Carbonic anhydrase IX as a diagnostic urinary marker for urothelial bladder cancer.

UNLABELLED: Urinary biomarkers are needed to improve the management and reduce the cost of urothelial bladder cancer (UBC); however, none have been recommended yet for clinical practice. This study evaluated carbonic anhydrase IX (CAIX) as a diagnostic urinary biomarker for UBC. CAIX was analyzed by quantitative polymerase chain reaction in urine samples of 196 patients with UBC and 123 controls with hematuria. Paired samples from urine and tumor tissue were evaluated in 16 cases. Data were validated in 155 independent samples. The sensitivity and specificity of CAIX for UBC detection were 86.2% and 95.1%, respectively (area under the curve [AUC]: 90.5%). There was a significant association of CAIX expression between the paired urine and tumor specimens (p=0.002). CAIX showed a significantly higher predictive accuracy than urinary cytology (90.5% vs 71.7%), specifically in low-grade tumors (90.0% vs 61.8%). CAIX expression decreased with increasing tumor stage and grade. Analyses in an independent validation cohort confirmed the high accuracy of CAIX for diagnosing UBC (AUC: 88.3%). PATIENT SUMMARY: We evaluated carbonic anhydrase IX (CAIX) as a urinary marker for bladder cancer (BCa) using a large series of patients from a single hospital. We found that urinary CAIX has a high sensitivity and specificity for diagnosing BCa.

Urinary carbonic anhydrase VI as a biomarker for kidney disease in pigs.

This study investigated whether carbonic anhydrase (CA)-VI has utility as a biomarker in swine kidney disease. Serum chemistry, histopathology, immunohistochemical staining and enzyme-linked immunosorbent assay (ELISA) analyses were performed. In the kidney of normal healthy pigs, CA-VI was localized in the epithelial cells of the renal distal straight tubules. CA-VI levels were 16 ± 35 ng/g wet tissue and 50 ± 66 ng/mL in normal pig kidney and urine, respectively, and 136 ± 173 ng/mL in the urine of pigs with kidney disease. CA-VI urinary concentration was not correlated with urinary urea nitrogen (UUN), urinary creatinine (Cre), or urinary albumin levels in pigs with kidney disease. However, UUN and Cre levels were positively correlated in the urine of pigs with kidney disease. These data suggest that urinary CA-VI may represent a biomarker for kidney disease in pigs, particularly for disorders affecting distal straight tubules.

Identification of novel translational urinary biomarkers for acetaminophen-induced acute liver injury using proteomic profiling in mice.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure. Currently, no adequate predictive biomarkers for DILI are available. This study describes a translational approach using proteomic profiling for the identification of urinary proteins related to acute liver injury induced by acetaminophen (APAP). Mice were given a single intraperitoneal dose of APAP (0-350 mg/kg bw) followed by 24 h urine collection. Doses of ≥275 mg/kg bw APAP resulted in hepatic centrilobular necrosis and significantly elevated plasma alanine aminotransferase (ALT) values (p<0.0001). Proteomic profiling resulted in the identification of 12 differentially excreted proteins in urine of mice with acute liver injury (p<0.001), including superoxide dismutase 1 (SOD1), carbonic anhydrase 3 (CA3) and calmodulin (CaM), as novel biomarkers for APAP-induced liver injury. Urinary levels of SOD1 and CA3 increased with rising plasma ALT levels, but urinary CaM was already present in mice treated with high dose of APAP without elevated plasma ALT levels. Importantly, we showed in human urine after APAP intoxication the presence of SOD1 and CA3, whereas both proteins were absent in control urine samples. Urinary concentrations of CaM were significantly increased and correlated well with plasma APAP concentrations (r = 0.97; p<0.0001) in human APAP intoxicants, who did not present with elevated plasma ALT levels. In conclusion, using this urinary proteomics approach we demonstrate CA3, SOD1 and, most importantly, CaM as potential human biomarkers for APAP-induced liver injury.

Vascular endothelial growth factor, carbonic anhydrase 9, and angiogenin as urinary biomarkers for bladder cancer detection.

