[Evaluation of urinary cystatin C as a marker of renal dysfunction].

BACKGROUND: Urinary excretion of some low molecular weight proteins (LMWPs) is used as an indicator of tubular dysfunction, since they are increased by the damage of tubular reabsorption. Although serum cystatin C is known to be a sensitive marker for GFR, the property of urinary cystatin C as a LMWP has not been fully observed. We evaluated the clinical utility of urinary cystatin C. METHODS: Urine samples were collected from 130 patients with various degree of renal dysfunction, 62 healthy subjects, and 2 patients with acute renal failure, one with renal acute renal failure, the other with prerenal acute renal failure. Urine levels of cystatin C, beta2-microglobulin (beta2mG), and alpha1-microglobulin (alpha1mG) were measured by immunonephelometry. Creatinine clearance(Ccr) tests were conducted on 130 patients with renal dysfunction. Creatinine(CRE) was measured by enzyme assay. RESULTS: The daily urinary excretions of cystatin C and alpha1mG were increased significantly in patients with Ccr<30 ml/min(group I), compared to those in patients with 30 < or = Ccr<70 ml/min(II), and Ccr > or = 70ml/min(III). Although the mean daily excretion of beta2mG increased as Ccr decreased, the significant difference was not observed. The rate of increase in the mean value between III and I was extremely high in cystatin C. Fractional excretions of cystatin C and beta2mG calculated in the same groups increased significantly in I compared to II and III. The rate of increase in the mean value was higher in cystatin C. Regression analyses between urine CRE and each three LMWP gave the best correlation coefficient for cystatin C in healthy subjects. While in one patient with renal acute renal failure, the rate of increase in urine cystatin C was higher than that of other LMWPs, in another patient with prerenal acute renal failure, the rate of increase in urine cystatin C was low. CONCLUSIONS: Although details of urinary movement of LMWPs in nephrons have not been clearly elucidated, the urinary cystatin C seems to have distinctive properties, and to be useful for the evaluation of renal injury.

Low-dose dual blockade of the renin-angiotensin system improves tubular status in non-diabetic proteinuric patients.

OBJECTIVE: Treatment with agents that inhibit the renin-angiotensin system is commonly regarded as a gold standard renoprotective strategy in patients with chronic kidney diseases. For maximum antiproteinuric effect, the dose titration of these agents is recommended. This therapeutic strategy is not used for proteinuric patients who are not able to receive high doses of angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonists. MATERIAL AND METHODS: In patients with primary glomerulonephritis (n=24), a randomized, triple-treatment, triple-period, cross-over study was performed to compare the effects of combined therapy with benazepril 5 mg and losartan 25 mg and monotherapy with either agent alone at a two-fold higher dose on the extent of tubular injury as assessed by alpha1-microglobulin (alpha1-m) excretion and the plasma level of transforming growth factor-beta1 (TGF-beta1). RESULTS: Combination therapy significantly reduced alpha1-m excretion compared to either agent used alone: 178.29+/-27.36 to 99.63+/-13.03 mg/g creatinine for losartan + benazepril vs 178.29+/-27.36 to 161.59+/-23.22 mg/g creatinine for benazepril alone (p<0.05; ANOVA) and 178.29+/-27.36 to 99.63+/-13.03 mg/g creatinine for losartan + benazepril vs 178.29+/-27.36 to 173.45+/-27.69 mg/g creatinine for losartan alone (p<0.05; ANOVA). There was a significant correlation between change in alpha1-m excretion and reduction in proteinuria (r=0.704; p=0.023). There were no differences in TGF-beta1 level between the studied treatments. Systemic blood pressure reduction did not differ among the therapies. CONCLUSIONS: Combination therapy with angiotensin-converting enzyme inhibitor and angiotensin II subtype 1 receptor antagonists at very small doses may be superior to monotherapy with these agents at higher doses as far as tubular injury is concerned. We speculate that such a therapeutic strategy may be a useful approach for patients who are known not to be capable of receiving optimal renoprotective doses of these regimens.

