A Pharmacologic "Stress Test" for Assessing Select Antioxidant Defenses in Patients with CKD.

BACKGROUND AND OBJECTIVES: Oxidative stress is a hallmark and mediator of CKD. Diminished antioxidant defenses are thought to be partly responsible. However, there is currently no way to prospectively assess antioxidant defenses in humans. Tin protoporphyrin (SnPP) induces mild, transient oxidant stress in mice, triggering increased expression of select antioxidant proteins (e.g., heme oxygenase 1 [HO-1], NAD[P]H dehydrogenase [quinone] 1 [NQO1], ferritin, p21). Hence, we tested the hypothesis that SnPP can also variably increase these proteins in humans and can thus serve as a pharmacologic "stress test" for gauging gene responsiveness and antioxidant reserves. DESIGN: , setting, participants, & measurementsA total of 18 healthy volunteers and 24 participants with stage 3 CKD (n=12; eGFR 30-59 ml/min per 1.73 m2) or stage 4 CKD (n=12; eGFR 15-29 ml/min per 1.73 m2) were injected once with SnPP (9, 27, or 90 mg). Plasma and/or urinary antioxidant proteins were measured at baseline and for up to 4 days post-SnPP dosing. Kidney safety was gauged by serial measurements of BUN, creatinine, eGFR, albuminuria, and four urinary AKI biomarkers (kidney injury molecule 1, neutrophil gelatinase-associated lipocalin, cystatin C, and N-acetyl glucosaminidase). RESULTS: Plasma HO-1, ferritin, p21, and NQO1 were all elevated at baseline in CKD participants. Plasma HO-1 and urine NQO1 levels each inversely correlated with eGFR (r=-0.85 to -0.95). All four proteins manifested statistically significant dose- and time-dependent elevations after SnPP injection. However, marked intersubject differences were observed. p21 responses to high-dose SnPP and HO-1 responses to low-dose SnPP were significantly suppressed in participants with CKD versus healthy volunteers. SnPP was well tolerated by all participants, and no evidence of nephrotoxicity was observed. CONCLUSIONS: SnPP can be safely administered and, after its injection, the resulting changes in plasma HO-1, NQO1, ferritin, and p21 concentrations can provide information as to antioxidant gene responsiveness/reserves in subjects with and without kidney disease. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Study with RBT-1, in Healthy Volunteers and Subjects with Stage 3-4 Chronic Kidney Disease, NCT0363002 and NCT03893799.

Urinary cobalt and ferritin in four-years-old children.

Cobalt (Co) is an essential trace element but may cause toxic effects upon occupational or environmental exposure. The present study is aimed to determine the urine concentrations of Co in four years-old children in the INMA-Asturias cohort (Spain) and to assess the factors determining the observed levels. This cohort is located in a heavily industrialized zone with strong potential for metal exposure. Some diet components such as consumption of sweets were meaningfully associated with higher urine Co concentrations. Traffic pollution also showed a noteworthy positive association with Co levels. Family tobacco consumption did not show substantial association with the urine concentrations of this metal in the INMA-Asturias children. A significant inverse association between urine Co and venous blood ferritin was found. Iron deficiency anemic children had significantly higher concentrations of Co than those with normal levels, e.g. median values 1.9 µg/g creatinine and 1.0 µg/g creatinine, respectively. This association could be explained by an increased expression of DMT1, a divalent metal transporter that captures higher levels of iron in deficiency states of this metal. This transporter is non-specific and not only captures iron but also other divalent metals such as Co. The presence of this metal in iron deficiency anemic children may represent an additional disturbing health factor that must be considered during treatment.

Parenterial iron sucrose-induced renal preconditioning: differential ferritin heavy and light chain expression in plasma, urine, and internal organs.

