Urine CA-2 as a biomarker for diagnosis urinary stone and prediction of its complications

PURPOSE Carbonic anhydrase-2 (CA-2) plays a role in mineralization and calcication in organism. Strong evidence suggests that CA-2 is associated with urolithiasis. However, the relationship between CA-2 and urinary stone remains unclear. The study aimed to assess the association of urine CA-2 (uCA-2) level and the potential risk of urinary stone. METHODS From to a prospective cohort study was conducted on patients with urinary stones and healthy subjects to determine the pretreatment uCA-2 level detection by Enzyme linked immunosorbent assay (ELISA). The difference of uCA-2 level between patients with urinary stones and healthy subjects was compared. Then comparison between stone patients with complications and without complications was carried out as well as correlation analysis to detect factors associated with biomarker expression. of calculi etc.) CT scan gender-matched, any known ectopic calcication. Detailed of gender, and study


Introduction
A number of potential metabolic 1-3 , genetic 4-6 , drug-induced 7,8 , and epidemiological 9,10 factors for urolithiasis have been reported. The knowledge of these factors contributes in determining the most appropriate therapy for each patient. Few studies have identi ed that urinary stone are related to carbonic anhydrase-2 (CA-2) 5,8 , thereby suggesting that the CA-2 is associated with urinary stone.
CA-2, as one of the multiple forms of human α-CA, exists as almost ubiquitous in the human body and have a relatively high expression level in the gastrointestinal tract, biliary tract, and kidneys 11 . The main function of CA-2 in human is to interconvert CO 2 and HCO 3 − to maintain the acid/base balance in blood and other tissues and help transport CO 2 out of the tissues. Several studies have demonstrated that CA-2 and its isoenzymes are involved in the calci cation in human tissues, including the blood vessel 12,13 and the brain 14 , though their mineralization mechanisms have not been fully understood. Similarly, CA isoenzymes are also involved in the calci cation process of many biological systems, including bacteriainduced calci cation, calcareous bone formation, and shell formation in shell-forming animals.
Furthermore, the occurrence of renal stone is frequently associated with ectopic calci cation, including vascular calci cation and gallstone [15][16][17] . Generally, ectopic calci cation follows the same pattern as physiological bone mineralization, with an increase in carbonated hydroxyapatite on brous collagen. CA-2 and its isoenzymes catalyze the reversible hydration of CO 2 and participate in the calci cation process in various biological systems. CA-2 and its isoenzymes also promote CaCO 3 formation, which in turn serves as the basis for Ca 3 (PO 4 ) 2 deposition (hydroxyapatite).
Although some relevant evidence supported the association of CA-2 and calci cation or mineralization, information on the role of CA-2 in the modi cation of the risk of urinary stone disease is scarce. In this study, we conducted a prospective clinical study to assess the association of the urine CA-2 (uCA-2) level and the potential risk of urinary stones.

Patients And Methods
A prospective study from March 2017 to November 2019 was conducted on patients with urinary stones were considered eligible to participate in the a liated hospital of Jiujiang University. Patients diagnosed with urinary stones whose age is between 20 and 70 were considered eligible to participate in the a liated hospital of Jiujiang University. All these objects correspond to the following conditions. (1) A non-contrast computerized tomography (CT) scan and color doppler ultrasound inspection proved an unilateral ureteral stone or kidney stone larger than 4 mm; (2) sCr was within normal limits and without disturbance of acid-base balance of water and electrolytes when diagnosed; (3)  Before medical intervention, serum and midstream urine samples were collected from study population and stored at 4°C during the entire sample collection period. Cell debris and particulate matter were removed from serum samples by 3000 r/min at 4°C for 15 minutes and urine samples by 1500 r/min at 4°C for 10 minutes. The midstream urine was utilized to analysis uCA-2 and urine kidney injury molecule-1 (uKIM-1). The uCA-2 and uKIM-1 levels were determined using commercially available enzyme-linked immunosorbent assay (ELISA) commercial kit following the 'manufacturer's protocol (Human CA-2 ELISA Kit (ab222881), Human KIM-1 ELISA Kit (ab235081), Abcam , Cambridge, UK). Measurements of creatinine (Cr), urea nitrogen (BUN) and Uric acid (UA) in plasma were performed at the department of clinical laboratory (A liated Hospital of Jiujiang College).
Continuous variables including age, body mass index index (BMI), serum biochemistry, uCA-2 and uKIM-1 were expressed as the mean±standard error of the mean. Enumeration data including gender was expressed as frequency (%). Statistical analysis was performed using Graphpad Prism 7.00 (Graphpad Software Inc., La Jolla, CA, USA). The intergroup differences were tested using One-way ANOVA and Student age, BMI, sCr, sBUN and sUA; Chi-square test was used to compare gender; Mann-Whitney U test and Kruskal-Wallis H test were used to compare uCA-2 and uKIM-1. Associations between uCA-2 and uKIM-1 and urinary stones complications were studied by Spearman correlations or partial correlations analysis. Rceiver operating curve (ROC) analysis was used to evaluate the ability of uCA-2 and uKIM-1to predict urinary stones complications. Area under the curve (AUC) was derived from receiver operating curve (ROC) analysis. Signi cance was considered at P<0.05.

