[Fundamental and clinical aspects of cystatin C].

Recent progress of fundamental and clinical studies on cystatin C was reviewed. Most of key studies are indebted to prof. Grubb A and his groups. International contributions from Japanese research work are included here. The protein is a basic low molecular weight protein of 13,300 with 120 amino acid residues and pI 9.3, functioning as a cysteine protease inhibitor. With an introduction of ERM-DA471, international reference material for serum cystatin C, global standardization for immunoassay systems has been much facilitated. No serious problems are present in the pre-analytical stage. Serum reference intervals are properly set in all Asian populations including Japanese with age and gender-related differences. The protein is a powerful serum intrinsic marker for glomerular filtration rate. Estimated glomerular filtration rate (eGFRcysC) in coupled with eGFRCr will definitely be a clinical routine for early detection and prevention of altered kidney function and cardiovascular events in general population. Genetic tests clinically indicated include hereditary cystatin C amyloid angiopathy (L68Q) and adult macular degeneration (A25T) although their frequency is extremely low.

Evaluation of adsorption of urine cystatin C to the polymer materials on the microplate by an antigen capture enzyme-linked immunosorbent assay.

BACKGROUND: Cystatin C is a low molecular weight protein of 13 kDa with an isoelectric point of 9.3. Its adsorption on the urine sampling containers may cause the underestimation of cystatin C levels. We newly developed an antigen capture enzyme-linked immunosorbent assay (ELISA) of sandwich method for measurement of adsorbed level. METHODS: We used a polystyrene microplates with 3 different polymers. These include high hydrophobic, low hydrophobic, and hydrophilic materials. Using the same microplate, the absorbed protein was measured by an antigen Capture ELISA, and calibration was conducted by an ordinary ELISA. RESULTS: In normal urine the concentrations of absorbed cystatin C levels to the 3 materials at day 1 were 0.50, 0.32-0.84 microg/l (median, interquartile range), 0.28, 0.21-0.37 microg/l, and <0.08, <0.08-0.09 microg/l in high hydrophobic, low hydrophobic, and high hydrophilic material, respectively. The absorption rate was 6%, 3%, and 1%, respectively. The adsorption is dependent on urine pH. It changes reciprocally with urine protein concentration. In pathologic urine, the absolute absorption level was <0.08 microg/l on the median, and the adsorption ratio (absorption level/urine level) was much less than 0.5% of that in normal urine. CONCLUSION: In the clinical setting, the absorption of cystatin C to sample containers is negligible since the rate of adsorption is low both in normal and pathologic urine. The material with high hydrophilic surface processing may be used for other proteins when interaction of the proteins with surface material affects the value to clinical decision.

Urinary protein 1/Clara cell 16 concentrations and lung functions in male subjects with pneumoconiosis.

BACKGROUND: Protein 1 (P1)/Clara cell 16 kDa protein (CC16, previously named CC10), a potentially immunosuppressive protein secreted by non-ciliated cells of the tracheobronchial epithelium, has been found to be a new useful lung-specific biomarker in several pathological lung conditions. Particularly, urinary P1 (uP1) may reflect the altered lung functions in pneumoconiosis. METHODS: We investigated the relationship between uP1 values and lung functions in 31 non-smoking pneumoconiotic males (mean age 73 years) with a history of dust exposure work in shipbuilding. The protein was measured using an originally prepared enzyme-linked immunosorbent assay system. The forced expiratory volume in 1 s % (FEV(1.0)%) and % vital capacity (%VC) were tested with a spirometer. RESULTS: The mean values of uP1 were 4.62 +/- 4.82 (mean +/- standard deviation) ng/mol creatinine. A univariable correlation test showed a significant positive correlation between uP1 and %VC (r = 0.356, P = 0.049). Also, a multiple regression analysis, when adjusted for age, disease duration, FEV(1.0)% and %VC, showed a significant correlation of uP1 with %VC (beta = 0.467, P = 0.030). CONCLUSION: The results suggest that a decreased uP1, corroborated by a decreased %VC, may be the result of damage to secretory cells. Measurement of uP1 may become a possible index of fibrotic changes in pneumoconiosis.

Expression of uteroglobin in normal and carcinogenic endometrium and influence of hormone replacement therapy.

Uteroglobin, first reported in 1968 as a steroid secreted in rabbit uterine fluid during early pregnancy, is a progesterone-regulated and progesterone-binding protein. There is evidence that indicates that uteroglobin is inversely correlated to neoplastic growth but its role to endometrial carcinogenesis is not known. Therefore we analyzed the expression of uteroglobin in 13 normal endometrium, 19 hyperplasia and 21 endometrial carcinoma samples and the relation to estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) by immunohistochemistry and Western blotting. We also analyzed the expression of uteroglobin in 15 menopausal women who received hormone replacement therapy (HRT). The expression of uteroglobin was higher during the secretory phase than in the proliferative phase; however, it was detected in endometrial hyperplasia as weakly as in the proliferative phase and decreased according to the loss of differentiation in endometrial carcinoma. The results were basically in accord with those for PR; however, the expression of uteroglobin was weak, though PR was most detected in endometrial hyperplasia. In menopausal endometrium, the group treated with estrogen plus progesterone exhibited higher expression of uteroglobin than the group treated only with estrogen. The evidence suggests that uteroglobin expression is regulated by progesterone in the normal endometrium but that the regulation by PR is lost in endometrial hyperplasia and carcinoma according to acquirement of tumorigenesis and that estrogen plus progesterone therapy reduces the risk for endometrial carcinoma by restoring uteroglobin.