A simple procedure for isolating microgram quantities of biologically active bikunin from human urine.

OBJECTIVE: To report a simple, relatively rapid protocol to isolate biologically active bikunin from human urine using ion-exchange-trypsin affinity chromatography. Bikunin is a protease inhibitor which has been shown to play a role in various processes, including inhibition of calcium oxalate crystallization, the regulation of proliferation and modulation of carcinogenesis. The unavailability of the purified protein has hampered studies on bikunin's expanding role in these processes. MATERIALS AND METHODS: Female human urine was dialysed (15 kDa threshold) and crudely fractionated with a double-saturated ammonium sulphate precipitation. The first precipitation was with 35% saturated ammonium sulphate, and the supernatant was harvested, and the second with 90% saturated ammonium sulphate, and the precipitate collected. The protein mixture was then passed over Sepharose SP-fast-flow cation exchange and Sepharose Q-fast-flow anion exchange columns connected in series. The final purification was with a trypsin-affinity column which selectively bound bikunin. RESULTS: This procedure could recover 1 microg of bikunin per 2 mL of urine, and the final product was essentially free of contaminating inter-alpha-trypsin inhibitor heavy chains or bikunin-heavy chain conjugates. Product purity was confirmed by two-dimensional polyacrylamide gel electrophoresis combined with silver staining or Western blot. All isolations contained the 17 kDa minimally glycoslyated/sulphated form of bikunin and the 28 kDa form of bikunin. Some preparations also contained 33-48 kDa forms of bikunin. The protein cores of all three proteins were confirmed to be bikunin by mass spectrometry and Western blot. Harvested bikunin retained its trypsin inhibitory activity (L-benzoylarginine-p-nitroanilide assay). Preparations containing the 33-45 kDa form had two to three times more trypsin inhibitory activity than preparations without this band. CONCLUSIONS: This novel ion exchange-trypsin affinity chromatography protocol uses only two chromatographic steps. The product consists of three isomers of biologically active bikunin, free of contaminating heavy chains or bikunin-heavy chain conjugates. The ready availability of purified bikunin should facilitate future studies of bikunin's emerging role in urolithiasis, proliferation and carcinogenesis.

Reproducibility of urinary cadmium, alpha1-microglobulin, and beta2-microglobulin levels in health screening of the general population.

The present study examined whether levels of cadmium, and alphal- and beta2-microglobulin in urine (Cd-U, ac-MG-U, and beta2-MG-U, respectively) were reproducible in urine samples collected from the same subjects on multiple occasions. For this purpose, two databases on background exposure to cadmium in Japan-one from study I between 2000 and 2001 and the other from study II in 2002-were revisited to find 231 apparently healthy, nonpregnant, nonlactating adult women who participated in both studies and thus had provided two urine samples. The databases contained information on Cd-U, alphal,-MG-U, and beta2-MG-U, creatinine (CR), and specific gravity (SG) as well as smoking and other lifestyle factors. Of the 231 women, 195 who had never smoked were selected for the present analysis. Cd-U as well as alpha1-MG-U were reproducible (e.g., with correlation coefficients [r] between study I and II results of 0.4 to 0.6) when measured on two occasions 9 to 10 months apart. The r values were lower for beta2-MG-U (r0.3). Exclusion of urine samples with inadequate urine density(i.e., CR <0.5 or >3.0 g/L or SG <1.010 or >1.030) resulted in substantial improvement of the agreements between the two measures (e.g., r = 0.6 to 0.7 for Cd-U and alpha1-MG-U). CR and SG correlated closely with each other, especially in low-density urine samples (r >0.9), and therefore the effects of CR and SG could not be evaluated separately. In the overall evaluation,single determination (i.e., without repeated urine sampling) of Cd-U and alpha1-MG-U should be acceptable, and it may also be acceptable for beta2-MG-U. Use of samples with adequate urine density rather than application of density correction to low-density urine samples in recommended.

