In vivo and in vitro Plasmodium falciparum Resistance to Chloroquine, Amodiaquine and Quinine in the Brazilian Amazon

In order to study the chemoresistance of Plasmodium falciparum to commonly used antimalarial drugs in Brazil the authors have studied ten patients with falciparum malaria, acquired in the Brazilian Amazon region. Patients were submitted to in vivo study of drug sensitivity, after chemotherapy with either 4-aminoquinolines (chloroquine or amodiaquine) or quinine. Adequate drug absorption was confirmed by standard urine excretion tests for antimalarials. Eight patients could be followed up to 28 days. Among these in vivo resistance (R I and R II responses) was seen in all patients who received 4-amino-quinolines. One patient treated with quinine exhibited a R III response. Peripheral blood samples of the same patients were submitted to in vitro microtests for sensitivity to antimalarials. Out of nine successful tests, resistance to chloroquine and amodiaquine was found in 100% and resistance to quinine in 11.11% of isolates. Probit analysis of log dose-response was used to determine effective concentrations EC50, EC90 and EC99 to the studied drugs. Good correlation between in vivo and in vitro results was seen in six patients. The results emphasize high levels of P. falciparum resistance to 4- aminoquinolines and suggest an increase in resistance to quinine in the Brazilian Amazon region, reinforcing the need for continuous monitoring of drug sensitivity to adequate chemotherapy according to the most efficacious drug regimens

Adrenoceptors of the medial septal area modulate water intake and renal excretory function induced by central administration of angiotensin II

We investigated the role of a-adrenergic antagonists and clonidine injected into the medial septal area (MSA) on water intake and the decrease in Na+, K+ and urine elicited by ANGII injection into the third ventricle (3rdV). Male Holtzman rats with stainless steel cannulas implanted into the 3rdV and MSA were used. ANGII (12 nmol/µl) increased water intake (12.5 ± 1.7 ml/120 min). Clonidine (20 nmol/µl) injected into the MSA reduced the ANGII-induced water intake (2.9 ± 0.5 ml/120 min). Pretreatment with 80 nmol/µl yohimbine or prazosin into the MSA also reduced the ANGII-induced water intake (3.0 ± 0.4 and 3.1 ± 0.2 ml/120 min, respectively). Yohimbine + prazosin + clonidine injected into the MSA abolished the ANGII-induced water intake (0.2 ± 0.1 and 0.2 ± 0.1 ml/120 min, respectively). ANGII reduced Na+ (23 ± 7 µEq/120 min), K+ (27 ± 3 µEq/120 min) and urine volume (4.3 ± 0.9 ml/120 min). Clonidine increased the parameters above. Clonidine injected into the MSA abolished the inhibitory effect of ANGII on urinary sodium. Yohimbine injected into the MSA also abolished the inhibitory effects of ANGII. Yohimbine + clonidine attenuated the inhibitory effects of ANGII. Prazosin injected into the MSA did not cause changes in ANGII responses. Prazosin + clonidine attenuated the inhibitory effects of ANGII. The results showed that MSA injections of a1- and a2-antagonists decreased ANGII-induced water intake, and abolished the Na+, K+ and urine decrease induced by ANGII into the 3rdV. These findings suggest the involvement of septal a1- and a2-adrenergic receptors in water intake and electrolyte and urine excretion induced by central ANGII.

High prevalence of the simultaneous excretion of polyomaviruses JC and BK in the urine of HIV-infected patients without neurological symptoms in São Paulo, Brazil

OBJECTIVE: To evaluate the prevalence of the urinary excretion of BKV and JCV in HIV-infected patients without neurological symptoms. METHODS: Urine samples from HIV-infected patients without neurological symptoms were tested for JC virus and BK virus by PCR. Samples were screened for the presence of polyomavirus with sets of primers complementary to the early region of JCV and BKV genome (AgT). The presence of JC virus or BK virus were confirmed by two other PCR assays using sets of primers complementary to the VP1 gene of each virus. Analysis of the data was performed by the Kruskal-Wallis test for numerical data and Pearson or Yates for categorical variables. RESULTS: A total of 75 patients were included in the study. The overall prevalence of polyomavirus DNA urinary shedding was 67/75 (89.3%). Only BKV DNA was detected in 14/75 (18.7%) urine samples, and only JCV DNA was detected in 11/75 (14.7%) samples. Both BKV and JCV DNA were present in 42/75 (56.0%) samples. CONCLUSION: In this study we found high rates of excretion of JCV, BKV, and simultaneous excretion in HIV+ patients. Also these results differ from the others available on the literature.

