The formation of calcium oxalate kidney stones is a multi-step and extremely complex process in which age, BMI, oxidative stress and trace elements play an important role. Age and BMI, as indicators of obesity, are causally associated with an increased risk of urolithiasis (8, 9). This study found that there were statistical differences in age and BMI between the two groups. 52% of patients in the calcium oxalate stone group were older than 60, and 29% were BMI > 25. Many trace elements participate in and affect the formation of calcium oxalate kidney stones, such as sodium and magnesium, which inhibit stone formation, while calcium and phosphorus promote stone formation (10). High concentration of Ca2+, oxalic acid and calcium oxalate crystals can induce oxidative stress response to damage renal tubular epithelial cells, resulting in the occurrence of kidney stones (11). High phosphorus caused by impaired blood phosphorus excretion may combine with oxalic acid in the body to form calcium oxalate complex (12). In this study, univariate analysis found that serum potassium, sodium, calcium, and magnesium were associated with calcium oxalate kidney stone development except for age and BMI. There may be coordination between serum sodium, potassium and calcium, which may participate in stone formation through coordination in renal tubules. This point needs to be proved by in vitro and in vivo experiments.
Oxidative stress injury is closely related to calcium oxalate kidney stone formation. Klotho is an anti-aging gene with antioxidant, anti-inflammatory and other effects. Klotho protein has two subtypes, membrane type and secreted Klotho protein, in which membrane type Klotho protein has a long extracellular domain and is cut into sKL protein by β-amyloid precursor protein lyase 1 and released into the blood. Currently, sKL, as a free product of Klotho, is believed to decrease serum levels in chronic and acute cardio-renal vascular diseases and serve as a marker of disease deterioration (13, 14).
Nrf2, as a regulator of oxidative stress, plays a key role in the balance between oxidation and antioxidant. After Nrf2 enters the nucleus, it activates the antioxidant reaction element ARE, and then promotes the expression of antioxidant proteins HO-1 and NQO1 (15, 16). Therefore, the objective of this study was to collect clinical data and blood samples. Serum levels of sKL, Nrf2, HO-1 and NQO-1 were decreased and serum levels of GSK3β were increased in patients with calcium oxalate kidney stones by comparing serum oxidation and antioxidant markers with healthy subjects. As we all know, sKL is a transmembrane protein, which is an important protein regulating blood phosphorus metabolism, and studies have found that it plays an important role in the calcification of blood vessels and heart valves (17). The study of Oh et al. (7) showed that the decrease of circulating blood sKL level in patients with end-stage renal disease was closely related to the enhancement of oxidative stress. The decrease of serum sKL level is related to the occurrence and development of cardiovascular diseases. The decrease of sKL level, BMI, smoking and drinking are risk factors for cardiovascular diseases. As the focus of this study, sKL was included in logistic regression analysis. It was found that the increase of serum sKL, HO-1 and NQO-1 levels was a protective effect of calcium oxalate kidney stones, which may be related to the antioxidant effect of sKL. NQO-1 and HO-1 are antioxidant enzymes that protect cells from oxidative stress (18–20). Animal experiments in the early stage of this study have confirmed (21) that after the mouse kidney stone model was established using glyoxylate, ELISA, Western Blot and Real-Time PCR results showed that the protein and mRNA expression levels of the key anti-oxidative stress proteins HO-1, Nrf2 and NQO-1 were significantly down-regulated. It suggested that oxidative damage occurred in mice. Overexpression of Klotho gene can reverse apoptosis and oxidative damage caused by glyoxylate, while knockout of Klotho gene can aggravate apoptosis and oxidative damage. It is suggested that Klotho plays an important role in the oxidative stress injury induced by calcium oxalate, which provides a new way to further study the formation mechanism of calcium oxalate kidney stones. Therefore, this study focused on sKL, the free product of Klotho, and found that serum sKL was positively correlated with NQO-1 and HO-1 by spearman correlation analysis of sKL and Nrf2 downstream related proteins. GSK3β, as a negative regulator of the Nrf2 pathway, enables nuclear rejection and degradation to promote oxidative stress damage (22). Some studies have suggested that GSK3β is a serum specific marker of cognitive impairment combined with diabetes, indicating that GSK3β aggravates oxidative damage and leads to deterioration of cognitive impairment (23). Kumari et al. (24) found that serum GSK3β and P53 are novel blood markers for predicting Alzheimer's disease, and the content of GSK3β in serum of patients was significantly increased. Mao et al. (25) found that Klotho activated autophagy by inhibiting GSK3β activity against coronary atherosclerotic heart disease caused by endothelial aging. Interestingly, the correlation analysis results of this study showed that serum sKL level was negatively correlated with serum GSK3β level. GSK3β acts as an oxidase to cause the increase of oxidation reactive substances, while sKL has antioxidant effects and can counteract oxidative damage reactions. The relationship between sKL and GSK3β has not yet been studied. In this study, the correlation between sKL and GSK3β was preliminarily detected in human serum. This will provide a theoretical basis for the team's future in vitro and in vivo research. Li et al. (26) found that serum Nrf2 level was related to lupus nephritis disease activity and renal function impairment. In addition, Eman et al. (27) found that serum Nrf2 level is an effective prognostic predictor of bronchial asthma in children, and is related to the severity of the disease. HO-1 is an intracellular enzyme that catalyzes the oxidation of heme to produce CO, biliverdin and iron. In one study (28), serum HO-1 levels were found to be inversely associated with coronary artery disease. In addition, Kata et al. (29) found that serum HO-1 was a specific marker for acute exacerbation of interstitial pneumonia. Although sKL, HO-1 and GSK3β have been shown to participate in oxidative stress and become disease-specific markers in other diseases, whether they play a role in calcium oxalate kidney stone formation has not been studied. However, the mechanism by which these markers of oxidative stress are involved in calcium oxalate kidney stone formation remains unclear and requires further study. In addition, sKL may participate in the formation of calcium oxalate kidney stones by activating oxidative stress markers such as HO-1 and GSK3β, which needs further study.
At present, the diagnosis of calcium oxalate kidney stones relies on CT and infrared spectrum detection (30), in addition to urine routine and 24-hour urine examination (31). Calcium oxalate kidney stones are commonly seen in patients with serologic abnormalities such as hyperphosphatemia, hypercalcemia, hyperoxaluria, and hyperparathyroidism (32). Urinary calcium, urinary oxalate, and urinary citrate levels are the initial criteria for the diagnosis of calcium oxalate stones (33), but there is no serological assessment of the diagnosis of the disease. Through the analysis of multi-index combined diagnostic experiments, we found that the AUC value of GSK3β + HO-1 + NQO-1 group was the largest (Fig. 2), and the diagnostic effect was the best. This result can provide an objective comprehensive analysis for determining the best joint indexes, and help to improve the diagnosis of calcium oxalate kidney stones. Therefore, sKL, GSK3β and HO-1 may be new targets for the prevention and treatment of calcium oxalate kidney stones.