Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.

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.

Limited-sampling strategy models for estimating the pharmacokinetic parameters of 4-methylaminoantipyrine, an active metabolite of dipyrone

Bioanalytical data from a bioequivalence study were used to develop limited-sampling strategy (LSS) models for estimating the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration (Cmax) of 4-methylaminoantipyrine (MAA), an active metabolite of dipyrone. Twelve healthy adult male volunteers received single 600 mg oral doses of dipyrone in two formulations at a 7-day interval in a randomized, crossover protocol. Plasma concentrations of MAA (N = 336), measured by HPLC, were used to develop LSS models. Linear regression analysis and a "jack-knife" validation procedure revealed that the AUC0-¥ and the Cmax of MAA can be accurately predicted (R²>0.95, bias <1.5%, precision between 3.1 and 8.3%) by LSS models based on two sampling times. Validation tests indicate that the most informative 2-point LSS models developed for one formulation provide good estimates (R²>0.85) of the AUC0-¥ or Cmax for the other formulation. LSS models based on three sampling points (1.5, 4 and 24 h), but using different coefficients for AUC0-¥ and Cmax, predicted the individual values of both parameters for the enrolled volunteers (R²>0.88, bias = -0.65 and -0.37%, precision = 4.3 and 7.4%) as well as for plasma concentration data sets generated by simulation (R²>0.88, bias = -1.9 and 8.5%, precision = 5.2 and 8.7%). Bioequivalence assessment of the dipyrone formulations based on the 90% confidence interval of log-transformed AUC0-¥ and Cmax provided similar results when either the best-estimated or the LSS-derived metrics were used.

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.

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.

Fecal estradiol and progesterone metabolite levels in the three-toed sloth (Bradypus variegatus)

The present study was carried out to assess the possibility of measuring fecal steroid hormone metabolites as a noninvasive technique for monitoring reproductive function in the three-toed sloth, Bradypus variegatus. Levels of the estradiol (E2) and progesterone (P4) metabolites were measured by radioimmunoassay in fecal samples collected over 12 weeks from 4 captive female B. variegatus sloths. The validation of the radioimmunoassay for evaluation of fecal steroid metabolites was carried out by collecting 10 blood samples on the same day as defecation. There was a significant direct correlation between the plasma and fecal E2 and P4 levels (P < 0.05, Pearson's test), thereby validating this noninvasive technique for the study of the estrous cycle in these animals. Ovulation was detected in two sloths (SL03 and SL04) whose E2 levels reached 2237.43 and 6713.26 pg/g wet feces weight, respectively, for over four weeks, followed by an increase in P4 metabolites reaching 33.54 and 3242.68 ng/g wet feces weight, respectively. Interestingly, SL04, which presented higher levels of E2 and P4 metabolites, later gave birth to a healthy baby sloth. The results obtained indicate that this is a reliable technique for recording gonadal steroid secretion and thereby reproduction in sloths.

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.

Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney, and evidence suggests that gender-related hormones participate in renal function. The aim of this study was to analyze the relationship of GAGs, gender, and proteinuria in male and female rats with chronic renal failure (CRF). GAGs were analyzed in total kidney tissue and 24-h urine of castrated (c), male (M), and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days of CRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAG quantification and composition were determined using agarose and polyacrylamide gel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM. CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria, while castration reduced these parameters. However, CRFF and CRFFc groups showed a decrease in renal GAGs concomitant with an increase in proteinuria. Our results suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainly HS, and possibly the glomerular filtration barrier, leading to proteinuria. The lack of this response in CRFMc, where HS did not increase, corroborates this theory. This pattern was not observed in females. Further studies of CRF are needed to clarify gender-dependent differences in HS synthesis.

Purple urine bag syndrome in end-stage chronic kidney disease

Introduction: When faced with violet, purple or purplish-blue urine, clinicians should consider urinary tract infection in their differential diagnosis. Case report: A 60-year-old woman with end-stage kidney disease and non-adherence to renal replacement therapy was admitted to our hospital for placement of hemodialysis catheter. During her hospitalization she had purple urine, and purple urine bag syndrome (PUBS) was diagnosed. She was effectively treated with antibiotics and her urine returned to a dark yellow color. Discussion: Although this condition is often easily treated, diagnosing PUBS in chronic renal patients probably means an increased serum concentration of indoxyl sulfate, metabolite that is involved in the progression of both CKD and cardiovascular disease. Conclusion: Hence, in the context of our renal patients, perhaps PUBS is not as benign as supposed.