Cell cycle arrest and cell death correlate with the extent of ischaemia and reperfusion injury in patients following kidney transplantation - results of an observational pilot study.

A prolonged cold ischaemia time (CIT) is suspected to be associated with an increased ischaemia and reperfusion injury (IRI) resulting in an increased damage to the graft. In total, 91 patients were evaluated for a delayed graft function within 7 days after kidney transplantation (48 deceased, 43 living donors). Blood and urine samples were collected before, immediately after the operation, and 1, 3, 5, 7 and 10 days later. Plasma and/or urine levels of total keratin 18 (total K18), caspase-cleaved keratin 18 (cc K18), the soluble receptor for advanced glycation end products (sRAGE), tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-binding protein-7 (IGFBP7) were measured. As a result of prolonged CIT and increased IRI, deceased donor transplantations were shown to suffer from a more distinct cell cycle arrest and necrotic cell death. Plasmatic total K18 and urinary TIMP-2 and IGFBP7 were therefore demonstrated to be of value for the detection of a delayed graft function (DGF), as they improved the diagnostic performance of a routinely used clinical scoring system. Plasmatic total K18 and urinary TIMP-2 and IGFBP7 measurements are potentially suitable for early identification of patients at high risk for a DGF following kidney transplantation from deceased or living donors.

UBC® Rapid Test for detection of carcinoma in situ for bladder cancer.

UBC® Rapid Test is a test that detects fragments of cytokeratins 8 and 18 in urine. We present results of a multicentre study measuring UBC® Rapid Test in bladder cancer patients and healthy controls with focus on carcinoma in situ (CIS) and high-grade bladder cancer. From our study with N = 452 patients, we made a stratified sub-analysis for carcinoma in situ of the urinary bladder. Clinical urine samples were used from 87 patients with tumours of the urinary bladder (23 carcinoma in situ, 23 non-muscle-invasive low-grade tumours, 21 non-muscle-invasive high-grade tumours and 20 muscle-invasive high-grade tumours) and from 22 healthy controls. The cut-off value was defined at 10.0 µg/L. Urine samples were analysed by the UBC® Rapid Test point-of-care system (concile Omega 100 POC reader). Pathological levels of UBC Rapid Test in urine are higher in patients with bladder cancer in comparison to the control group (p < 0.001). Sensitivity was calculated at 86.9% for carcinoma in situ, 30.4% for non-muscle-invasive low-grade bladder cancer, 71.4% for nonmuscle-invasive high grade bladder cancer and 60% for muscle-invasive high-grade bladder cancer, and specificity was 90.9%. The area under the curve of the quantitative UBC® Rapid Test using the optimal threshold obtained by receiveroperated curve analysis was 0.75. Pathological values of UBC® Rapid Test in urine are higher in patients with high-grade bladder cancer in comparison to low-grade tumours and the healthy control group. UBC® Rapid Test has potential to be more sensitive and specific urinary protein biomarker for accurate detection of high-grade patients and could be added especially in the diagnostics for carcinoma in situ and non-muscle-invasive high-grade tumours of urinary bladder cancer.

Urinary Intestinal Fatty Acid-Binding Protein Can Distinguish Necrotizing Enterocolitis from Sepsis in Early Stage of the Disease.

Necrotizing enterocolitis (NEC) is severe disease of gastrointestinal tract, yet its early symptoms are nonspecific, easily interchangeable with sepsis. Therefore, reliable biomarkers for early diagnostics are needed in clinical practice. Here, we analyzed if markers of gut mucosa damage, caspase cleaved cytokeratin 18 (ccCK18) and intestinal fatty acid-binding protein (I-FABP), could be used for differential diagnostics of NEC at early stage of disease. We collected paired serum (at enrollment and week later) and urine (collected for two days in 6 h intervals) samples from 42 patients with suspected NEC. These patients were later divided into NEC (n = 24), including 13 after gastrointestinal surgery, and sepsis (n = 18) groups using standard criteria. Healthy infants (n = 12), without any previous gut surgery, served as controls. Both biomarkers were measured by a commercial ELISA assay. There were no statistically significant differences in serum ccCK18 between NEC and sepsis but NEC patients had significantly higher levels of serum and urinary I-FABP than either sepsis patients or healthy infants. Urinary I-FABP has high sensitivity (81%) and specificity (100%) and can even distinguish NEC from sepsis in patients after surgery. Urinary I-FABP can be used to distinguish NEC from neonatal sepsis, including postoperative one, better than abdominal X-ray.