OBJECTIVE: To investigate whether elevated urinary levels of vascular endothelial growth factor (VEGF), carbonic anhydrase 9 (CA9), and angiogenin are associated with bladder cancer (BCa). METHODS: This was a case-control study in which voided urine samples from 127 patients (63 control subjects and 64 patients with BCa) were analyzed. The urinary concentrations of VEGF, CA9, angiogenin, and bladder tumor antigen (BTA) were assessed using enzyme-linked immunosorbent assays. We used the area under the curve of receiver operating characteristic curves to determine the ability of VEGF, CA9, and angiogenin to detect BCa in voided urine samples. Data were also compared with the findings from a commercial enzyme-linked immunosorbent assay-based BCa detection assay (BTA-Trak). The sensitivity, specificity, and positive and negative predictive values were calculated. RESULTS: The urinary concentrations of VEGF, CA9, angiogenin, and BTA were significantly elevated in those with BCa. VEGF was the most accurate urinary biomarker (area under the curve 0.886, 95% confidence interval 0.8301-0.9418). Furthermore, multivariate regression analysis highlighted VEGF (odds ratio 5.90, 95% confidence interval 2.60-13.40, P < .0001) as an independent variable. The sensitivity and specificity for VEGF (83% sensitivity and 87% specificity) outperformed those for BTA (80% sensitivity and 84% specificity). CONCLUSION: VEGF could be a valuable addition to voided urine sample analysis for the detection of BCa. Larger, prospective studies are needed to determine the clinical utility of urinary VEGF and angiogenin as biomarkers in the noninvasive evaluation of patients with BCa.

Splicing variants of carbonic anhydrase IX in bladder cancer and urine sediments.

OBJECTIVE: In human cancers, carbonic anhydrase IX (CAIX) influences cell proliferation and tumor progression, maintaining intracellular and extracellular pH under hypoxic conditions. An alternative CAIX isoform, lacking of exons 8-9 (AS) and independent from the levels of hypoxia, was recently demonstrated in cancer cells. AS-CAIX competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study, we evaluated mRNA expression of the 2 CAIX isoforms and their clinical relevance in bladder cancers and urine sediments. MATERIALS AND METHODS: We measured mRNA expression of FL- and AS-CAIX isoforms in tumor tissues and benign mucosa from 45 patients with bladder transitional cell carcinoma. The expression of the 2 isoforms was also measured in urine sediment of 81 bladder cancer patients and 93 control subjects. RESULTS: Expression of FL-CAIX mRNA was lower than AS-CAIX in benign mucosa (P = 0.006) whereas in paired bladder cancers FL-CAIX mRNA was higher (P = 0.007). Consequently, the percentage of FL-CAIX in bladder cancers [median: 62.6%] was significantly higher than in benign mucosa [15.0%] (P < 0.0001). In the urinary sediments of bladder cancer patients FL-CAIX mRNA was significantly higher in comparison with normal controls (P = 0.003). FL-CAIX percentage appeared dramatically higher in urine sediments of bladder cancer patients [64.5%] in comparison with controls [7.5%] (P < 0.0001). In addition, FL-CAIX% was significantly different in sediments from pTa-pT1 and ≥ pT2 patients [51.5% and 91.7%, respectively] (P = 0.016). Stratification according tumor grade indicated that FL-CAIX% was significantly lower in G1 bladder cancers [33.3%] in comparison with G2-G3 [88.6%] (P = 0.005) The clinical sensitivity for FL-CAIX% in urine sediments was 0.93, with a 0.76 specificity. Using the same cut-off positive predictive value (PPV) was 0.78, whereas negative predictive value (NPV) was 0.93. CONCLUSIONS: Our results seem to indicate that in bladder cancers and related urine sediments, FL-CAIX is the prevalent and is the most accurate clinically relevant variant surrogate of hypoxic stress.

Carbonic anhydorase isoenzyme I (CA-I) concentration in feces and urine as a temporary marker of occult blood in beagle dogs.

This study was undertaken to investigate whether the concentration of carbonic anhydorase isoenzyme I (CA-I) in canine feces and urine is useful as a temporary marker of occult blood. Concentrations of CA-I were measured by enzyme-linked immunosorbent assay (ELISA). Fecal CA-I concentrations in 113 healthy beagle dogs (50 male and 63 female) of various ages ranged from 4.3 to 16.7 ng/g feces (mean; 7.0 +/- 2.9 ng/g feces). One milliliter of blood from 3 healthy beagle dogs was found to contain 1,047, 1,062 and 1,150 microg CA-I. The fecal CA-I concentrations of dogs receiving intragastric infusions of autologous blood (10 ml) were very low. However, the fecal CA-I concentrations of dogs receiving infusion of autologous blood (5 ml) into the ascending colon were very high. Detection of fecal CA-I would be useful for identifying dogs with hemorrhaging of the large intestine. Of 55 urinary samples collected from healthy beagle dogs by catheter, chemical tests for occult blood were negative in 44, but CA-I concentrations ranged from 1.8 to 12.6 ng/ml (mean; 6.9 +/- 5.4 ng/ml) by ELISA. The CA-I concentrations of the other 11 samples, which tested positive for occult blood on chemical testing, ranged from 41.2 to 525.0 ng/ml by ELISA. Although CA-I is not a specific marker of erythrocytes, CA-I may be used to detect occult blood in canine feces and urine until a specific immunological test kit using antibody for Hb is developed.

Soluble form of carbonic anhydrase IX (CA IX) in the serum and urine of renal carcinoma patients.