Experimental data suggest that iron sucrose (FeS) injection, used either alone or in combination with other prooxidants, can induce "renal preconditioning," in part by upregulating cytoprotective ferritin levels. However, the rapidity, degree, composition (heavy vs. light chain), and renal ferritin changes after FeS administration in humans remain to be defined. To address these issues, healthy human volunteers (n = 9) and patients with stage 3-4 chronic kidney disease(n = 9) were injected once with FeS (120, 240, or 360 mg). Plasma ferritin was measured from 0 to 8 days postinjection as an overall index of ferritin generation. Urinary ferritin served as a "biomarker" of renal ferritin production. FeS induced rapid (≤2 h), dose-dependent, plasma ferritin increases in all study participants, peaking at approximately three to five times baseline within 24-48 h. Significant urinary ferritin increases (~3 times), without dose-dependent increases in albuminuria, neutrophil gelatinase-associated lipocalin, or N-acetyl-ß-d-glucosaminidase excretion, were observed. Western blot analysis with ferritin heavy chain (Fhc)- and light chain (Flc)-specific antibodies demonstrated that FeS raised plasma Flc but not Fhc levels. Conversely, FeS increased both Fhc and Flc in urine. To assess sites of FeS-induced ferritin generation, organs from FeS-treated mice were probed for Fhc, Flc, and their mRNAs. FeS predominantly raised hepatic Flc. Conversely, marked Fhc and Flc elevations developed in the kidney and spleen. No cardiopulmonary ferritin increases occurred. Ferritin mRNAs remained unchanged throughout, implying posttranscriptional ferritin production. We conclude that FeS induces rapid, dramatic, and differential Fhc and Flc upregulation in organs. Renal Fhc and Flc increases, in the absence of nephrotoxicity, suggest potential FeS utility as a clinical renal "preconditioning" agent.

Ferritin in serum and urine: A pilot study.

Serum ferritin reflects total body iron stores, thus a low serum ferritin is used as a parameter of iron deficiency. In healthy adults in Japan, urine ferritin levels were about 5% of serum ferritin levels, with a correlation coefficient of 0.79. It is not known whether a low urine ferritin could serve as a non-invasive screen for iron deficiency. If so, this might be useful for neonates and young children, avoiding phlebotomy to screen for iron deficiency. However, for urinary ferritin screening to be feasible, ferritin must be measurable in the urine and correlate with serum ferritin. Testing should also clarify whether the iron content of ferritin in serum and urine are similar. In this pilot feasibility study we measured ferritin in paired serum and urine samples of healthy adult males, healthy term neonates, growing preterm neonates, and children who had very high serum ferritin levels from liver disorders or iron overload. We detected ferritin in every urine sample, and found a correlation with paired serum ferritin (Spearman correlation coefficient 0.78 of log10-transformed values). These findings suggest merit in further studying urinary ferritin in select populations, as a potential non-invasive screen to assess iron stores.

Screening, identification of prostate cancer urinary biomarkers and verification of important spots.

Prostate-specific antigen (PSA) has been widely used as the unique serum biomarker for the diagnosis of prostate cancer (PCa). When PSA is moderately increased (e.g., 4-10 ng/ml), it is difficult to differentiate benign prostatic hyperplasia (BPH) from cancer. The diagnostic test (i.e., prostate biopsy) is invasive, adding pain and economic burden to the patient. Urine samples are more convenient, non-invasive and readily available than blood. We sought to determine whether ferritin might be the potential urinary biomarker in prostate cancer diagnosis. Using two-dimensional electrophoresis (2DE) followed by mass spectrometry (MS), differentially expressed urinary proteins among patients with PCa, BPH and normal controls were obtained. The ferritin heavy chain (FTH) gene, ferritin light chain (FTL) gene and protein expression of BPH-1 cells and PC-3 cells were analyzed by real-time quantitative PCR and Western blotting, respectively. Stable FTH or FTL silenced cell lines were generated by small hairpin(sh) RNA lentiviral transfection. The function of the cell lines was evaluated by the colony formation assay, transwell assay, and flow cytometry. Compared with BPH and normal controls, 15 overexpressed proteins, including FTH and FTL, were identified in the urine of the PCa group. FTH and FTL were also highly expressed in PC-3 cell lines compared with BPH-1 cells. FTH-silenced cells showed reduced cell proliferation, migration and increased cell apoptosis. FTL-silenced cells showed increased proliferation and migration abilities. There are differences in urinary proteins among patients with PCa, BPH and normal controls. FTH and FTL play different roles in PCa cells and are potential biomarkers for PCa.

High urinary ferritin reflects myoglobin iron evacuation in DMD patients.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the dystrophin gene leading to the absence of the normal dystrophin protein. The efforts of many laboratories brought new treatments of DMD to the reality, but ongoing and forthcoming clinical trials suffer from absence of valuable biomarkers permitting to follow the outcome of the treatment day by day and to adjust the treatment if needed. In the present study the levels of 128 urinary proteins including growth factors, cytokines and chemokines were compared in urine of DMD patients and age related control subjects by antibody array approach. Surprisingly, statistically significant difference was observed only for urinary ferritin whose level was 50 times higher in young DMD patients. To explain the observed high urinary ferritin content we analysed the levels of iron, iron containing proteins and proteins involved in regulation of iron metabolism in serum and urine of DMD patients and their age-matched healthy controls. Obtained data strongly suggest that elevated level of urinary ferritin is functionally linked to the renal management of myoglobin iron derived from leaky muscles of DMD patients. This first observation of the high level of ferritin in urine of DMD patients permits to consider this protein as a new urinary biomarker in muscular dystrophies and sheds light on the mechanisms of iron metabolism and kidney functioning in DMD.

