Astroglial cells derived from lateral and medial midbrain sectors differ in their synthesis and secretion of sulfated glycosaminoglycans

Astroglial cells derived from lateral and medial midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in cocultures. These different properties of the two types of cells may be related to the composition of their extracellular matrix. We have studied the synthesis and secretion of sulfated glycosaminoglycans (GAGs) by the two cell types under control conditions and ß-D-xyloside-stimulated conditions, that stimulate the ability to synthesize and release GAGs. We have confirmed that both cell types synthesize and secrete heparan sulfate and chondroitin sulfate. Only slight differences were observed between the proportions of the two GAGs produced by the two types of cells after a 24-h labeling period. However, a marked difference was observed between the GAGs produced by the astroglial cells derived from lateral and medial midbrain sectors. The medial cells, which contain derivatives of the tectal and tegmental midline radial glia, synthesized and secreted ~2.3 times more chondroitin sulfate than lateral cells. The synthesis of heparan sulfate was only slightly modified by the addition of ß-D-xyloside. Overall, these results indicate that astroglial cells derived from the two midbrain sectors have marked differences in their capacity to synthesize chondroitin sulfate. Under in vivo conditions or a long period of in vitro culture, they may produce extracellular matrix at concentrations which may differentially affect neuritic growth.

Scintigraphy and Doppler ultrasonography for the evaluation of obstructive urinary calculi

Forty-seven patients with unilateral obstructive calculi (12 males and 35 females) were submitted to 99mTc-diethylene triamine pentaacetic acid (DTPA) or 99mTc-dimercaptosuccinic acid (DMSA) scans for assessment of renal function. The scans revealed unilateral functional deficit in 68 and 66% of the patients, respectively. A calculus size of 1.1 to 2.0 cm was significantly associated with deficit detected by DTPA, but duration of obstruction and calculus localization were not. After relief of the obstruction, the mean percent renal function of the affected kidney was found to be significantly increased from 25 ± 12% to 29 ± 12% in DTPA and from 21 ± 15% to 24 ± 12% in DMSA. Initial Doppler ultrasonography performed in 35 patients detected an increased resistive index in 10 (29%). In the remaining patients with a normal resistive index, ureteral urinary jet was observed, indicating partial obstruction. The high frequency of renal function impairment detected by DTPA and of tubulointerstitial damage detected by DMSA as well as the slight amelioration of unilateral renal function after relief of obstruction suggest that scintigraphy assessment may help evaluate the unilateral percentage of renal function and monitor renal function recovery when it occurs. The presence of a urinary jet detected by Doppler ultrasonography further indicates the severity of obstruction and the recovery prognosis.

Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term "family" is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.

Comparison of methods for urinary albumin determination in patients with type 1 diabetes

We tested the correlation of the albumin-to-creatinine ratio (A/C) in an early-morning urine sample, measured with a commercial kit (DCA 2000®), with the conventional immunoturbidimetric determination in the laboratory and with overnight albumin excretion rate (reference method). Fifty-five type 1 diabetic adolescents had their first-morning urine collected on the 1st and 8th day of the period. Urinary albumin and creatinine were determined immediately using the DCA 2000® kit. Samples were also stored for laboratory analysis. To evaluate the correlation between early-morning urinary A/C ratio and overnight albumin excretion rate, 16 subjects had a timed overnight urine collection. A/C ratios determined with the DCA 2000® kit and by the laboratory method were 13.1 ± 20.5 and 20.4 ± 46.3 mg/g, respectively. A/C results by both methods proved to be strongly correlated (r = 0.98, P<0.001). DCA 2000®-determined A/C showed 50% sensitivity and 100% specificity when compared to the reference method. Spot urinary A/C of the subset of 16 subjects significantly correlated with their overnight albumin excretion rate (r = 0.98, P<0.001). Intraindividual variation ranged from 17 to 32% and from 9 to 63% for A/C and overnight albumin excretion rate, respectively. In conclusion, an early-morning specimen should be used instead of timed overnight urine and the A/C ratio is an accurate, reliable and easily determined parameter for the screening of diabetic nephropathy. Immediate measurement of the A/C ratio is feasible using the DCA 2000® kit. Intraindividual variability indicates the need for repeated determinations to confirm microalbuminuria and the diagnosis of incipient diabetic nephropathy.