Tumour-associated protein carbonic anhydrase IX (CA IX) has two major forms. One is a cell-associated, transmembrane protein seen on Western blots as a twin band of 54/58 kDa, expressed in gastric mucosa and in several types of cancer. The other is a soluble protein s-CA IX of 50/54 kDa, which is released into the culture medium or into the body fluids, most likely by proteolytic cleavage of the extracellular part from transmembrane and intracellular sequences. While TC media of CA IX-positive tumour cell lines or short-term cultures of tumour explants contain a relatively high concentration of s-CA IX (20-50 ng ml(-1)), the level of this antigen in blood serum and urine of renal clear cell carcinoma patients is about 1000 x lower. The concentration of CA IX in the blood and in urine varies within wide limits and there is no obvious correlation with tumour size. After nephrectomy, s-CA IX is cleared from the blood within a few days. Only an extremely low concentration of CA IX was detectable in the sera and in urine of control individuals.

The potential role of fecal carbonic anhydrase II in screening for colorectal cancer.

Several studies have demonstrated a relationship between mucosal carbonic anhydrase (CA) isoenzymes, particularly CA II, and cancer of the large intestine. Recent work has suggested the potential usefulness of fecal CA assay for colorectal cancer screening. This clinical study examined the accuracy of fecal CA II as a marker of adenocarcinoma of the colon and rectum. An enzyme-linked immunosorbent assay was used to measure CA II in urine, serum, and stool samples from 31 colorectal cancer patients and 26 control subjects. An immunochemical fecal occult blood test was also performed in all study participants. Urine and serum CA II were similar in the two study groups. However, both the prevalence and the mean level of fecal CA II in the cancer patients were significantly higher than those in the control group. The detection rate for CA II in the stool was 65 per cent for the cancer patients versus 4 per cent for the control population. The fecal CA II test was similar in sensitivity and specificity to the immunochemical fecal occult blood test (65 vs 48%; 96 vs 100%). Measurement of fecal CA II might be useful in screening for colorectal cancer.

Evaluation of patterns of urinary proteins by SDS-PAGE in rats of different ages.

The patterns of urinary proteins in rats of different ages were examined on SDS gradient polyacrylamide gel electrophoresis coupled with silver staining. Proteins were fractionated into at least 26 bands. Densitometric measurements were used to characterize protein excretion patterns. The results showed that proteinuria in newborn, young and adult rats is predominantly tubular, consisting of low molecular-weight species. Conversely, late adults and old rats had a mixed glomerular pattern, with a steadily increasing excretion of albumin, IgG and transferrin, as was the case of other high molecular-weight proteins. Fragments of both immunoglobulins and albumin were found in all urine samples assayed. In 1 month old rats the percentage of Tamm-Hörsfall (T-H) protein was higher (P < 0.01) than in the remaining groups studied. In newborns, relatively high albumin, IgG and transferrin percentages were detected, as well as an alpha 1-acid glycoprotein and carbonic anhydrase excretion (P < 0.05 and P < 0.01 respectively) higher than that observed in the other age groups studied.

The excretion of carbonic anhydrase isozymes CA I and CA II in the urine of apparently healthy subjects and in patients with kidney disease.

Carbonic anhydrase isozymes CA I and CA II were assayed by a radio-immunosorbent technique in the plasma and urine of apparently healthy subjects and of patients with renal disease. The concentrations (mean +/- SD, n = 8) of CA I and CA II in the plasma of healthy subjects were 2.3 +/- 2.3 and 0.8 +/- 0.5 mg/l, respectively. The urinary excretion values were 3.8 +/- 2.0 and 3.5 +/- 1.9 micrograms/24 h, and the apparent renal clearances were 21 +/- 17 and 52 +/- 44 microliters/min, respectively, values that are similar to those of other low molecular weight proteins. CA I and CA II have mol. wt of 28,850 and 29,300, respectively, they are globular in shape and have a Stoke-Einstein radius of 25 A. They could, therefore, be expected to be filtered at the glomeruli and thereafter reabsorbed by the proximal tubules. CA II is also present in the cytoplasm of renal proximal and distal tubular cells. A study of the pattern of urinary excretion of CA I and CA II could permit detection of damage to renal tubular cells in two ways--either from defective reabsorption of filtered CA I and CA II by the proximal tubular cells, or from leakage of CA II from the proximal or distal tubules into the urine. Some patients with hypercalcuria and renal tubular acidosis showed increased excretion of these enzyme proteins and of beta 2-microglobulin (BMG) into the urine, but the prevalence was rather low (27%). Further studies of patients with more severely damaged kidneys are required.

Carbonic anhydrase III in Duchenne muscular dystrophy.

Plasma carbonic anhydrase III (CAIII) levels determined by radioimmunoassay have been compared, in detail, with creatine kinase (CK) as indices of Duchenne muscular dystrophy (DMD). CAIII levels were markedly elevated in all patients but variability of levels in a number of individual patients was higher than CK.