Renal tubular proteinuria in patients with irritable bowel syndrome.

OBJECTIVE: Irritable bowel syndrome (IBS) is a common condition that is poorly understood. We have previously demonstrated tubular protinuria in patients with inflammatory bowel disease. This study examined whether tubular proteinuria was a feature of IBS. METHODS: Eighty control subjects (male:female, 28:52; age range 20-65 years) and 21 patients with IBS (male:female, 9:12; age range 16-64 years) (not significant) were recruited. Patients with known renal disease, hypertension, diabetes or microbiological evidence of urinary infection were excluded. The IBS patients all fulfilled the ROME II criteria. None had preceding gastroenteritis. Urinary alpha1-microglobulin (alpha1-M) was measured in a second-voided morning urine sample and corrected for urinary concentration by measurement of creatine. Blood samples were analysed for haematochemical indices including C-reactive protein. Statistical analysis was by unpaired t test. RESULTS: None of the IBS patients were reclassified with inflammatory bowel disease over a 5-year follow up period. All had normal haematochemical parameters. Mean +/- standard deviation urinary alpha1-M concentrations were significantly higher in IBS patients than controls (IBS patients, 1.17 +/- 0.65 mg/mmol; controls, 0.75 +/- 0.36 mg/mmol; P < 0.01) and exceeded 1.5 mg/mmol (the upper reference limit) in seven patients. There was no difference in urinary alpha1-M concentrations in the diarrhoea-predominant and constipation-predominant groups (mean +/- standard deviation, 1.342 +/- 0.65 versus 0.76 +/- 0.48 mg/mmol; P = 0.062). CONCLUSIONS: Urinary alpha1-M concentration is commonly increased in IBS, suggesting the presence of renal proximal tubular injury.

A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings.

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.

Effects of smoking and lead exposure on proximal tubular integrity among Egyptian industrial workers.

BACKGROUND: The hazards of occupational lead exposure are well documented. Tissue damage produced by lead is slow and progressive. The renal system is one of the systems primarily affected by lead. The present study aimed to evaluate renal proximal tubular functional and structural integrity among lead-exposed and cigarette-smoking male Egyptian workers. This study was extended to investigate the effect of lead exposure and cigarette smoking on urinary excretion of copper (Cu) and zinc (Zn). METHODS: Participants included in the present study were 156 male workers grouped as follows: i) 75 lead non-exposed workers subgrouped into G1 that consisted of 36 non-smokers (age, 39.08+/-6.65 years) and G2, which included 39 smokers (age, 43.87+/-9.93 years); ii) 45 lead-exposed workers (work duration <20 years) subgrouped into G3 that consisted of 25 non-smokers (age, 37.40+/-3.76 years) and G4, which included 20 smokers (age, 38.40+/-4.95 years), and iii) 36 lead-exposed workers (work duration > or =20 years) subgrouped into G5 that consisted of 16 non-smokers (age, 45.94+/-4.19 years) and G6 which included 20 smokers (age, 45.70+/-2.25 years). Functional integrity of proximal tubules was studied by determining urinary level of low-molecular-weight protein alpha1-microglobulin (alpha1M), and structural integrity was investigated by measuring urinary activities of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (NAG) and the cytoplasmic enzyme glutathione S-transferase (GST). Urinary concentrations of Pb, Cu, Zn, and creatinine were also determined. RESULTS: Data of the present investigation showed increased urinary excretion of all measured urinary parameters among lead-exposed workers in comparison with non-exposed workers whether they were non-smokers or smokers (G3 vs. G1, G5 vs. G1, G4 vs. G2, and G6 vs. G2), with greater elevation among lead-exposed smokers than among lead-exposed non-smokers (G4 vs. G3, G6 vs. G5). In addition, there was a greater increase in levels of all urinary parameters among workers with work duration > or =20 years than in those with <20 years' work duration (G6 vs. G4, G5 vs. G3). CONCLUSIONS: Lead exposure in Egyptian workers causes damage to renal proximal tubules and results in its dysfunction. Cigarette smoking has a nephrotoxic effect and also is synergistic to lead nephrotoxicity on urinary excretion of GST and NAG, as well as Pb.

