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PubMed1986

Association of BK viruria with hemorrhagic cystitis in recipients of bone marrow transplants.

Fifty-three recipients of bone marrow transplants were monitored prospectively for urinary excretion of human polyomaviruses by enzyme-linked immunosorbent assays of urinary supernatants and DNA hybridization assays of urinary cells. Excretion of BK virus was demonstrated in 47 percent of the transplant recipients and was the result of the reactivation of latent virus. Hemorrhagic cystitis of long duration (greater than or equal to 7 days) was associated with BK viruria. The disease occurred four times more frequently in patients who excreted BK virus than in those who did not, and the virus was identified in 55 percent of the urine specimens during episodes of cystitis as compared with 8 to 11 percent of the specimens during cystitis-free periods. BK viruria often preceded or coincided with the onset of the disease. Among 19 patients with BK viruria lasting seven days or longer, hemorrhagic cystitis occurred in 15. Occurrence of the disease was related to the source of marrow. The disease occurred in 50 percent of 38 recipients of allogeneic marrow and in 7 percent of 15 recipients of syngeneic or autologous marrow. Among recipients of allogeneic marrow, the disease was observed in 71 percent of the 21 patients excreting BK virus and in 24 percent of the 17 not excreting the virus. An association of BK virus with hemorrhagic cystitis was demonstrated in 16 of the 18 cases of the disease that were adequately characterized. We conclude that reactivation of BK virus may account for a substantial proportion of late-onset, long-lasting hemorrhagic cystitis in recipients of bone marrow transplants.

BK Virus↗

PubMed2025

Absolute Quantification of Cellular and Cell-Free Mitochondrial DNA Copy Number from Human Blood and Urinary Samples Using Real Time Quantitative PCR.

Mitochondrial DNA copy number (mtDNA-CN) in human body fluids is widely used as a biomarker of mitochondrial dysfunction in common metabolic diseases. Here we describe protocols to measure cellular and/or cell free (cf)-mtDNA-CN in human peripheral blood and urine. Cellular mtDNA is located inside the mitochondria where it encodes key subunits of the respiratory complexes in mitochondria and is usually normalized with reference to the nuclear genome as the mitochondrial genome to nuclear genome ratio (Mt/N) in either whole blood, peripheral blood mononuclear cells (PBMCs), or whole urine. Cf -mtDNA is usually found outside of the mitochondria, often released following mitochondrial damage, can trigger inflammatory pathways, and is usually measured as mtDNA-CN per volume of the starting material. Here we describe how to (1) separate whole blood into PBMCs, plasma, and serum fractions and whole urine into urinary supernatant and pellet, (2) prepare DNA from each of these fractions, (3) prepare reference standards for absolute quantification, (4) carry out qPCR for either relative or absolute quantification from test samples, (5) analyze qPCR data, and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high throughput use and can be modified to quantify mtDNA from other body fluids, human cells, and tissues.

Humans↗

PubMed1992

Partition of free dolichol in human urine.

We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol.

Adult↗

PubMed1991

The diagnostic significance of lactate dehydrogenase isoenzymes in urinary cytology.

Lactate dehydrogenase (LDH) isoenzyme distribution was examined in 106 urine samples being tested cytologically for evidence of bladder cancer; the samples were selected to have less than 20 leucocytes and erythrocytes per high power field and the LDH pattern determined by electrophoresis. The Papanicolaou stained-smears showed 68 negative, 17 suspicious and 21 positive. The LDH M-fraction of the urinary supernatant in cytologically positive cases was significantly greater than in negative cases, although the latter included a few false negative samples. Some of the false negatives gave positive results for the LDH M-fraction; these results suggest that the determination of LDH isoenzymes in the urine is useful in diagnosing urinary tract cancers, including early stage, and for follow-up of patients with bladder cancers after surgical resection.

Carcinoma, Transitional Cell↗

PubMed1983

Crystal formation induced by uropathogenic bacteria. An in vitro study in human urine.

