Chronic kidney disease (CKD) is one of the major microvascular complications of type 2 diabetes mellitus (T2DM) (1–3) and a leading cause of end-stage renal disease (ESRD) in patients with T2DM (4). CKD is associated with an increasing risk of adverse outcomes including cardiovascular disease, infection and death, posing a significant threat to healthcare systems and having negative impacts on quality of life (5, 6). Official figures showed that CKD affected between 8% and 16% of the population worldwide and was a leading cause of death (7). A national survey conducted in China in 2012 revealed that the overall prevalence of CKD was 10.8% (10.2–11.3) (8), which indicated that CKD has become an important public health problem in China.

Growing evidence from clinical research suggests that interventions such as glycemic control, blood pressure control, lipid-lowering measures, use of RAS blockers, weight control and lifestyle modifications can delay the progression of CKD, but the residual risk of CKD remains substantial. Therefore, it is essential to explore novel potential biomarkers that can be used to predict CKD in patients with T2DM.

Over recent decades, rapid advances in bioassay technology have led to the detection of a growing number of metabolites, which have provided new insights into pathways and biomarkers related to diabetes and its complications (9–12). It is worth noting that many studies have found that plasma amino acid levels are significantly altered in patients with T2DM or CKD. Amino acids involved in urea cycle might play an important role in inflammatory markers and oxidative stress (13, 14), many researchers took efforts to examine the relationship between such amino acids and T2DM or CKD. The complete urea cycle is mainly expressed in liver and the products and substrates in the cycle include arginine, citrulline and ornithine. An animal experiment showed that abnormal amino acid metabolism in the urea cycle might be associated with insulin resistance (15). Nokhoijav et al. found that glutamine metabolism, urea cycle, and beta-oxidation make up crucial parts of the metabolic changes in T2DM. In addition, it has been demonstrated that CKD mice had high levels of arginine and citrulline (16). Strong evidence supports the hypothesis that citrulline is a possible biomarker of kidney metabolism (17, 18). However, the association between amino acids related to urea cycle and CKD in T2DM was largely unknown.

We conducted a cross-sectional survey in a Chinese population with T2DM to investigate any associations between serum levels of amino acids involved in urea cycle and the risk of CKD.

In this cross-sectional survey, we found that a higher concentration of citrulline was positively associated with increased risk of CKD in T2DM. The ratio of ornithine to citrulline was also associated with a markedly decreased risk of CKD in T2DM.

The progression of CKD increased the risk of all-cause mortality in T2DM patients (4, 25). Unfortunately, CKD in T2DM develops silently until severe damage has occurred. It is therefore crucial to find biomarkers that can detect decreased kidney function early within T2DM. The pathogenesis of CKD is complex and multifactorial, age, gender and duration of T2DM were typically unmodifiable risk factors for CKD; glycemic control, hypertension, lipid abnormalities, smoking and physical activity were modifiable risk factors for CKD (26). Insufficient insulin secretion was one of the risk factor of diabetic nephropathy (27); 5-methoxytryptophan (5-MTP), acetylcarnitine, taurine and tiglylcarnitine, were strongly correlated with the development of CKD (28). A prospective cohort study found that blood pressure levels, diabetes status, serum lipid status, obesity, smoking, and alcohol consumption affected the development of CKD (29). However, these markers are not sufficient to fully understand the pathogenesis of CKD, especially in T2DM. In our current study, we found that citrulline significantly increased the risk of CKD in T2DM. Some previous studies observed that citrulline was associated with diabetes. Research performed by Zhou Yong et al. revealed that plasma citrulline levels elevated in diabetes (30). A population-based study showed that plasma citrulline levels correlated with HbA1c levels (31). An animal experiment concluded that plasma citrulline and ornithine levels were elevated in obese and insulin-deficient mice, and further suggested that citrulline could be an early indicator of obesity-dependent metabolic impairment (32). Some encouraging data showed that citrulline could be a candidate predictor of renal injury, which was consistent with our findings. A GC-MS study showed that urinary metabolites differed between individuals with and without CKD in diabetes (33). A previou experiment comprising twelve mice showed that citrulline and arginine levels were elevated in CKD mice (16). Results derived from Framingham Heart Study (FHS) demonstrated that citrulline increased the risk of CKD (OR:1.48; 95%CI:1.19–1.83), indicating that the elevation of citrulline level in plasma might be a signal of underlying renal dysfunction (18). The results of an eight-year follow-up study from Korea showed that high levels of citrulline were strongly associated with the development of CKD (34). In addition, there were several studies documented that citrulline was associated with CKD progression or decreasing eGFR (17, 35, 36).

Interestingly, our study also found that the ornithine/citrulline ratio was reduced in CKD, which was in line with the results of some researchers. A study conducted in France in 2014 reported that citrulline/ornithine ratio increased in the late stages of CKD (36). A population-based study from South Africa showed that citrulline/ornithine ratio was significantly higher in the diabetic group compared to the non-diabetic group in stage 1 of CKD, indicating that this ratio may predict early CKD (37). It is worth noting that a small case-control study found that the ratio of citrulline to ornithine elevated in T2DM (38).