Morphological evidence by scanning electron microscopy of excretion of metacyclic forms of Trypanosoma cruzi in vector's urine

Comparision by scanning electron microscopy (SEM) of Trypanosoma cruzi flagellates attached to the cuticle of the rectal gland of infected Dipetalogaster maxima nymphs, showed marked differences before amd after feeding. Before feeding numerous metacyclic trypomastigotes were observed among the abundant epimastigotes that formed the carpet of flagellates. On the other hand, in insects that were allowed to urinate for 24 hours after a meal, the metacyclics were scarce,indicating that they had been detached by the urine flow. An asymetric type of cell division, probably originating both an epi-and a trypomastigote, was occasionally observed. The occurrence of swellings at different levels of the flagella of epimastigotes suggests that secondary sites of attachment may be common.

Correlation between sodium and potassium excretion in 24- and 12-h urine samples

Low-sodium and high-potassium diets have been recommended as an adjunct to prevention and treatment of hypertension. Analysis of these nutrients in 24-h urine has been considered the reference method to estimate daily intake of these minerals. However, 24-h urine collection is difficult in epidemiological studies, since urine must be collected and stored in job environments. Therefore, strategies for shorter durations of urine collection at home have been proposed. We have previously reported that collecting urine during a 12-h period (overnight) is more feasible and that creatinine clearance correlated strongly with that detected in 24-h samples. In the present study, we collected urine for 24 h divided into two 12-h periods (from 7:00 am to 7:00 pm and from 7:00 pm to 7:00 am next day). A sample of 109 apparently healthy volunteers aged 30 to 74 years of both genders working in a University institution was investigated. Subjects with previous myocardial infarction, stroke, renal insufficiency, and pregnant women were not included. Significant (P < 0.001) Spearman correlation coefficients (r s) were found between the total amount of sodium and potassium excreted in the urine collected at night and in the 24-h period (r s = 0.76 and 0.74, respectively). Additionally, the 12-h sodium and potassium excretions (means ± SD, 95% confidence interval) corresponded to 47.3 ± 11.2%, 95%CI = 45.3-49.3, and 39.3 ± 4.6%, 95%CI = 37.3-41.3, respectively, of the 24-h excretion of these ions. Therefore, these findings support the assumption that 12-h urine collected at night can be used as a reliable tool to estimate 24-h intake/excretion of sodium and potassium.

Human polyomaviruses JC and BK in the urine of Brazilian children and adolescents vertically infected by HIV

The aim of this study was to characterize the urinary excretion of the BK (BKV) and JC (JCV) human polyomaviruses in a cohort of human immunodeficiency virus (HIV)-infected children and adolescents. One hundred and fifty-six patients were enrolled: Group I included 116 HIV-infected children and adolescents [median age = 11.4 years (y); range 1-22 y]; Group II included 40 non-HIV-infected healthy controls (median age = 11.37 y; range 7-16 y). Single urine samples from both groups were screened for the presence of JCV and BKV DNA by polymerase chain reaction at enrolment. The overall rate of JCV and BKV urinary excretion was found to be 24.4% and 40.4%, respectively (n = 156). Group I had urinary excretion of JCV and BKV in 27.6% and 54.3% of subjects, respectively. In contrast, Group II showed positive results for JCV in 17.5% of subjects and for BKV in 12.5% of subjects (p Pearson JCV = 0.20; p Pearson BKV < 0.0001). In Group I, there was no association between JCV/BKV shedding and age, gender or CD4 values. Patients with an HIV viral load < 50 copies/mL had a lower excretion of BKV (p < 0.001) and a trend of lower JCV excretion (p = 0.07). One patient in Group I (1/116, 0.9%) showed clinical and radiological features consistent with progressive multifocal leukoencephalopathy, suggesting that children with HIV/polyomavirus coinfection should be kept under surveillance.

Transforming growth factor beta activity in urine of patients with type 2 diabetes and diabetic nephropathy

Diabetic nephropathy (DN) is characterized structurally by progressive mesangial deposition of extracellular matrix (ECM). Transforming growth factor-ß (TGF-ß) is considered to be one of the major cytokines involved in the regulation of ECM synthesis and degradation. Several studies suggest that an increase in urinary TGF-ß levels may reflect an enhanced production of this polypeptide by the kidney cells. We evaluated TGF-ß in occasional urine samples from 14 normal individuals and 23 patients with type 2 diabetes (13 with persistent proteinuria >500 mg/24 h, DN, 6 with microalbuminuria, DMMA, and 4 with normal urinary albumin excretion, DMN) by enzyme immunoassay. An increase in the rate of urinary TGF-ß excretion (pg/mg UCreat.) was observed in patients with DN (296.07 ± 330.77) (P<0.001) compared to normal individuals (17.04 ± 18.56) (Kruskal-Wallis nonparametric analysis of variance); however, this increase was not observed in patients with DMMA (25.13 ± 11.30) or in DMN (18.16 ± 11.82). There was a positive correlation between the rate of urinary TGF-ß excretion and proteinuria (r = 0.70, a = 0.05) (Pearson's analysis), one of the parameters of disease progression.