Human urine was studied in regard to precipitation of crystals under various experimental conditions. These included alkalization of the urine by adding NaOH or by inoculation with Proteus bacteria. The contents of calcium and magnesium in the urinary supernatant after incubation for 24 hours at 37 degrees C were significantly lower after inoculation with Proteus bacteria than in noninoculated urine. Microscopy of the sediments showed distinct crystalline aggregations after the bacterial inoculation. Inoculation with killed Proteus organisms did not lead to crystalline precipitation in otherwise identical experiments. The effect of other live bacteria was also studied. The mechanism of crystal formation in bacteria-inoculated urine is discussed.

Adolescent↗

PubMed1994

[Urinary tests for inflammation in glomerulonephritis].

Boyden camera tests, chemiluminescence of urinary supernatant, passive hemagglutination tests were used in measuring chemotactic properties of the urine, intensity of free-radical processes, fibronectin urine concentrations, respectively, in 31 glomerulonephritis (GN) patients. The above parameters were closely related to nephritis activity. These were much lower in patients with nephritis remission, than in subjects with nephrotic syndrome or exacerbation of latent nephritis. Chemotactic activity and intensity of free-radical processes were unrelated to protein urea which, however, correlated with fibronectin concentrations (r = 0.35, p < 0.05). The authors discuss the role of the mediators' values in evaluation of local renal inflammation and feasibility of their use as criteria of nephritis activity and prognosis.

Adolescent↗

PubMed1987

Evaluation of gamma-glutamyl transpeptidase-to-creatinine ratio from spot samples of urine supernatant, as an indicator of urinary enzyme excretion in dogs.

gamma-Glutamyl transpeptidase activity was measured accurately in canine urine supernatant without gel filtration and was relatively stable at 4 C for at least 4 days after collection. The urinary gamma-glutamyl transpeptidase-to-creatinine ratio in spot samples was simple and quick to measure and was correlated with the 24-hour enzyme excretion. However, the usefulness of this ratio may be limited by within-day variation, and a questionable theoretical basis for its validity.

Animals↗

PubMed1982

Studies on protein binding of antibiotics. IV. Effect of the binding of drug to 100,000 X g supernatant fluid of rabbit liver homogenates on urinary excretion.

The extent of the binding of beta-lactam antibiotics to 100,000 X g supernatant fluid of rabbit liver homogenates was investigated. Urinary excretion tended to decrease as the extent of the binding to 100,000 X g supernatant fluid increased. Predicting urinary excretion was achieved using the binding to 100,000 X g supernatant fluid and other physicochemical factors. A good coincidence resulted from multiple regression analysis. [14C]Benzylpenicillin binding proteins in rabbit liver were investigated and revealed that the affinity of cefoperazone was much stronger than that of cefazolin.

Animals↗

PubMed1989

Urinary neopterin as a new biochemical marker for the monitoring of disease activity and prognosis in membranous nephropathy associated with hepatitis B surface antigenemia.

In an attempt to find a parameter for the differentiation of membranous nephropathy with hepatitis B surface antigenemia (HBVMN) and other types of nephrotic syndrome (NS) in HBsAg (+) carriers, we measured the urinary neopterin in 20 HBVMN patients, other types of NS in HBsAg (+) carriers, asymptomatic HBsAg (+) carriers and HBsAg (-) controls, during the active nephrotic phase and in remission. The urinary neopterin excretion was found to be significantly elevated only in the HBVMN during NS or heavy proteinuria. It progressively decreased and corresponded with the amount of proteinuria. In patients with deteriorated renal function, initially, urinary neopterin excretion was significantly higher than in others and it remained significantly elevated during the follow-up period. In vitro when a patient's mononuclear cells were stimulated with lymphoblastoid cell line, the release of neopterin content in supernatant was increased during the acute phase. There was a significant correlation between supernatant and urinary neopterin level. These results suggest that urinary neopterin may be a new biochemical marker for the differential diagnosis of HBVMN and may be used to monitor the course of HBVMN and serve as a prognostic indicator. However, the specificity is not clear.

Biomarkers↗

PubMed2006

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery.

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.

Biomarkers↗

PubMed1982

Studies on protein binding of antibiotics. V. Effect of the binding of drug to 100,000 X g supernatant fluid of human liver homogenates on urinary excretion.

