids change depending on the primary disease of CKD, making strict evaluation of GFR difficult. The usefulness of d-amino acid clearance as a method of evaluating renal function we have reported has been studied in renal transplant patients, whose kidneys are considered to be less damaged by the primary disease, and therefore, it may be necessary to investigate this method in CKD patients with other primary diseases. Additionally, principal component analysis of various l- and d-amino acid concentrations in the blood of patients with CKD demonstrated that d-serine and d-asparagine form a cluster group similar to conventional renal function markers, such as creatinine [18, 19]. The results suggest that d-amino acids, but not l-amino acids, are useful as biomarkers of kidney disease.

6 Evaluation of Renal Function by d-Serine and d-Asparagine

On the basis of these studies, we explored the potential of d-amino acids as markers for renal function evaluation, replacing creatinine and inulin. To assess the GFR, it is necessary to evaluate its clearance into urine. We measured the clearance of d-serine and d-asparagine in renal transplant donors and found that their clearance correlated strongly with inulin clearance. Additionally, these d-amino acids exhibited very similar dynamics to inulin [19, 20] (Figure 2a–c).

This strong correlation suggests that the clearance of d-serine and d-asparagine can be directly used to measure the GFR. Moreover, unlike creatinine clearance, the slope of d-serine and d-asparagine clearance closely matches that of measured inulin clearance. In other words, d-serine and d-asparagine clearance can mitigate slope bias in GFR measurement, which cannot be avoided when using creatinine clearance.

7 Why d-Amino Acids Are Useful in Assessing Renal Function

The theoretical basis for why d-amino acids are useful for renal function assessment has been discussed. The three conditions mentioned above are essential for evaluating renal function.

7.3 Condition 3: Stable Dynamics Regardless of GFR

The difference between the clearance of d-serine and d-asparagine and that of inulin clearance does not vary with GFR (Figure 3a–c). A comparison of urinary excretion per unit time also revealed that interindividual differences are as small as those observed with creatinine (Figure 3d–f) [10]. In other words, their clearance remains consistent even when renal function deteriorates.

These characteristics indicate that d-serine and d-asparagine satisfy all three criteria and are valuable for renal function evaluation. Furthermore, d-serine and d-asparagine are endogenous factors and serve as useful molecules for assessing renal function.

8 Summary and Outlook

We demonstrated the potential usefulness of d-amino acids as biomarkers of renal function. d-Serine and d-asparagine, as endogenous molecules, fulfill the requirements for biomarkers of renal function and GFR and are anticipated to see broader application in the future. Currently, our studies have primarily focused on renal transplant patients. However, we believe their utility as biomarkers of renal function across diverse patient profiles warrants further evaluation.

In particular, unlike creatinine, d-amino acids are less influenced by muscle mass, making them promising candidates for use as renal function biomarkers in pediatric patients, where this issue is especially challenging [18, 21].

Recently, d-alanine, a d-amino acid, has been identified as a biomarker for predicting the severity of influenza and COVID-19 infections [22, 23]. In the field of malignancy, d-asparagine, d-alanine, and d-proline have been reported as biomarkers for the development of bladder cancer [24].

Additionally, studies have shown that the ratio of d-amino acids in feces is lower in patients with ulcerative colitis. Moreover, in vivo d-amino acid supplementation has been demonstrated to improve colitis, hepatitis, and cholangitis in mice [25], suggesting its potential use as both a biomarker and a therapeutic target. Functionally, d-alanine can correct the circadian rhythm and its associated physiological processes, suggesting the potential to improve life style-related diseases [26]. Recent findings suggest that d-alanine plays a role in glucose metabolism and viral infection [27].

We believe that further elucidation of the dynamics of d-amino acids in the body, alongside research in the field of nephrology, will contribute to the accumulation of new knowledge and the development of treatments for a wide range of diseases currently affecting humanity.

Conflicts of Interest

The authors declare no conflicts of interest.