Effect of calcium intake on urinary oxalate excretion in calcium stone-forming patients

Dietary calcium lowers the risk of nephrolithiasis due to a decreased absorption of dietary oxalate that is bound by intestinal calcium. The aim of the present study was to evaluate oxaluria in normocalciuric and hypercalciuric lithiasic patients under different calcium intake. Fifty patients (26 females and 24 males, 41 ± 10 years old), whose 4-day dietary records revealed a regular low calcium intake (<=500 mg/day), received an oral calcium load (1 g/day) for 7 days. A 24-h urine was obtained before and after load and according to the calciuria under both diets, patients were considered as normocalciuric (NC, N = 15), diet-dependent hypercalciuric (DDHC, N = 9) or diet-independent hypercalciuric (DIHC, N = 26). On regular diet, mean oxaluria was 30 ± 14 mg/24 h for all patients. The 7-day calcium load induced a significant decrease in mean oxaluria compared to the regular diet in NC and DIHC (20 ± 12 vs 26 ± 7 and 27 ± 18 vs 32 ± 15 mg/24 h, respectively, P<0.05) but not in DDHC patients (22 ± 10 vs 23 ± 5 mg/24 h). The lack of an oxalate decrease among DDHC patients after the calcium load might have been due to higher calcium absorption under higher calcium supply, with a consequent lower amount of calcium left in the intestine to bind with oxalate. These data suggest that a long-lasting regular calcium consumption <500 mg was not associated with high oxaluria and that a subpopulation of hypercalciuric patients who presented a higher intestinal calcium absorption (DDHC) tended to hyperabsorb oxalate as well, so that oxaluria did not change under different calcium intake.

Urinary iron excretion induced by intravenous infusion of deferoxamine in ß-thalassemia homozygous patients

The purpose of the present study was to identify noninvasive methods to evaluate the severity of iron overload in transfusion-dependent ß-thalassemia and the efficiency of intensive intravenous therapy as an additional tool for the treatment of iron-overloaded patients. Iron overload was evaluated for 26 ß-thalassemia homozygous patients, and 14 of them were submitted to intensive chelation therapy with high doses of intravenous deferoxamine (DF). Patients were classified into six groups of increasing clinical severity and were divided into compliant and non-compliant patients depending on their adherence to chronic chelation treatment. Several methods were used as indicators of iron overload. Total gain of transfusion iron, plasma ferritin, and urinary iron excretion in response to 20 to 60 mg/day subcutaneous DF for 8 to 12 h daily are useful to identify iron overload; however, urinary iron excretion in response to 9 g intravenous DF over 24 h and the increase of urinary iron excretion induced by high doses of the chelator are more reliable to identify different degrees of iron overload because of their correlation with the clinical grades of secondary hemochromatosis and the significant differences observed between the groups of compliant and non-compliant patients. Finally, the use of 3-9 g intravenous DF for 6-12 days led to a urinary iron excretion corresponding to 4.1 to 22.4% of the annual transfusion iron gain. Therefore, continuous intravenous DF at high doses may be an additional treatment for these patients, as a complement to the regular subcutaneous infusion at home, but requires individual planning and close monitoring of adverse reactions.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 µg/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 µM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 µM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 µM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Effect of intracerebroventricularly injected insulin on urinary sodium excretion by cerebroventricular streptozotocin-treated rats

Recent evidence suggests that insulin may influence many brain functions. It is known that intracerebroventricular (icv) injection of nondiabetogenic doses of streptozotocin (STZ) can damage insulin receptor signal transduction. In the present study, we examined the functional damage to the brain insulin receptors on central mechanisms regulating glomerular filtration rate and urinary sodium excretion, over four periods of 30 min, in response to 3 µl insulin or 0.15 NaCl (vehicle) injected icv in STZ-treated freely moving Wistar-Hannover rats (250-300 g). The icv cannula site was visually confirmed by 2% Evans blue infusion. Centrally administered insulin (42.0 ng/µl) increased the urinary output of sodium (from 855.6 ± 85.1 to 2055 ± 310.6 delta%/min; N = 11) and potassium (from 460.4 ± 100 to 669 ± 60.8 delta%/min; N = 11). The urinary sodium excretion response to icv insulin microinjection was markedly attenuated by previous central STZ (100 µg/3 µl) administration (from 628 ± 45.8 to 617 ± 87.6 delta%/min; N = 5) or by icv injection of a dopamine antagonist, haloperidol (4 µg/3 µl) (from 498 ± 39.4 to 517 ± 73.2 delta%/min; N = 5). Additionally, insulin-induced natriuresis occurred by increased post-proximal tubule sodium rejection, despite an unchanged glomerular filtration rate. Excluding the possibility of a direct action of STZ on central insulin receptor-carrying neurons, the current data suggest that the insulin-sensitive response may be processed through dopaminergic D1 receptors containing neuronal pathways.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Urinary NO3 excretion and renal failure in indinavir-treated patients