To characterize the effect of the binding of antibiotics to tissue protein on urinary excretion, we examined the extent of the binding of 9 penicillins and 9 cephems to 100,000 X g supernatant fluid of human liver homogenates. The correlation analysis revealed a significant simple correlation between the binding to 100,000 X g supernatant fluid of liver homogenates and urinary excretion. Drugs with lesser binding were mainly excreted in the urine, conversely, urinary excretion of highly bound drugs exhibited lower values. The prediction with regard to urinary excretion of healthy subjects was done by multiple regression analysis using the binding to 100,000 X g supernatant fluid and other physicochemical factors. The values predicted for urinary excretion coincided well with the observed values.

Anti-Bacterial Agents↗

PubMed2002

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes.

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

Animals↗

PubMed1994

Determination of interleukin 6 in human urine and epithelial cell supernatants.

Interleukin 6 (IL-6) is a cytokine with many biological functions. It is produced by different tissues in response to infection and is secreted into the local body fluids. The aim of this study was to find a suitable assay to measure IL-6 in human urine. IL-6 was quantitated by a bioassay and by immunoassays based on neutralizing or nonneutralizing antibodies. The effect of human urine on the quantitation of IL-6 by these assays was analyzed using pooled human urine with added recombinant or natural human IL-6. Urine was found to disturb the growth of the B9 cells. When fractions from gel-filtered human urine were tested, a fraction corresponding to a protein molecular weight range of 10,000-1,000 was found to have a strong inhibitory effect in the B9 assay. In contrast, the low molecular weight fractions containing salts and pigments were not found to disturb the assay. The inhibitory effect of urine was avoided by diluting the samples > 80 times (final dilution in the test plate) or by dialysis. Furthermore, we analyzed IL-6 in urine samples from patients with urinary tract infection and supernatants from epithelial cells stimulated with bacteria in vitro. The B9 assay and the immunoassay based on non-neutralizing anti-IL-6 antibodies were more sensitive than the immunoassays based on neutralizing antibodies. While most of the B9 activity in the urine samples and supernatants could be neutralized by anti-IL-6 antibodies, some samples contained unneutralizable activity. These components remain to be defined. The results demonstrate considerable variation between assays used to quantitate natural IL-6.

B-Lymphocytes↗

PubMed1987

Chemical characterization of urinary glycopeptides.

We prepared human urinary glycopeptides from the supernatant liquid remaining after precipitation of the nondialyzable fraction with cetylpyridinium chloride. Using cation exchange and affinity chromatographies and gel filtration, we obtained 28 glycopeptide subfractions. By compositional analyses of sugar and amino acid, and by reducing-terminal analyses after reduction with NaBH4, we determined the size of the carbohydrate moiety and the types of carbohydrate-peptide linkage involved. We isolated several glycopeptides not previously described: six with sialic acid, two with fucose, two with glucose, and one with N-acetylgalactosamine. The sialic acid glycopeptides had a short carbohydrate chain of the O-glycoside type. The fucose-containing glycopeptides were fucosyllactosaminoglycans. The glucose glycopeptides were polymers linked to a small peptide moiety. The N-acetylgalactosamine-rich glycopeptide was found in an N-glycoside-type fraction, with N-acetylgalactosamine at the nonreducing terminal.

Acetylglucosamine↗

PubMed1993

Impaired cytokine production in whole blood cell cultures of patients with urological carcinomas.

The production of the cytokines interferon gamma (IFN gamma), interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha) was investigated in the mitogen-stimulated whole blood cell culture media from 51 patients with urinary bladder carcinomas, 52 patients with renal carcinomas, 31 patients with prostatic carcinomas and 360 healthy controls. The cytokines were measured 4 days after induction by a sensitive enzymo-immunological assay. In the blood cell culture supernatants of the patients with urinary bladder carcinomas significant lower levels of IFN gamma (P < or = 0.001), IL-2 (P < or = 0.01) and TNF alpha (P < or = 0.05) were found as compared to the controls. Blood cells of patients with renal carcinomas had lower production of IFN gamma (P < or = 0.01), IL-2 (P < or = 0.001) and IL-1 alpha (P < or = 0.01), whereas the values of the total group of patients with prostatic carcinomas were not significantly different from those of the controls. Lymphocyte and monocyte counts were almost identical in the control and all tumor patient groups. When the patients with renal carcinomas and prostatic carcinomas were analyzed according to their different clinical stages we could show a gradual depression of the IFN-gamma levels, which was related to tumor burden.