[A study on urine microglobulin change after interventional therapy of renal artery in patients with coronary heart disease].

OBJECTIVE: To evaluate the urine microglobulin change after interventional therapy in patients with renal artery stenosis. METHODS: According to the results of renal artery angiography, 69 consecutive patients with coronary heart disease were divided into 2 groups, including 44 patients with severe renal artery stenosis RAS (luminal narrowing > 70%, group I) and 25 patients with atherosclerotic lesion in renal artery (luminal narrowing > 50% but < 70%, group II). The urine alpha(1), beta(2)-microglobulin (alpha(1), beta(2)-MG) were measured respectively. The successful procedural rates, rates of complication, serum creatitine and the urine alpha(1), beta(2)-MG 3 months after procedure were also recorded. The acute results and long-term follow-up outcomes were compared between the 2 groups. RESULTS: Urine alpha(1)-MG [(5.2 +/- 2.5) microg/L vs (3.0 +/- 2.7) microg/L, P > 0.001] and beta(2)-MG [(377 +/- 173) microg/L vs (202 +/- 184) microg/L, P > 0.001] were significantly higher in group I than in group II. However, the urine alpha(1)-MG of the 2 groups did not exceed the normal range (<6 microg/L). The urine beta(2)-MG [(237 +/- 187) microg/L vs (377 +/- 173) microg/L, P > 0.01] 3 months after stenting procedure in group I were significantly decreased. There were no significance change in urine alpha(1)-MG in group I and both the microglobulins in group II during follow-up. There was significant difference in improvement of blood pressure between the 2 groups (62.5% vs 9.1%, P > 0.01). As compared with group II, group I patients had more re-admission (22.5% vs 9.1%), renal failure (5% vs 0) and higher mortality (5% vs 0). However, these measurements did not differ significantly. Multivariate analysis revealed that persistent elevation of urine beta(2)-MG was an independent predictor of severe events (including re-admission and renal failure) after renal artery stenting (OR = 3.01, 95% CI 1.01 - 8.95, P = 0.036). CONCLUSIONS: Severe RAS causes damage of tubular reabsorption. Renal artery stenting improves tubular function, but in patients with persistent elevation of beta(2)-MG, the rates of re-admission and renal failure remain high.

alpha1-Microglobulin as a promising marker of cadmium-induced tubular dysfunction, possibly better than beta2-microglobulin.

The purpose of the present study was to evaluate the validity of alpha1-microglobulin (alpha1-MG) in comparison with popularly used beta2-microglobulin (beta2-MG). A database on 8975 cases of never-smoking adult women was revisited; the data were based on spot urine samples from the women in 10 prefectures all over Japan. The validity of alpha1-MG was examined following essentially the same protocol as beta2-MG was examined in a previous study. Comparisons were made for alpha1-MG as observed (e.g. alpha1-MG(ob)), as corrected for creatinine (CR or cr) (e.g. alpha1-MGcr) and as corrected for a specific gravity (SG or sg) of 1.016 (e.g. alpha1-MGsg). A cut-off value of 5.0 mg alpha1-MG/g cr or l was deduced from 400 microg beta2-MG/g cr taking advantage of the regression equation between alpha1-MG and beta2-MG. The prevalence of alph1-microglobulinuria as corrected for a specific gravity of 1.016 (or alpha1-MGsg-uria in short) was essentially unchanged irrespective of SG, except for in very dense or very thin urine samples. alpha1-MGcr-uria prevalence decreased at higher CR. Comparison of the present observation with previous findings on beta2-MG-uria prevalence showed that the variation in prevalence of MG-uria as a function of urine density was smaller for alpha1-MGsg whereas it was substantially larger for beta2-MGcr, and thus it appeared prudent to consider alpha1-MGsg rather than beta2-MGcr as a marker of tubular dysfunction.