Association of ferritin with prostate cancer.

PURPOSE: Prostate specific antigen (PSA) has been widely used as the unique serum biomarker for the diagnosis and/ or pre-diagnosis of prostate cancer (PCa). However, the diagnostic value of PSA is a subject of ongoing debate owing to its lack of specificity, especially when the PSA level is moderately increased (e.g., 4-10 ng/ml). Thus, we suggest the need for identification of a new biomarker to discriminate PCa cases from benign prostatic hyperplasia (BPH) and normal controls (N). METHODS: Urine or tissue samples of PCa patients, BPH patients, and normal controls were systematically collected. The expression of ferritin light chain (FTL) and ferritin heavy chain (FTH) was verified by immunohistochemistry in the tissue. The concentration of urinary ferritin was measured by Access Immunoassay. The level of creatinine in the urine was detected on a HITACHI 7080 system to calculate the ferritin-creatinine ratio (FCR). The data were statistically analyzed using the rank sum test. RESULTS: Immunohistochemical characterization of tissues in patients with PCa and BPH was conducted. We found representative immunohistochemical expression of FTL and FTH, with strong staining intensity in PCa and weak staining intensity in BPH. Furthermore, there were differences in urinary FCR among the three groups, with significant differences in the PCa group (134.46±47.01) compared to both the BPH (24.18±3.17, p = 0.009) and control (6.42±0.82, p = 0.003) groups. In contrast, there was no significant difference between the BPH and N groups (p = 0.649). CONCLUSIONS: Ferritin is a potential urinary biomarker to discriminate between PCa and BPH patients.

Urinary ferritin and cystatin C concentrations at different stages of kidney disease in leishmaniotic dogs.

Traditional analytes do not detect early renal disease; therefore there is a need to find new early markers of chronic kidney disease (CKD) in dogs to avoid the progression to irreversible renal damage. Our objective was to evaluate the presence of ferritin and cystatin C in urine of dogs with CKD and to relate their concentrations with the severity of the disease. Samples obtained from dogs naturally infected with Leishmania infantum were classified into four groups on the basis of the results of urinary protein/creatinine ratio and serum creatinine. This study shows that ferritin and cystatin C concentrations were increased in the urine of dogs with renal damage. Cystatin C value in urine only increased in severe stages of CKD with serum creatinine values >1.4 mg/dL, while the urinary ferritin concentration increased in dogs with proteinuria and serum creatinine <1.4 mg/dL, being, therefore, a renal biomarker earlier than creatinemia.

The influence of iron stores on cadmium body burden in a Thai population.

Cadmium is a toxin of increasing public health concern due to its presence in most human foodstuffs and in cigarette smoke. Exposure to cadmium leads to tissue bioaccumulation and, in particular, has nephrotoxic effects. The aim of the present study was to investigate the association between cadmium body burden and iron stores in a Thai population. A total of 182 healthy adult Thai subjects of both genders (89 males, 93 females) aged between 18 and 57 years and weighing 40-95 kg were included in this study. The total amounts of cadmium excreted in urine over 2 h (microg/g creatinine) were used as an index of long-term cadmium exposure. Quantitation of cadmium was performed using electrothermal (graphite furnace) atomic absorption spectrometry. The urinary cadmium excreted displayed a normal frequency distribution. The average urinary cadmium level did not exceed the WHO maximum tolerable internal dose for the non-exposed population (2 microg/g creatinine). Body iron stores reflected by serum ferritin levels did not show any correlation with cadmium burden in both males and females, although a relatively stronger influence of body iron store status on cadmium burden was shown in females. When the levels of serum ferritin were stratified into five levels (<20, 20-100, 101-200, 201-300, and >300 microg/l), a significant difference in total cadmium body burden was observed between females and males only in the group with a low level of serum ferritin of <20 microg/l. The cadmium body burden in females was about twice that in males in this group.

Immunoassay for human serum hepcidin.