Polymorphism of Clara cell 10-kD protein gene of sarcoidosis.

Clara cell 10-kD protein (CC10) exhibits potent antiinflammatory properties. G38A polymorphism was found in the CC10 gene. We investigated the genetic influence of the allele on the development of sarcoidosis using case control analysis in a Japanese population (265 sarcoidosis cases and 258 control subjects). The A allele frequency in sarcoidosis cases (45.1%) was significantly higher than healthy control subjects (34.9%, p = 0.0002). According to outcomes, we divided 223 patients with follow-up periods of 3 years or more into two subgroups (55 progressive and 168 regressive disease). The A allele frequency in patients with progressive disease was significantly higher than control subjects (odds ratio = 4.55; 95% confidence interval, 2.97-6.97; p < 0.0001), whereas that of regressive disease was not. The A/A genotypes had significantly lower bronchoalveolar lavage fluid CC10 levels than the G/G (nonsmokers, p = 0.0054, and smokers, p = 0.0045) and G/A genotypes (nonsmokers, p = 0.0022, and smokers, p = 0.0402). The reporter gene assay showed significantly lower reporter activities in the presence of interferon-gamma for the 38A construct than the 38G construct (p = 0.0177). The G38A polymorphism in the CC10 gene may influence protein expression and be associated with the development of progressive sarcoidosis.

Clinical aspects of Clara cell 10-kDa protein/ uteroglobin (secretoglobin 1A1).

Clara cell 10-kDa protein (CC10)/ uteroglobin (UG) is a nonglycoprotein with a molecular mass of 16 kilodaltons, which is produced by mucosal epithelial cells in the lung (Clara cells), uterus and prostate. Like other low molecular weigh proteins it is catabolized in renal proximal tubules. Structurally it is a homodimer of subunits of 70 amino acids covalently bound in an antiparallel manner. It belongs to secretogobin (SCGB) family and is assigned as subgroup 1A1. The function of the protein so far elucidated is immunoregulatory and anti-inflammatory in innate immunity. The knockout mouse of UG gene resulted in aggravation of inflammation by allergic and hyperoxic stimuli. It also showed very similar pathological features with human IgA nephropathy. The value is changed in the lung fluid and serum of various inflammatory and allergic lung diseases. Several kinds of single nucleotide polymorphisms (SNPs) in human CC10/UG gene were recently discovered; Adenine allele accumulation in G38A SNP has possible association with asthma and IgA nephropathy, being paralleled with disease severity of IgA nephropathy. Its expression is enhanced by some transcriptional factors induced by cytokines such as interferon-gamma. For cancer cells, the protein functions as an antagonist of neoplastic phenotype. CC10/UG forms one of intra- and intercellular regulators involved in inflammation and malignant transformation in the respiratory and urogenital fields.

Association of the uteroglobin gene polymorphism with IgA nephropathy.

Immunoglobulin A (IgA) nephropathy results from the abnormal deposition of IgA in the renal mesangium. Genetic factors may be involved in the development and progression of IgA nephropathy. Uteroglobin (UG) is a steroid-inducible, cytokine-like, multifunctional protein with anti-inflammatory and immunomodulatory properties. The knockout or antisense mouse of the UG gene develops renal disease similar to IgA nephropathy. We analyzed the UG gene as a candidate for a predisposing factor in 61 Japanese patients with IgA nephropathy (23 children, 38 adults) and detected only the G38A mutation. The gene frequency of the G38A mutation in patients was 0.43, not significantly different from the frequency of 0.36 in healthy controls. However, the frequency of patients homozygous for G38A was twice that of controls, and a significant increase was seen in child patients. We measured serum UG levels in patients and healthy adults. A significant decrease in serum UG levels in homozygotes of G38A compared with homozygotes of G38 was detected only in adult women patients and controls. There is no information on where serum UG is produced or how UG may work in association with IgA nephropathy. However, it is possible that the effect of G38A may be apparent under such stimulation as sex steroids or infections, and homozygotes of the G38A mutation cannot produce sufficient UG in response to stimulation and may be predisposed to IgA nephropathy, especially in childhood.

[Present and future prospects of individualized medical services by genetic tests].

With progress of human genome projects many gene mutations and their expression profiles have been much identified. Laboratory tests have become essential to achieve proper individualized medical services. In particular single nucleotide polymorphisms (SNPs) and gene expression profiles are of high interests. As one of these ongoing mmodels, we here present the SNPs of human Protein 1' also called as human Clara cell 10-kd protein (CC10). We initially found six different SNPs (A-908G, G38A, G118A, C1225T, G1226A, C4777G). For laboratory use multiplex PCR was developed to determine frequencies in normal control. The -908G and 38A genotypes were more frequent in sarcoidosis than those in controls. The SNPs of in the human protein 1 gene may be the genetic susceptibility factors for Sarcoidosis.