Urinary fatty acid-binding protein as a new clinical marker of the progression of chronic renal disease.

Previous studies have indicated that in massive proteinuria, free fatty acids (FFAs) bound to albumin were overloaded in the proximal tubule and exacerbated tubulointerstitial damage. Liver-type fatty acid-binding protein (L-FABP) is an intracellular carrier protein of FFAs that is expressed in the proximal tubule of human kidney. We sought to evaluate urinary L-FABP as a clinical marker in chronic renal disease. Urinary L-FABP was measured in patients with nondiabetic chronic renal disease (n = 120) with the use of a newly established ELISA method. We then monitored these patients for 15 to 51 months. Clinical data were analyzed with multivariate analysis. Urinary L-FABP was correlated with urinary protein, urinary alpha(1)-microglobulin, and serum creatinine concentrations. Urinary L-FABP at the start of follow-up (F = 17.1, r =.36, P <.0001) was selected as a significant clinical factor correlated with the progression rate, defined as a slope of a reciprocal of serum creatinine over time. We next selected the patients with mild renal dysfunction (n = 35) from all 120 patients and divided them into 2 groups according to progression rate: the progression group (n = 22) and the nonprogression group (n = 13). Serum creatinine and urinary protein concentrations and blood pressure at the start of follow-up were higher in the progression group than in the nonprogression group, although we detected no significant difference between the 2 groups. Urinary L-FABP was significantly higher in the former group than in the latter (P <.05). The results showed that urinary L-FABP reflected the clinical prognosis of chronic renal disease. Urinary L-FABP may be a clinical marker that can help predict the progression of chronic glomerular disease.

Identification of bikunin isolated from human urine inhibits calcium oxalate crystal growth and its localization in the kidneys.

BACKGROUND: We previously reported a high molecular weight substance purified from human urine that strongly inhibited calcium oxalate (CaOx) crystal growth in vitro. In the study present herein, we identified and investigated a protein purified from human urine that strongly inhibits CaOx crystal growth using a column chromatography series. METHODS: The protein was identified by amino acid sequencing and was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and immunohistochemical staining. RESULTS: The molecular weight of this protein was approximately 35 KDa, and it also had another band around 20 KDa. We determined that the amino acid sequence of the protein was homologous with that of bikunin, the light chain of the inter-alpha-trypsin inhibitor, which is known as a strong CaOx crystallization inhibitor in vitro. On western blotting analysis, the molecular weight was also found to be around 35K Da, the same as that of bikunin. Immunohistochemical staining revealed that it was mainly located in the epithelial cells of the proximal tubules and the thin descending segment near the loop of Henle, but not in the glomeruli, distal tubules or the collecting ducts. CONCLUSION: In the present study, the protein extracted from human urine was identical to bikunin, which may be expressed mainly in the proximal tubules and the thin descending segment near the loop of Henle, and which prevents CaOx crystallization in vitro.

In acute inflammation, the chondroitin-4 sulphate carried by bikunin is not only longer, it is also undersulphated.