Results
Among the 160 participants in the present study, including 103 (64.37%) were males and 57 (35.63%) females. 118 patients with urinary stones were classifed into urinary stones group and 42 healthy subjects were classifed into healthy control group. All the 118 urinary stones patients, In terms of detailed calculi location, 29 (24.58%) were ureteral stones and 89 (75.42%) were kidney stones; The average stone size in the urinary stones group was 10.86±4.24 mm (5-32mm). Total mean age of all participants was 41.93±8.33 years (20-68 years), the demographic and clinical characteristics of patients and healthy subjects are reported in Table 1. There were no signi cant differences between patients and healthy controls in age, gender, BMI, sCr, sBUN, and sUA. Table 1 also shows nonparametric univariate analysis of the relationship of pretreatment uCA-2 and uKim-1 between the two groups. Participants of urinary stones group and healthy control group did not signifcantly differ in pretreatment uKim-1 level. Moreover, the mean pretreatment uCA-2 level was signi cantly higher in patients with urinary stones group than healthy controls group (Fig. 1).
The prevalence of stones complications of urinary stones group had 40.67% hematuria, 24.57% pain and 35.59% hydronephrosis. Almost 41.52% patients were detected absent stone complications. Stones complications was associated with a high uCA-2 level excretion, whereas no association was observed with uKim-1 level excretion (Fig. 2) The uCA-2 level was positive correlation with urinary stones complications (R=0.379, P=0.000), especially pain complications (R=0.524, P=0.000) and hematuria complications (R=0.374, P=0.000). Spearman correlations or partial correlation analysis showed that there was no statistical correlation between uKim-1 level and urinary stones complications.
The predictive value of uCA-2 and uKIM-1 levels to predict urinary stones complications was analyzed using a ROC curve (Fig. 3). ROC analysis of uCA-2 yielded an area under the cure (AUC) of 0.786 for total complications, 0.720 for hematuria, 0.852 for pain and 0.683 for hydronephrosis (respectively, each P=0.000); There were no signi cant correlation in ROC analysis of uKIM-1 yielded for any complications. These values between uCA-2 and uKIM-1 predicted urinary stones complications were signi cantly different. Table 2 shows the sensitivity and speci city of uCA-2 level to predict urinary stones complications. The optimal cut-off value of the uCA-2 level was chosen based on the maximum value of the Jordan index, and the indicated that uCA-2 level was a reliable predictor. Furthermore, we determined that a uCA-2 level threshold of 10.94 ng/mL had 83.67% sensitivity and 68.12% speci city for predicting urinary stones complications.