Keratins are novel markers of renal epithelial cell injury.

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress.

Urinary Biomarkers Indicative of Apoptosis and Acute Kidney Injury in the Critically Ill.

BACKGROUND: Apoptosis is a key mechanism involved in ischemic acute kidney injury (AKI), but its role in septic AKI is controversial. Biomarkers indicative of apoptosis could potentially detect developing AKI prior to its clinical diagnosis. METHODS: As a part of the multicenter, observational FINNAKI study, we performed a pilot study among critically ill patients who developed AKI (n = 30) matched to critically ill patients without AKI (n = 30). We explored the urine and plasma levels of cytokeratin-18 neoepitope M30 (CK-18 M30), cell-free DNA, and heat shock protein 70 (HSP70) at intensive care unit (ICU) admission and 24h thereafter, before the clinical diagnosis of AKI defined by the Kidney Disease: Improving Global Outcomes -creatinine and urine output criteria. Furthermore, we performed a validation study in 197 consecutive patients in the FINNAKI cohort and analyzed the urine sample at ICU admission for CK-18 M30 levels. RESULTS: In the pilot study, the urine or plasma levels of measured biomarkers at ICU admission, at 24h, or their maximum value did not differ significantly between AKI and non-AKI patients. Among 20 AKI patients without severe sepsis, the urine CK-18 M30 levels were significantly higher at 24h (median 116.0, IQR [32.3-233.0] U/L) than among those 20 patients who did not develop AKI (46.0 [0.0-54.0] U/L), P = 0.020. Neither urine cell-free DNA nor HSP70 levels significantly differed between AKI and non-AKI patients regardless of the presence of severe sepsis. In the validation study, urine CK-18 M30 level at ICU admission was not significantly higher among patients developing AKI compared to non-AKI patients regardless of the presence of severe sepsis or CKD. CONCLUSIONS: Our findings do not support that apoptosis detected with CK-18 M30 level would be useful in assessing the development of AKI in the critically ill. Urine HSP or cell-free DNA levels did not differ between AKI and non-AKI patients.

Preliminary Results of a Multicentre Study of the UBC Rapid Test for Detection of Urinary Bladder Cancer.

BACKGROUND/AIM: UBC Rapid is a test detecting fragments of cytokeratins 8 and 18 in urine. These are cytokeratins frequently overexpressed in tumor cells. We present the first results of a multi-centre study using UBC Rapid in patients with bladder cancer and healthy controls. MATERIALS AND METHODS: Clinical urine samples from 92 patients with tumors of the urinary bladder (45 low-grade and 47 high-grade tumors) and from 33 healthy controls were used. Urine samples were analyzed by the UBC Rapid point-of-care (POC) system and evaluated both visually and quantitatively using a concile Omega 100 POC reader. For visual evaluation, different thresholds of band intensity for considering a test as positive were applied. Sensitivities and specificities were calculated by contingency analyses. RESULTS: We found that pathological concentrations by UBC Rapid are detectable in urine of patients with bladder cancer. The calculated diagnostic sensitivity of UBC Rapid in urine was 68.1% for high-grade, but only 46.2% for low-grade tumors. The specificity was 90.9%. The area under the curve (AUC) after receiver-operated curve (ROC) analysis was 0.733. Pathological levels of UBC Rapid in urine are higher in patients with bladder cancer in comparison to the control group (p<0.0001). CONCLUSION: UBC rapid can differentiate between patients with bladder cancer and controls. Further studies with a greater number of patients will show how valuable these results are.

Significance of suppressor of cytokine signaling-3 expression in bladder urothelial carcinoma in relation to proinflammatory cytokines and tumor histopathological grading.