Aged↗

PubMed1997

Clofibrate feeding to Sprague-Dawley rats increases endogenous biosynthesis of oxalate and causes hyperoxaluria.

The effects of clofibrate feeding (5 g/kg diet) on oxalate metabolism were investigated in male and female rats. Following clofibrate feeding, 24-hour urinary excretion of oxalate increased until 4 days and then reached a plateau. Whereas the contribution of dietary oxalate (1.4 g/kg diet, as potassium salt) to urinary oxalate was less than 5% in both control and clofibrate-treated male rats, the contribution of dietary glycolate (1.0 g/kg diet, as sodium salt) to urinary oxalate was six times higher in clofibrate-treated male rats compared with controls, indicating that the clofibrate-induced hyperoxaluria is due to increased endogenous biosynthesis of oxalate. This was supported by the increased lactate dehydrogenase (LDH) activity observed in liver supernatants of clofibrate-treated rats compared with controls, and the increased rate of conversion of glycolate and glyoxylate to oxalate by clofibrate-treated male rat liver supernatants. Female rats had lower excretion of urinary oxalate and lower levels of liver glycolic acid oxidase (GAO) as compared with males. Clofibrate-treated female rat liver supernatants had higher LDH levels and produced more oxalate from glyoxylate. Thus, it can be concluded that the increase in LDH activity may be the cause of the increased endogenous biosynthesis of oxalate leading to increased urinary excretion of oxalate in male and female rats treated with clofibrate.

Alcohol Oxidoreductases↗

PubMed1995

[The effect of peripheral blood mononuclear cell products from children with nephrotic syndrome on thromboxane A2 metabolism].

To confirm that the soluble factors produced by peripheral blood mononuclear cells (PBMC) from children with minimal change nephrotic syndrome (MCNS) enhance thromboxane (TX) A2 synthesis, we studied the urinary albumin and TXA2 metabolite excretions of rats intravenously injected with PBMC culture supernatant (PCS). Furthermore, we studied which does mainly effect on urinary albumin excretion, TXA2 metabolism in kidney or in platelet. In this study, the urinary albumin, TXB2, and 11-dehydro-TXB2 excretions of rats injected with PCS from MCNS children were remarkably increased, but this change was not observed in rats injected with PCS from children with MCNS in remission, glomerulonephritis, or healthy controls. The urinary albumin excretion of rats administered with acetylsalitylic acid before injection of PCS from MCNS children did not increase. We concluded that the soluble factors produced by PBMC from MCNS children increase urinary albumin excretion, and that the TXA2 metabolism is closely related to this effect.

Albuminuria↗

PubMed2001

Augmented stretch activated adenosine triphosphate release from bladder uroepithelial cells in patients with interstitial cystitis.

PURPOSE: Extracellular adenosine triphosphate (ATP) has been shown to mediate inflammation and nociception and, therefore, it may have a role in symptoms associated with interstitial cystitis. We theorized that the bladder uroepithelium releases ATP in response to stretch and, furthermore, this process is augmented in interstitial cystitis. MATERIALS AND METHODS: We quantitated ATP using the luciferin-luciferase assay. Urinary ATP levels were compared in 35 patients with interstitial cystitis and in 33 normal controls after pH correction. Cultured interstitial cystitis and normal urothelial cells from the bladder biopsies of 5 patients each were stretched with the Flexcell 2000 machine (Flexcell International Corp., McKeesport, Pennsylvania) and supernatant ATP concentrations were measured. RESULTS: Mean urinary ATP plus or minus standard error of mean was significantly higher in patients with interstitial cystitis than in controls (L value 985 +/- 161 versus 377 +/- 27, p = 0.0007). Supernatant ATP released by stretched interstitial cystitis cells was stretch intensity dependent when comparing 0%, 10% and 20% elongation, and was also significantly higher in stretched interstitial cystitis than in stretched normal cells. CONCLUSIONS: Adenosine triphosphate was significantly elevated in the urine of individuals with interstitial cystitis and the stretch activated release of ATP was augmented in interstitial cystitis urothelium. Increased extracellular ATP may have a role in mechanosensory transduction and to our knowledge it represents a novel hypothesis.

Adenosine Triphosphate↗