Treatment with indinavir (IDV), a protease inhibitor, is frequently associated with renal abnormalities. We determined the incidence of renal failure (creatinine clearance <80 mL min-1 1.73 (m²)-1) in HIV patients treated with highly active antiretroviral therapy, including IDV, and investigated the possible mechanisms and risk factors of IDV nephrotoxicity. Thirty-six patients receiving IDV were followed for 3 years. All were assessed for age, body weight, duration of infection, duration of IDV treatment, sulfur-derivative use, total cholesterol, triglycerides, magnesium, sodium, potassium, creatinine, and urinalysis. We also determined renal function in terms of creatinine clearance, urine osmolality and fractional excretion of sodium, potassium, and water. Urinary nitrate (NO3) excretion was measured in 18 IDV-treated patients and compared with that of 8 patients treated with efavirenz, a drug without renal side effects. Sterile leukocyturia occurred in 80.5% of the IDV-treated patients. Creatinine clearance <80 mL min-1 1.73 (m²)-1 was observed in 22 patients (61%) and was associated with low body weight and the use of sulfur-derivatives. These patients also had lower osmolality, lower urine volume and a higher fractional excretion of water compared to the normal renal function group. Urinary NO3 excretion was significantly lower in IDV-treated patients (809 ± 181 µM NO3-/mg creatinine) than in efavirenz-treated patients (2247 ± 648 µM NO3-/mg creatinine, P < 0.01). The lower NO3 excretion suggests that IDV decreases nitric oxide production.

Determination of renal function in long-term heart transplant patients by measurement of urinary retinol-binding protein levels

Significant improvements have been noted in heart transplantation with the advent of cyclosporine. However, cyclosporine use is associated with significant side effects, such as chronic renal failure. We were interested in evaluating the incidence of long-term renal dysfunction in heart transplant recipients. Fifty-three heart transplant recipients were enrolled in the study. Forty-three patients completed the entire evaluation and follow-up. Glomerular (serum creatinine, creatinine clearance measured, and creatinine clearance calculated) and tubular functions (urinary retinol-binding protein, uRBP) were re-analyzed after 18 months. At the enrollment time, the prevalence of renal failure ranged from 37.7 to 54% according to criteria used to define it (serum creatinine > or = 1.5 mg/dL and creatinine clearance <60 mL/min). Mean serum creatinine was 1.61 ± 1.31 mg/dL (range 0.7 to 9.8 mg/dL) and calculated and measured creatinine clearances were 67.7 ± 25.9 and 61.18 ± 25.04 mL min-1 (1.73 m²)-1, respectively. Sixteen of the 43 patients who completed the follow-up (37.2%) had tubular dysfunction detected by increased levels of uRBP (median 1.06, 0.412-6.396 mg/dL). Eleven of the 16 patients (68.7%) with elevated uRBP had poorer renal function after 18 months of follow-up, compared with only eight of the 27 patients (29.6%) with normal uRBP (RR = 3.47, P = 0.0095). Interestingly, cyclosporine trough levels were not different between patients with or without tubular and glomerular dysfunction. Renal function impairment is common after heart transplantation. Tubular dysfunction, assessed by uRBP, correlates with a worsening of glomerular filtration and can be a useful tool for early detection of renal dysfunction.

Multiple factor analysis of metachronous upper urinary tract transitional cell carcinoma after radical cystectomy

Transitional cell carcinoma (TCC) of the urothelium is often multifocal and subsequent tumors may occur anywhere in the urinary tract after the treatment of a primary carcinoma. Patients initially presenting a bladder cancer are at significant risk of developing metachronous tumors in the upper urinary tract (UUT). We evaluated the prognostic factors of primary invasive bladder cancer that may predict a metachronous UUT TCC after radical cystectomy. The records of 476 patients who underwent radical cystectomy for primary invasive bladder TCC from 1989 to 2001 were reviewed retrospectively. The prognostic factors of UUT TCC were determined by multivariate analysis using the COX proportional hazards regression model. Kaplan-Meier analysis was also used to assess the variable incidence of UUT TCC according to different risk factors. Twenty-two patients (4.6%). developed metachronous UUT TCC. Multiplicity, prostatic urethral involvement by the bladder cancer and the associated carcinoma in situ (CIS) were significant and independent factors affecting the occurrence of metachronous UUT TCC (P = 0.0425, 0.0082, and 0.0006, respectively). These results were supported, to some extent, by analysis of the UUT TCC disease-free rate by the Kaplan-Meier method, whereby patients with prostatic urethral involvement or with associated CIS demonstrated a significantly lower metachronous UUT TCC disease-free rate than patients without prostatic urethral involvement or without associated CIS (log-rank test, P = 0.0116 and 0.0075, respectively). Multiple tumors, prostatic urethral involvement and associated CIS were risk factors for metachronous UUT TCC, a conclusion that may be useful for designing follow-up strategies for primary invasive bladder cancer after radical cystectomy.