BACKGROUND: Bladder cancer is among the five most common malignancies worldwide. Altered expression of suppressor of cytokine signaling -3 (SOCS-3) has been implicated in various types of human cancers; however, its role in bladder cancer is not well established. AIM: The present study was undertaken to investigate the mRNA expression of SOCS-3 in normal and cancerous bladder tissue and to explore its correlation with urinary levels of some proinflammatory cytokines, cytokeratin-18 (CK -18) and with tumor histopathological grading, in order to evaluate their role as potential diagnostic markers. MATERIALS AND METHODS: SOCS3 mRNA expression levels were evaluated using quantitative real time PCR. Urinary levels of interleukins 6 and 8 were estimated by enzyme linked immunosorbent assay (ELISA). Cytokeratin-18 expression was analyzed by immuunohistochemistry then validated by ELISA. RESULTS: SOC3 m RNA expression levels were significantly lower in high grade urothelial carcinoma (0.36±0.12) compared to low grade carcinoma (1.22±0.38) and controls (4.08±0.88), (p<0.001). However, in high grade urothelial carcinoma the urinary levels of IL-6, IL-8, total CK-18(221.33±22.84 pg/ml, 325.2±53.6 pg/ ml, 466.7±57.40 U/L respectively) were significantly higher than their levels in low grade carcinoma (58.6±18.6 pg/ ml, 58.3±50.2 pg/ml, 185.5±60.3 U/L respectively) and controls (50.9±23.0 pg/ml, 7.12±2.74 pg/ml, 106.7±47.3U/L respectively), (p<0.001). CONCLUSIONS: Advanced grade of urothelial bladder carcinoma is significantly associated with lowered mRNA expression of SOC3 as well as elevated urinary levels of proinflammatory cytokines and CK-18. Furthermore, our results suggested that urinary IL-8, IL-6 and CK-18 may benefit as noninvasive biomarkers for early detection as well as histopathological subtyping of urothelial carcinoma.

Use of monitoring levels of soluble forms of cytokeratin 18 in the urine of patients with superficial bladder cancer following intravesical Ad-IFNα/Syn3 treatment in a phase l study.

A phase l study using intravesical Ad-IFNαSyn3 for patients with bacillus Calmette-Guérin-resistant superficial bladder cancer showed a complete remission (CR) of 43% at 90 days after treatment with high levels of interferon-α (IFNα) being produced. Ad-IFNα kills bladder cancer cells by two apoptotic and one necrotic mechanism that can be measured by soluble forms of cytokeratin 18 (CK18) using M30 and M65 ELISAs, assays for caspase-cleaved (apoptotic) and uncleaved (necrotic) cell death, respectively. Therefore, we determined whether M30 and M65 levels in the urine after treatment could document all three mechanisms of cancer cell kill and also predict having a CR. High levels of both M30 and M65 were found in all patients within 24 h after treatment with all three types of cancer cell death occurring. Moreover, the return of both M30 and M65 levels in the urine to normal levels within 5 days or more after treatment was strongly associated with obtaining a CR (P=0.003). This is the first time that such assays have been used to study response to therapy in the urine of patients with bladder cancer and in the future may prove valuable in predicting clinical outcome.

Evaluation of a new quantitative point-of-care test platform for urine-based detection of bladder cancer.

OBJECTIVE: Several commercial point-of-care (POC) tests are available for urine-based detection of bladder cancer (BC). However, these tests are restricted to dichotomized results (positive or negative), which limits their diagnostic value. Quantitative protein-based tests offer improved risk stratification but require complex methods restricted to specialized centers. Recently, the first quantitative POC system based on the detection of cytokeratin fragments became available. The aim of the study was to evaluate the diagnostic accuracy of this quantitative POC test. PATIENTS AND METHODS: A total of 198 patients having symptoms suspicious for BC were included. All patients received urethrocystoscopy and upper-tract imaging. Urine samples were analyzed by the urine BC antigen (UBC) rapid POC system and evaluated both visually and quantitatively using the concile Omega 100 POC reader. For visual evaluation, different thresholds of band intensity for considering a test positive were applied. Moreover, the UBC enzyme-linked immunosorbent assay (ELISA), urine cytology, and the nuclear matrix protein 22 BladderChek were performed. Sensitivities and specifities were calculated by contingency analyses. Optimal cutoffs of quantitative tests were determined by receiver operating characteristic curves. RESULTS: A total of 61 patients (30.8%) were diagnosed with BC. Visual evaluation of the UBC revealed sensitivities of 38.1% to 71.4% with corresponding specificities of 54.1% to 89.1%, dependent on the threshold of band intensity applied. The quantitative UBC rapid showed a sensitivity of 60.7% and a specificity of 70.1% at optimal cutoff (area under the curve = 0.68). A constant increase of both the probability of BC and high-risk BC with increasing UBC rapid values was observed. UBC concentrations determined by the reader significantly correlated with the UBC ELISA (P<0.001). The UBC ELISA, the nuclear matrix protein22 BladderChek and cytology showed sensitivities of 48.3%, 16.4%, and 51.7% with specificities of 71.3%, 95.3%, and 78.1%, respectively. CONCLUSION: The UBC rapid in combination with a quantitative POC-reader system for the first time enables quantitative determination of a BC marker under POC conditions. Diagnostic accuracy is at least equivalent to elaborate ELISA-based measurement. The quantitative use of the UBC rapid test facilitates risk prediction compared with conventional nonquantitative dichotomized POC testing.

