In this study, we simultaneously measured nine trace elements from patients with different stages of DKD and evaluated the association between them, both in single and mixtures. Mn and Cu are positively correlated in all DKD stages, and Mn is still the most positively correlated element with the largest contribution and weight in the mixture model. Ni and Fe are the two elements negatively correlated in both mid and end DKD, Ni ranks first in the weight of the mixture, while Fe ranks second and has a significant correlation only in the end DKD. At the end DKD, there was a negative correlation trend between nine element mixtures and DKD. When the total mixture was lower than 50%, trace elements had a certain risk, which was mainly driven by Mn and Cu.

Recent epidemiological studies on kidney function suggest that besides genetic and lifestyle factors, sex and age show significant differences in CKD.27 The prevalence of DKD in this study was 48%, with no significant difference between men and women. This study used a case–control approach and employed the PSM method to individually match age and sex in mid and end DKD. Urine is a non-invasive and usually preferred biological monitoring medium, especially for some water-soluble trace elements.28 In the process of producing urine, the kidney is constantly exposed to and filters many toxins and pollutants, which makes it vulnerable to adverse effects. The levels of these trace elements in urine indirectly reflect the decline in kidney function, influencing the filtration and reabsorption of these elements.29 Fe deficiency anemia is a common comorbidity in CKD, found in 44% of our study. Clinically, Fe is often used to treat anemia associated with CKD,28 but excessive Fe storage can lead to infections and subsequently impair kidney function, causing abnormal Fe excretion.30 Experimental studies have observed that high exposure to Fe leads to increased urinary Fe excretion and kidney damage.31 In this study, Fe levels were found to be significantly lower in subjects with DKD, yet Fe served as a stronger protective factor against ESRD than for mid DKD. This might be due to kidney disease reducing the body’s overall Fe absorption.32 However, it is important to note that Fe²+, the active form involved in ferroptosis, can promote oxidative damage in DKD by triggering renal fibrosis and cell death. In contrast, Fe³+, stored in ferritin, is less harmful but may contribute to imbalances when improperly regulated. Ferroptosis has been proven to be a potential mechanism in the progression of DKD. Fe overload was found in DKD mice, leading to ferroptosis, which in turn triggers renal fibrosis and mediates renal cell death.33

Previous epidemiological and mechanistic studies have indicated that high exposure to Cu in both serum and urine can increase the risk of CKD and proteinuria, leading to poor kidney function.34 35 Previous studies have shown that excessive urinary Cu can also affect the development of DKD.36 Cu’s toxicity is largely due to its oxidative properties, especially in its Cu²+ form, which can catalyze the production of ROS, leading to oxidative stress and further kidney damage. The increase in urinary Cu excretion may be due to the dissociation of Cu from its carrier protein due to glomerular damage caused by acidification and kidney damage. An imbalance in Cu homeostasis may lead to impaired antioxidant capacity and the progression of DKD. Fe and Cu are both important biological oxidizing agents. They function as essential elements of SOD, which helps cells resist oxidative damage.37 However, when in overload, they can act as ROS and cause damage to renal tubular epithelial cells.34 However, the accumulation or reduction of element ions does not directly reflect ferroptosis or Cu death. The oxidation valence state of elements, such as Fe2+, promotes ferroptosis, while Fe3+ is usually an inert storage in ferritin. The toxicity of Cu also has different forms of toxicity compared with Cu2+. Therefore, future research needs to pay more attention to the determination of element valence states and the assessment of toxicity risks.

Cu is a cofactor of Cu, Zn-SOD, where Cu²+ directly participates in the catalytic cycle and reduces oxidative damage to cells, while Zn²+ plays a structural role and enhances stability. Zn plays a key role in regulating numerous cellular and subcellular processes in humans, such as DNA replication, energy metabolism, protein structure maintenance, and growth.38 In mid DKD, Zn levels are elevated, potentially as a protective response to renal stress, leveraging Zn’s antioxidant properties. Conversely, in end DKD, Zn levels decrease, signaling severe kidney dysfunction and impaired reabsorption of essential minerals.39 In advanced kidney disease, reduced renal function hampers Zn reabsorption, leading to lower urine Zn levels. Urinary Zn concentrations mirror kidney damage and dysfunction in DKD, with high levels possibly signifying compensatory responses in early stages and low levels indicating severe impairment in advanced stages.

A study has shown that supplementation with Se positively affects some inflammatory and oxidative stress markers in patients with DKD.40 41 Se is an essential trace element or metalloid that helps produce a variety of enzymes with antioxidant properties like glutathione peroxidase.42 A double-blind randomized controlled trial for Swedish elderly put forward a possible mechanism for the association between Se exposure and CKD that kidney function was dramatically improved with the supplemented effect of Se and coenzyme Q10.43

Mn is essential for enzymes like Mn SOD, protecting against oxidative stress in CKD and DKD, but its levels rise due to impaired kidney function, especially during dialysis, becoming a consistent risk factor for DKD.44 Conversely, Ni, despite its toxic potential in excess, plays a protective role at appropriate levels by modulating pathways related to renal disease development.45 and enhancing cellular defenses against oxidative stress and inflammation via the Nrf2/NLRP3 pathway.46 The pathway supports cellular resilience against environmental and metabolic stresses, which is critical in chronic conditions like DKD where oxidative stress and inflammation are prevalent. In an animal experiment, kidney damage indicators such as serum urea and creatinine were significantly increased in mice exposed to Ni alone, indicating that the kidneys were unable to eliminate these waste products, resulting in a decrease in the concentration of Ni in the urine.47 Research indicates that urinary metal levels, including Ni, are influenced by kidney function. In the middle and late stages of DKD, reduced glomerular filtration and tubular reabsorption may lead to lower urinary excretion of elements like Ni, which might not directly reflect a systemic reduction in body burden but rather impaired kidney function.48 Furthermore, Ni has been shown to activate antioxidant defenses, which can reduce oxidative stress, a key factor in the progression of DKD. This is consistent with studies suggesting that oxidative stress and inflammation are central to kidney damage in DKD, and protective effects from elements like Ni could vary across disease stages depending on the body’s oxidative stress response. Additionally, animal models and in vitro studies have shown that other elements and antioxidants play protective roles in reducing oxidative stress in CKD and diabetes-related conditions.49 50

Our study benefits from a large sample size and the use of PSM matching to assess the impact of nine urinary trace elements across various DKD stages. For the first time, we examine stage-specific variations in these elements and their disease progression risks. However, the cross-sectional design precludes establishing causality, and we did not differentiate between element valence states or metabolites. Although the recovery rates of trace elements Zn, Ni, and Se in urine were slightly below or above the ideal range, this study employed rigorous quality control measures, including within-day and between-day precision evaluations (with coefficients of variation, all within acceptable ranges) to ensure the robustness of the data.51 Moreover, urinary elements may not be ideal biomarkers for all substances, and our single measurement may not capture fluctuating levels. The hospital-based recruitment could introduce selection and confounding biases. Future research should use prospective cohorts and control for occupational exposures.