Bikunin (Bk) is a Künitz-type serine proteinase inhibitor, which occurs in human plasma, mainly as covalent complexes with one or two of the three peptide heavy chains. The leading member of this glycoprotein family is inter-alpha-inhibitor (I alpha I), which consists of two heavy chains (H1 and H2) linked to Bk. Bk carries a glycosaminoglycan (GAG) chain, which is linked by ester bonds to the heavy chains of I alpha I. Furthermore, Bk, I alpha I and related components such as pre-alpha-inhibitor (P alpha I), all together making up the I alpha I family, present antiinflammatory and antimetastatic effects that hinge on this GAG chain. Recently (Eur. J. Biochem. 268 (2001) 2717), we provided evidence that, during acute phase response, the GAG chain of Bk, which is a low-sulphated chondroitin-sulphate, increases in size according to the severity of the inflammatory disease. This increase affects Bk-containing proteins in circulating blood as well as Bk excreted in higher amounts in urine of these patients. In this work, we have more extensively analysed the GAG chain of Bk isolated from urine collected from a unique patient with septic shock. Using MALDI-TOF-MS and HPLC analyses of chondrodisaccharides released by enzymatic digestion, we have demonstrated that the GAG chain is clearly modified; it consists of 20 +/- 5 disaccharide units vs. 14 +/- 3 for reference Bk originating from healthy donors. Among them, only 3 +/- 2.5 units are 4-sulphated for patient's Bk vs. 5 +/- 1.5 for reference Bk. Therefore, the non-sulphated region of the GAG chain, which is located towards its non-reducing end, where the heavy chains are positioned, is lengthened from 9 for reference Bk to 17 disaccharide units. We suggest that the biological effects of Bk-proteins may hereby be modulated during inflammatory diseases.

Urinary excretion of N-acetyl-beta-D-glucosaminidase and alpha1-microglobulin in children with proliferative blood diseases.

The purpose of the study was to estimate the urinary excretion of NAG and alpha-1M among children who suffer from proliferative blood diseases. The group of the examined children included those who went through a viral hepatitis (VH) and who are or were treated by means of cytostatic drugs. The study comprised 73 children aged from 4 to 18 (average 11.7+/-3.5. There were 70 children with the diagnosis of leukemia and 3 with the diagnosis of non-Hodgkin lymphoma. The examined group was divided according to the stage of treatment of a basic disease. Group I--22 children who are treated currently or whose treatment has been completed recently. Group II--51 children whose treatment was completed over two years ago. In group II there were 4 subgroups distinguished depending on positive antigenemia HBs and the presence of HCV antibodies. There were no clinical or biochemical features of damage of renal function observed among any of the children. The testing group consisted of 70 healthy children who were selected regarding age and sex. The urinary excretion of NAG and alpha-1M was estimated in the second morning portion of urine and it was presented as NAG/creatinine and alpha-1M/creatinine ratio. The results of the research underwent the statistical analysis by means of a t-Student test. It was stated that the urinary excretion of NAG and alpha-1M was higher among children who currently are or were treated by means of cytostatics drugs. It was also stated that the urinary excretion of NAG was higher among the children who went through viral hepatitis C in comparison with HBs antigen carriers. Similarly, the urinary excretion of alpha-1M was higher among children with positive markers of viral hepatitis B and C markers in comparison with a group of HBs antigen carriers.

Disposition characteristics of protein drugs in the perfused rat kidney.

The renal disposition characteristics of 111In-labeled neocarzinostatin (NCS), soybean trypsin inhibitor (STI), and superoxide dismutase (SOD) were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery profiles and tissue accumulation of proteins were determined under filtering or nonfiltering conditions. In the nonfiltering kidney perfusion experiment, no significant tissue accumulation was observed, suggesting minimal uptake from the glomerular and peritubular capillary sides. Therefore, tissue recovery corresponded to that with tubular reabsorption after glomerular filtration. The total amount of NCS or STI being filtrated through glomeruli, the sum of tissue and urinary recoveries, was similar to that of inulin, but that of SOD was about half. Similarly, the steady-state distribution volumes (Vd) of NCS and STI obtained by moment analysis of their venous outflow curves were similar to that of inulin, while the Vd value of SOD was significantly lower. These results suggest the restricted passage of SOD through the glomerular and postglomerular capillary wall. The tubular reabsorption ratio of proteins against the total filtrated amount decreased with an increase in the administered dose, suggesting nonlinearity of reabsorption. SOD had the largest reabsorption ratio. Thus, this experimental system is useful for quantitative analysis of renal disposition of proteins.