Keratin 18 and heat-shock protein in chronic kidney disease.

Chronic kidney disease (CKD) is an affliction associated with increased systemic stress and cell death. We will review the role of keratin 18 (K-18) and caspase-cleaved CK-18 (ccK-18) as markers for increased apoptosis and necrosis during renal failure progression. The importance of preventative expression of heat-shock proteins (HSPs) in response to cell stress will also be discussed. The frequent development of CKD leads to serious complications. The potential of use of K-18 and HSP as early biomarkers of renal failure will be reviewed. Also, the role of these proteins with respect to dialysis regimes and in acute ischemic kidney injury following renal transplantation will be discussed.

[Molecular markers for the diagnosis of non-muscle-invasive bladder cancer].

The diagnostic efficiency of oncomarkers, such as cytokeratins 8, 18 (TPA, TPS, UBC) and vascular endothelial growth factor, was evaluated to diagnose non-muscle-invasive bladder cancer. The serum and urinary levels of the markers were studied using enzyme immunoassay; their diagnostic properties were assessed by the ROC-analysis. Comparison of the groups of apparently healthy individuals, patients with chronic cystitis, and those with noninvasive bladder cancer showed that the oncomarkers might potentially serve as screening parameters of the early diagnosis of bladder cancer and, in a number of cases, permit the differential diagnosis with chronic cystitis.

Increased total cytokeratin-18 serum and urine levels in chronic kidney disease.

BACKGROUND: Increased cell death in chronic kidney disease (CKD) by either necrosis or apoptosis has been confirmed by a variety of studies. Possible sources are an inadequate persistent inflammation and ischemia as a consequence of CKD or caused by the underlying renal disease. Detection of total or caspase cleaved cytokeratin 18 (CK-18) is a novel and elegant method to determine necrosis or apoptosis of epithelial cells in the patients' sera and urine. METHODS: 120 patients with CKD stages 1 to 5 were included in the study. Twenty healthy volunteers served as controls. Total and caspase cleaved CK-18 urine and serum concentrations were determined by ELISA. RESULTS: The concentration of serum total CK-18 was significantly higher in CKD stages 3-5 as compared to the healthy controls. Urinary total CK-18 excretion was increased in patients with CKD 5 compared to controls. A significant correlation between urine total CK18 and urine protein and albumin levels was found. Moreover, ROC curve analysis showed the potential of serum and especially urine total CK-18 levels to predict various CKD stages. CONCLUSIONS: We provide evidence for increased total CK-18 serum and urine levels in CKD patients, possibly indicating that epithelial cell necrosis is prevalent in CKD.

Tubular epithelial injury and inflammation after ischemia and reperfusion in human kidney transplantation.

OBJECTIVE: To provide an integrated insight into the kinetics of tubular injury, inflammation, and oxidative stress after human kidney transplantation. BACKGROUND: Tissue injury due to ischemia and reperfusion is an inevitable consequence of kidney transplantation. Tubular epithelial injury, inflammation, and oxidative stress play major roles in the pathophysiology of acute kidney injury in small animals, but it remains to be established whether this paradigm holds true for human kidney transplantation. METHODS: Markers of tubular injury, inflammation, and oxidative stress were compared between recipients of kidneys from donors after cardiac death (DCD; N = 8) with prolonged ischemia and recipients of living donor kidneys with minimal ischemia (N = 8). RESULTS: In the early postoperative period, creatinine clearance and tubular sodium reabsorption were profoundly reduced in DCD kidneys, coinciding with significantly increased urinary concentrations of tubular injury markers (neutrophil gelatinase-associated lipocalin, N-acetyl-ß--glucosaminidase, and cystatin C) and an 18-fold increase in renal production of cytokeratin-18, indicating extensive necrotic cell death. Tubular injury in DCD kidneys was followed by greater systemic inflammatory activity and oxidative stress in the postoperative period (measured with 17-plex cytokine arrays and as plasma F2-isoprostanes, respectively). In contrast, no evidence of oxidative damage to either of the 2 kidney types was found in the early reperfusion period. CONCLUSIONS: These findings establish the relevance of observations in animal models for human kidney transplantation and form the basis for development of novel therapies to improve early graft function and expand the use of donor kidneys with prolonged ischemia.