Is there an association between T102C polymorphism of the serotonin receptor 2A gene and urinary incontinence?

The regulation of bladder function is influenced by central serotonergic modulation. Several genetic polymorphisms related to serotonin control have been described in the literature. T102C polymorphism of the serotonin receptor 2A gene (5-HT2A) has been shown to be associated with certain diseases such as non-fatal acute myocardial infarction, essential hypertension, and alcoholism. In the present study, we examined the association between 5-HT2A gene polymorphism and urinary incontinence in the elderly. A case-control study was performed in 298 elderly community dwellers enrolled in the Gravataí-GENESIS Project, Brazil, which studies gene-environmental interactions in aging and age-related diseases. Clinical, physical, biochemical, and molecular analyses were performed on volunteers. 5-HT2A genotyping was determined by PCR-RFLP techniques using the HpaII restriction enzyme. The subjects had a mean age of 68.05 ± 6.35 years (60-100 years), with 16.9% males and 83.1% females. The C allele frequency was 0.494 and the T allele frequency was 0.506. The CC genotype frequency was 21.78%, the CT genotype frequency was 55.24% and the TT genotype frequency was 22.98%. We found an independent significant association between the TT genotype (35.7%) and urinary incontinence (OR = 2.06, 95%CI = 1.16-3.65). Additionally, urinary incontinence was associated with functional dependence and systolic hypertension. The results suggest a possible genetic influence on urinary incontinence involving the serotonergic pathway. Further investigations including urodynamic evaluation will be performed to better explain our findings.

Association of urinary 90 kDa angiotensin- converting enzyme with family history of hypertension and endothelial function in normotensive individuals

We described angiotensin-I-converting enzyme (ACE) isoforms with molecular masses of 190, 90, and 65 kDa in the urine of normotensive offspring of hypertensive subjects. Since they did not appear in equal amounts, we suggested that 90 kDa ACE might be a marker for hypertension. We evaluated the endothelial response in normotensive offspring with or without family history of hypertension and its association with the 90 kDa ACE in urine. Thirty-five normotensive subjects with a known family history of hypertension and 20 subjects without a family history of hypertension, matched for age, sex, body weight, and blood pressure, were included in the study. Endothelial function was assessed by ultrasound and a sample of urine was collected for determination of ACE isoforms. In the presence of a family history of hypertension and detection of 90 kDa ACE, we noted a maximal flow mediated dilation of 12.1 ± 5.0 vs 16.1 ± 6.0% in those without a previous history of hypertension and lacking urinary 90 kDa ACE (P < 0.05). In subjects with a family history of hypertension and presenting 90 kDa ACE, there were lower levels of HDL-cholesterol (P < 0.05) and higher levels of triglycerides (P < 0.05). Subjects with 90 kDa ACE irrespective of hypertensive history presented a trend for higher levels of triglycerides and HDL-cholesterol (P = 0.06) compared to subjects without 90 kDa ACE. Our data suggest that the 90 kDa ACE may be a marker for hypertension which may be related to the development of early atherosclerotic changes.

Cotransfected human chondrocytes: over-expression of IGF-I and SOX9 enhances the synthesis of cartilage matrix components collagen-II and glycosaminoglycans

Damage to cartilage causes a loss of type II collagen (Col-II) and glycosaminoglycans (GAG). To restore the original cartilage architecture, cell factors that stimulate Col-II and GAG production are needed. Insulin-like growth factor I (IGF-I) and transcription factor SOX9are essential for the synthesis of cartilage matrix, chondrocyte proliferation, and phenotype maintenance. We evaluated the combined effect of IGF-I and SOX9 transgene expression on Col-II and GAG production by cultured human articular chondrocytes. Transient transfection and cotransfection were performed using two mammalian expression plasmids (pCMV-SPORT6), one for each transgene. At day 9 post-transfection, the chondrocytes that were over-expressing IGF-I/SOX9 showed 2-fold increased mRNA expression of the Col-II gene, as well as a 57% increase in Col-II protein, whereas type I collagen expression (Col-I) was decreased by 59.3% compared with controls. The production of GAG by these cells increased significantly compared with the controls at day 9 (3.3- vs 1.8-times, an increase of almost 83%). Thus, IGF-I/SOX9 cotransfected chondrocytes may be useful for cell-based articular cartilage therapies.