Discussion
Our prospective study included 118 patients with urinary stone and 42 healthy individuals. Substantial evidence indicated that patients with urinary stone exhibited increased uCA-2 level was found in urine by ELISA. The key nding in this study is that an signi cant difference in uCA-2 excretion by ELISA analysis could discriminate patients with urinary stones from healthy controls. If our results are supported by many prospective validation studies, such a test might be clinically bene cial in the surveillance of high-risk patients, such as patients with metabolic syndrome 1-4 , in ammatory bowel disease 18 and accepted bariatric surgery 19 et al. High risk of stone recurrence patients may also reap the bene ts of our results.
The small, hard mineral deposits that characterize urinary stone can cause severe pain, hematuria, obstruction or (and) hydronephrosis. The diagnosis of urinary stone depends on imaging examination and lacks effective biomarker, which means it is di cult to predict the risk recurrence of urinary stone. The AUA guideline has proposed that millions of patients newly diagnosed with urinary stone could be advised to a screening evaluation consisting to reduce the risk of stone recurrence 20 . To achieve this goal, more advanced diagnostic approaches have to be developed and applied to earlier detection of urinary stone. The available clinical data today support the conclusion that leads to a decreased risk of stones recurrence of asymptomatic patients, because of an early dietary or drug intervention 21,22 .
To improve understanding on the signi cance of the excessive production of uCA-2 in patients with urinary stone, we evaluated data from our prospective database on patients with urinary stone and assigned uCA-2 level to patients with or without stone-related complications according to appropriate symptoms and signs. Our data showed that uCA-2 were signi cantly increased excretion in patients with urinary stone develop one or more complications. All 3 complications showed a high level of concordance and pain complication demonstrated superiority over the others with regard to predicting uCA-2 excretion.
To trace the phenomenon of higher uCA-2 level in patients with urinary stone develop one or more complications, our goal in this study was to describe the unclear effect of stone formation. The results of uCA-2 excretion and stone complications interactions had emphasized potential factors to pathophysiological mechanism, such as crystal-membrane interaction, microbial-stone interaction, or other possible interactions. The data show that the non-signi cantly higher uKIM-1 level excretion was found in hydronephrosis complications among the other two groups. Our results are seemingly at variance with a previous study evaluated that uKIM-1 as a biomarkers of acute renal injury was signi cantly higher in stone patients with hydronephrosis compared to without hydronephrosis 23 . The difference results may be explained by the variation of participants inclusion criteria.
The excretion of uCA-2 by the bacteria in patients with urinary stone is also a potentially important factor. Twenty bacterial genera have signi cant differences in relative abundance between urinary stone patients and healthy subjects. These ndings may provide new and non-invasive potential biomarkers for the diagnosis of kidney stone 24 . Escherichia coli is one of most commonly detected pathogenic microorganisms attached to renal stone 25 , which also actively secrete PH-dependent CA-2 26 . Both CA and urease in bacteria play a synergic role in promoting CaCO 3 precipitation 27 . Microbial urease is a Nicontaining enzyme found in various microorganisms that hydrolyzes urea to NH4 + and CO 2 , whereas carbonic anhydrase transforms CO 2 into HCO 3 − . In Bacillus megaterium, calcite precipitation is driven by the coupled activity of both urease and carbonic anhydrase enzymes. The precipitation of CaCO 3 , which predominantly consists of calcite crystals, by microbial carbonic anhydrase has been documented 28 . CA-2 may decrease the stone inhibitory protein through its potential effect on the g-glutamyl carboxylase reaction by providing CO 2 . CA-2 may also supply CO 2 to g-glutamyl carboxylase that catalyzes glutamate residue carboxylation in some proteins, thereby enhancing their biological activity. Several proteins that require this post-translational carboxylation to become biologically active may be involved in the calci cation processes in human tissues, including matrix Gla protein, growth arrest-speci c protein 6, Gla-rich protein, and osteocalcin 29 . The relationship between CA-2 and other stone-promoting orinhibiting factor will be the focus of our study in the future.
Although we have a larger number of samples, the overall sample size is still small. The study did not follow up healthy participants with a relatively increased uCA-2 excretion. Stone formation is a dynamic process that cannot easily be captured by a midstream urine collection. Finally, the relationship among uCA-2, Ca, oxalic acid and P excretion is di cult to evaluate in urine because the 24 h urine biochemical analysis was not measured.

Conclusion
Excessive CA-2 excretion in urine is a major risk factor for urinary stone. Our ndings suggested that urinary CA-2 may be used as an unappreciated biomarker for the diagnosis renal stone in patients and to predict its complications.  Figure 1 uCA-2 and uKIM level detected in patients with urinary stones and healthy subjects by ELISA. *P 0.05 as evaluated using Mann-Whitney U test.