[Expression and clinical significance of nuclear matrix protein 22 and cytokeratin 18 in transitional cell carcinoma of the bladder].

OBJECTIVE: To evaluate the expression and clinical significance of urinary nuclear matrix protein (NMP22) and cytokeratin 18 (CK18) for transitional cell carcinoma of the bladder. METHODS: Urinary NMP22 and CK18 levels of 293 patients with transitional cell carcinoma of the bladder, 400 patients with non-transitional cell carcinoma of the bladder, and 105 bladder benign disease were analysed by enzyme-linked-immunosorbent assay (ELISA). RESULTS: The levels of urinary NMP22 and CK18 in the patients with transitional cell carcinoma of the bladder (M = 17.3 U/ml, M(CK18) = 484.2 U/L) were significantly higher than those in the non-transitional cell carcinoma of the bladder (M = 6.8 U/ml, M(CK18) = 156.0 U/L) and the benign disease group (M(NMP22) = 2.3 U/ml, M(CK18) = 66.6 U/L) (P < 0.001). The sensitivity and specificity of urinary NMP22 and CK18 were 79.2%, 88.6% and 78.2%, 82.9%, respectively, for transitional cell carcinoma of the bladder before any treatment. The joint sensitivity of the two markers was 91.7%. The NMP22 and CK18 levels were significantly lower in the recovered patients after surgical operation (P < 0.01), while in patients with recurrence or metastasis the levels of the markers were significantly higher (P < 0.01). There was a significant relationship between NMP22 and CK18, (r = 0.689, P < 0.01). The levels of urinary nmp22 and CK18 were significantly different among pathological grade G1, G2, G3, and stage Ta, T1, T2, T3 (P < 0.01). CONCLUSION: NMP22 and CK18 are useful tumor marker for diagnosis of transitional cell carcinoma of the bladder and for monitoring the state of illness. The joint use of the two markers can improve the sensitivity of cancer detection. NMP22 and CK18 may become a new class of tumor markers, and to be the basis for development of a new assay with an increased efficacy for the detection and treatment of bladder cancer.

Telomerase activity, cytokeratin 20 and cytokeratin 19 in urine cells of bladder cancer patients.

AIM OF THE STUDY: This work aims to search for markers suitable for the screening of bladder cancer, which should be specific, sensitive, reproducible, non-invasive and at acceptable cost. PATIENTS AND METHODS: The study included 50 patients diagnosed as bladder cancer (35 TCC, 15 SCC) of different stages and grades, 30 patients with various urothelial diseases, besides 20 apparently healthy subjects of matched age and sex to the malignant group. A random midstream urine sample was collected in a sterile container for the determination of telomerase by RT-PCR, keratin 19 by ELSA CYFRA 21-1 IRMA kit, keratin 20 by RT-PCR and immunohistochemical staining, and urine cytology. RESULTS: For all parameters (telomerase, K19, K20 and cytology) the malignant group was significantly different from both the benign and the control groups. None of the four studied parameters was correlated to the stage of the disease, and when it comes to grade, only K19 showed a significant positive correlation with grade both in TCC and SCC. When ROC curves for all parameters were compared, K19 had the largest area under the curve, and then comes K20. CONCLUSION: K 19 may be used as a biological marker for the diagnosis of bladder cancer. K19 could not be used for differential diagnosis of different types of bladder cancer, meanwhile it could be a marker for differentiation that decreases in less differentiated tumors. As a tumor marker, K20 reflects inability to differentiate tumor type or grade in TCC, while in SCC of the bladder it is correlated with the grade. As a method, RT-PCR is superior to immunostaining for the detection of bladder cancer, meanwhile K20 immunohistochemistry (IHC) results were much better than urine cytology as a bladder cancer screening test. Haematuria and inflammation reduced the specificity of telomerase assay, which reduced its validity as a tumor marker of bladder cancer. K19 and K20 are the best candidates as screening tests for the diagnosis of bladder cancer, representing the highest sensitivity and specificity, beside the radiological and histopathology. Meanwhile, telomerase, although it was a sensitive enough marker, it reflected a high false positive rate.