We developed and validated the first serum enzyme-linked immunosorbent assay for hepcidin, the principal iron-regulatory hormone that has been very difficult to measure. In healthy volunteers, the 5% to 95% range of hepcidin concentrations was 29 to 254 ng/mL in men (n = 65) and 17 to 286 ng/mL in women (n = 49), with median concentrations 112 versus 65 (P < .001). The lower limit of detection was 5 ng/mL. Serum hepcidin concentrations in 24 healthy subjects correlated well with their urinary hepcidin (r = 0.82). Serum hepcidin appropriately correlated with serum ferritin (r = 0.63), reflecting the regulation of both proteins by iron stores. Healthy volunteers showed a diurnal increase of serum hepcidin at noon and 8 pm compared with 8 am, and a transient rise of serum hepcidin in response to iron ingestion. Expected alterations in hepcidin levels were observed in a variety of clinical conditions associated with iron disturbances. Serum hepcidin concentrations were undetectable or low in patients with iron deficiency anemia (ferritin < 10 ng/mL), iron-depleted HFE hemochromatosis, and juvenile hemochromatosis. Serum hepcidin concentrations were high in patients with inflammation (C-reactive protein > 10 mg/dL), multiple myeloma, or chronic kidney disease. The new serum hepcidin enzyme-linked immunosorbent assay yields accurate and reproducible measurements that appropriately reflect physiologic, pathologic, and genetic influences, and is informative about the etiology of iron disorders.

Blunted hepcidin response to oral iron challenge in HFE-related hemochromatosis.

Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P = .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P < .001) and heterozygotes (P = .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P < .001 for C282Y/C282Y and P = .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P = .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( > or = 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.

Comparison between desferrioxamine and combined therapy with desferrioxamine and deferiprone in iron overloaded thalassaemia patients.

Desferrioxamine (DFX) alone (40-50 mg/kg/d s.c. over 8-12 h, five times weekly) was compared with combined DFX twice weekly and deferiprone (75 mg/kg/d) over 12 months in previously poorly chelated thalassaemia patients. Serum ferritin fell from 5506 +/- 635 microg/l (mean +/- SEM) to 3998 +/- 604 microg/l (P < 0.001; n = 14) in the DFX group and from 4153 +/- 517 microg/l to 2805 +/- 327 microg/l in the combined group (P < 0.01; n = 11). Deferiprone plus DFX produced a greater mean urine iron excretion (1.01 mg/kg/24 h) than iron intake from blood transfusion in each patient. Main side-effects were skin reactions (DFX alone), nausea and arthralgia (combined therapy). As chelation therapy, the combined protocol was as effective as DFX five times weekly.

Elevated tumour markers in patients with Balkan endemic nephropathy.

Balkan nephropathy (BEN) is commonly associated with urothelial cancer. Urothelial cancer is manifested in the advanced stage of disease. The aim of this study was to facilitate early detection of urothelial cancer in BEN patients and their family members living in an endemic region by using tumour markers, carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA), and a putative marker, ferritin. Fifteen BEN patients with normal renal function, 17 with renal failure (BEN-RF), 13 healthy members of their families (HFM), 14 patients with glomerulonephritis (GN) and 12 healthy controls (C) were studied. Serum CEA levels in BEN patients were within normal limits, however, in BEN-RF patients they were significantly increased over HFM (p<0.05). Serum TPA levels in BEN and BEN-RF patients were significantly higher than in the C and HFM groups (p<0.05). Urinary CEA was not significantly different between the groups studied. Urinary TPA levels in HFM (median 125 U/l, BEN (236 U/l) and BEN-RF (275 U/l) were significantly increased over C (30 U/l), however, TPA levels were increased also in GN patients (437 U/l). None of the BEN patients studied developed urothelial cancer during the ten years' follow-up. Markedly elevated urinary TPA-like levels in all patients studied (HFM, BEN, BEN-RF, GN) suggest that urinary TPA may not be a reliable tumour marker. However, the clinical relevance of high TPA levels in BEN patients should be evaluated.

[Follow up control of stage IV neuroblastoma: 123I-MIBG scintigraphy, bone scintigraphy and catecholamine metabolites]. / Verlaufskontrolle des Neuroblastoms Stadium IV: 123I-mIBG-Szintigraphie, Knochenszintigraphie und Katecholaminmetabolite.