Citrulline is a non-essential amino acid, synthesized mainly in the intestine by the conversion of glutamine (39, 40). In kidney, the enzyme dimethyl arginine dimethyl amino hydrolase (DDAH) metabolizes asymmetric dimethylarginine (ADMA) to generate dimethylamine and citrulline, and subsequently citrulline is converted to arginine, a process that requires catalysis by argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) (16, 41). This could, to some extent, explain our findings that renal injury probably inhibits the activity of ASS or ASL, thereby affecting the conversion of citrulline to arginine (de novo synthesis of arginine) and resulting in abnormal arginine metabolism.

As we have described previously, citrulline and ornithine are two intermediates in arginine metabolism and an abnormal ratio of these two amino acids may reflect abnormal arginine metabolism. Abnormal ADMA metabolism is a manifestation of disorders of arginine metabolism. ADMA is produced by methylation of arginine residues in intracellular proteins by protein arginine N-methyltransferases (PRMTs), and over the past decades ADMA has been revealed to have biological properties that inhibit nitric oxide (NO) synthase, reduce NO bioavailability and lead to endothelial dysfunction (42, 43). NO is a potent endothelial vasodilator that balances the constrictor and regulates vascular tone and blood pressure. Some findings showed that plasma ADMA levels increased in the early stage of CKD (44–47) and that ADMA was negatively associated with eGFR (48, 49). Moreover, two cohort studies conducted in patients with CKD noted that ADMA favors CKD progression and renal function decline (50, 51). ADMA, therefore, explains well many pathophysiological aspects of CKD. We speculate that elevated plasma citrulline levels or abnormal ratio of ornithine to citrulline may be a consequence of the massive accumulation of ADMA in the vivo, which inhibits the synthesis of NO, a deficiency of which is a major feature of CKD.

There are two plausible conjectures regarding the results of our study: (1) renal injury interferes with arginine metabolism, resulting in abnormal citrulline and the ratio of ornithine to citrulline; (2) impaired citrulline metabolism may limit the bioavailability of arginine for nitric oxide synthesis, leading to endothelial dysfunction affecting renal function. Either explanation, however, previous studies were consistent in suggesting that citrulline or the ratio of ornithine to citrulline was closely associated with renal function. Our study drew the same conclusion: in patients with CKD, citrulline levels were significantly elevated and the ratio of ornithine to citrulline decreased. Large cohort studies and animal experiments are needed to validate the actual link between these metabolites and CKD.

Our research had critical implications for both public health and clinical practice. We found that patients with CKD had higher levels of citrulline and lower levels of ornithine/citrulline ratio. Our current study discovered an association between amino acids related to the urea cycle and CKD, which dramatically widens our appreciation of pathological mechanisms of CKD. Furthermore, our findings shed light on the prevention of CKD and the delay of its progression.

There were several limitations in our current study. First, because our study was a retrospective cross-sectional investigation, the true causality could not be validated. However, our findings were consistent with some earlier studies, which might support our results. Second, given the nature of cross-sectional study, only statistical associations between citrulline, the ratio of ornithine to citrulline and CKD could be revealed, not causal relationships. Diabetes was not only accompanied by aberrations in amino acid metabolism, but also accelerated kidney damage, and we cannot determine whether urea cycle disturbances resulted from CKD in our study. These findings need to be confirmed in prospective cohort studies. Third, our study involved T2DM patients who were hospitalized and likely had more severe CKD and T2DM. Therefore, it is important to be cautious when extending our results to other populations. Forth, LDL-C, HDL-C, TG, and HbA1c had a large number of missing values. Taking into account that amino acids related to urea cycle rather than glucose and lipids were the main factors in this study, we regarded the missing values as a category. Finally, diet may affect citrulline levels (52), however, information on diet was not collected and not available to the analysis.

In conclusion, we found that high serum citrulline was significantly associated with increased risk of CKD and ornithine/citrulline ratio was inversely associated with risk of CKD in T2DM. Prospective cohort studies from different populations with T2DM are required to replicate our findings and mechanistic investigations are also warranted to understand molecular mechanisms underlying the biological link between serum amino acids involved in urea cycle and risk of CKD.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author/s.

The studies involving human participants were reviewed and approved by First Affiliated Hospital of Liaoning Medical University. The ethics committee waived the requirement of written informed consent for participation. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

QZ and Z-ZF conceived the project, designed experiments. WZ wrote the manuscript and analyzed data. JuZ collected the information and contributed to the writing of this manuscript. XY conceived the project, designed experiments and interpretated the data. NL contributed to the data interpretation and data analysis. JiZ collect the information and contributed to the data interpretation and wrote the manuscript and analyzed data. All authors edited the final version of the manuscript. All authors contributed to the article and approved the submitted version.