AIM: The diagnostic value of 123I-mIBG-scintigraphy, bone scintigraphy and catecholamine metabolites in the follow up of neuroblastoma stage IV will be evaluated. METHODS: Nineteen children suffering from neuroblastoma were analysed retrospectively by 123I-mIBG-scintigraphy, bone scintigraphy and measurement of homovanillic acid, vanillic acid, neuronspecific enclose, lactate dehydrogenase, and ferritine. Follow up was 7-132 (median 36) months. RESULTS AND CONCLUSION: The significance of the methods was dependent on the time of diagnostic use. In principal, 123I-mIBG-scintigraphy has the highest diagnostic impact. For initial staging and diagnosis of recurrence a combination of all three methods can be used. On the contrary, follow up during chemotherapy is best documented by 123I-mIBG-scintigraphy, whereas bone scintigraphy is of limited and measurement of catecholamine metabolites of less diagnostic value.

Clinical significance of urinary ferritin excretion in patients with transitional cell carcinoma.

1. The serum ferritin level provides a valuable index of the body iron store. An increase in serum ferritin has often been observed in patients with neoplastic disease and correlates well with the stage of cancer. A few studies have suggested the potential of urinary ferritin as a marker for transitional cell carcinoma. The rationale of the measurement, however, has not been investigated in detail. 2. Urinary ferritin levels were evaluated in patients with diverse urological diseases to investigate their potential clinical implications. 3. Analysis of logarithmic transformed values (ng/mg creatinine) showed that patients with both neoplastic and non-neoplastic urological diseases had significantly higher ferritin levels than normal control subjects (P = 0.02). There was no apparent difference between subgroups of patients with urological disease (P > 0.5). For patients with urothelial carcinoma, univariate analysis revealed a strong positive relationship between urinary ferritin levels and the density of lymphoid cells in tumour stroma (P = 0.0001), while no important association was observed with tumour grade (P = 0.32), stage (P = 0.29) or urinary cytology detection (P = 0.33). Patients with muscle-invasive tumour had significantly higher ferritin levels than those with papillary, superficial cancer (P < 0.05). For patients with non-neoplastic urological disease (n = 19), urinary ferritin levels tend to correlate with the severity of tissue inflammation (P = 0.03). 4. The results suggest that urinary ferritin may reflect the degree of local inflammatory reaction in the urinary tract.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous determination of urinary CEA, ferritin and TPA in Egyptian bladder cancer patients.

Urinary carcinoembryonic antigen (CEA), ferritin (Fer) and tissue polypeptide antigen (TPA) were determined in 328 cases (106 with bladder cancer, 152 with non-malignant urinary tract disease and 70 healthy controls). CEA was determined by the kit supplied by Roche Diagnostica (CEA EIA Doumab 60), ferritin by the Tandem-E Fer kit supplied by Hybritech and TPA by the Prolifigen TPA-IRMA kit supplied by Sangtec Medical. The results of this work revealed that combined determination of urine CEA and Fer, CEA and TPA or Fer and TPA showed higher sensitivity than determination of the individual markers. There was no significant difference between combined and individual marker determination with respect to false positivity in non-malignant urinary tract diseases. At 97% specificity, the sensitivities of urine CEA, Fer and TPA were 82.1%, 71.7% and 90.6%, respectively, while combined urine CEA & Fer, CEA & TPA and Fer & TPA showed sensitivities of 92.5%, 99.1% and 98.1%, respectively. When the specificity was related to the entire non-cancer group (patients with benign urinary tract diseases and normal controls), some reduction in the sensitivities of the combined markers was noted compared to the normal group only. In conclusion, combined determination of urine markers is superior to determination of individual markers in the diagnosis of bladder cancer.

Clinical significance of urine ferritin determination in urologic malignancies.

Urine and serum ferritin levels were measured by a double-antibody radio-immunoassay in 96 normal adults and 57 patients with urologic malignancies. In the 96 adults, the urine ferritin levels ranged from 0 to 28 ng/ml. Elevated urine ferritin levels were observed in all 7 patients with renal pelvic carcinoma, 4 of 13 patients with renal carcinoma, 1 of 13 patients with well-differentiated (Grade I) bladder carcinoma, 8 of 19 patients with median-differentiated (Grade II) bladder carcinoma, and all 5 patients with poorly-differentiated (Grades III, IV) bladder carcinoma. These results indicated that urine ferritin assay was helpful in discriminating well-differentiated bladder carcinoma from the poorly-differentiated (P less than 0.01) in addition to differentiating renal pelvic carcinoma from renal carcinoma (P less than 0.05).