A micromethod for quantitation of debrisoquine and 4-hydroxydebrisoquine in urine by liquid chromatography

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred µl of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 min (D) and 16.6 min for the internal standard, metoprolol (20 µg/ml). The 5-µm CN-reverse-phase column (Shimpack, 250 x 4.6 mm) was eluted with a mobile phase consisting of 0.25 M acetate buffer, pH 5.0, and acetonitrile (9:1, v/v) at 0.7 ml/min with detection at lexcitation = 210 nm and lemission = 290 nm. The method, validated on the basis of measurements of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 390-6240 ng/ml (D) and 750-12000 ng/ml (4-OHD) linearity, and 5.7/8.2% (D) and 5.3/8.2% (4-OHD) intra/interassay precision. The method was validated using urine of a healthy Caucasian volunteer who received one 10-mg tablet of Declinax®, po, in the morning after an overnight fast. Urine samples (diuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretion of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and excreted into urine, was 6.4% and 31.9%, respectively. The hydroxylation capacity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person investigated and can be compared to reference limits of >12.5 for poor metabolizers (PM) and <12.5 for extensive metabolizers (EM). In parallel, the recovery ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: SD + 4-OHD) versus reference limits of RR <0.12 for PM and RR >0.12 for EM. The healthy volunteer was considered to be an extensive metabolizer on the basis of the debrisoquine test.

Familial predisposition to hypertension and the association between urinary sodium excretion and blood pressure in a population-based sample of young adults

The reasons for the inconsistent association between salt consumption and blood pressure levels observed in within-society surveys are not known. A total of 157 normotensive subjects aged 18 to 35 years, selected at random in a cross-sectional population-based survey, answered a structured questionnaire. They were classified as strongly predisposed to hypertension when two or more first-degree relatives had a diagnosis of hypertension. Anthropometric parameters were obtained and sitting blood pressure was determined with aneroid sphygmomanometers. Sodium and potassium excretion was measured by flame spectrophotometry in an overnight urine sample. A positive correlation between blood pressure and urinary sodium excretion was detected only in the group of individuals strongly predisposed to hypertension, both for systolic blood pressure (r = 0.51, P<0.01) and diastolic blood pressure (r = 0.50, P<0.01). In a covariance analysis, after controlling for age, skin color and body mass index, individuals strongly predisposed to hypertension who excreted amounts of sodium above the median of the entire sample had higher systolic and diastolic blood pressure than subjects classified into the remaining conditions. The influence of familial predisposition to hypertension on the association between salt intake and blood pressure may be an additional explanation for the weak association between urinary sodium excretion and blood pressure observed in within-population studies, since it can influence the association between salt consumption and blood pressure in some but not all inhabitants.

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 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.

GABA in the central amygdaloid nucleus modulates the electrolyte excretion and hormonal responses to blood volume expansion in rats

We investigated the involvement of GABAergic mechanisms of the central amygdaloid nucleus (CeA) in unanesthetized rats subjected to acute isotonic or hypertonic blood volume expansion (BVE). Male Wistar rats bearing cannulas unilaterally implanted in the CeA were treated with vehicle, muscimol (0.2 nmol/0.2 µL) or bicuculline (1.6 nmol/0.2 µL) in the CeA, followed by isotonic or hypertonic BVE (0.15 or 0.3 M NaCl, 2 mL/100 g body weight over 1 min). The vehicle-treated group showed an increase in sodium excretion, urinary volume, plasma oxytocin (OT), and atrial natriuretic peptide (ANP) levels compared to control rats. Muscimol reduced the effects of BVE on sodium excretion (isotonic: 2.4 ± 0.3 vs vehicle: 4.8 ± 0.2 and hypertonic: 4.0 ± 0.7 vs vehicle: 8.7 ± 0.6 µEq·100 g-1·40 min-1); urinary volume after hypertonic BVE (83.8 ± 10 vs vehicle: 255.6 ± 16.5 µL·100 g-1·40 min-1); plasma OT levels (isotonic: 15.3 ± 0.6 vs vehicle: 19.3 ± 1 and hypertonic: 26.5 ± 2.6 vs vehicle: 48 ± 3 pg/mL), and ANP levels (isotonic: 97 ± 12.8 vs vehicle: 258.3 ± 28.1 and hypertonic: 160 ± 14.6 vs vehicle: 318 ± 16.3 pg/mL). Bicuculline reduced the effects of isotonic or hypertonic BVE on urinary volume and ANP levels compared to vehicle-treated rats. However, bicuculline enhanced the effects of hypertonic BVE on plasma OT levels. These data suggest that CeA GABAergic mechanisms are involved in the control of ANP and OT secretion, as well as in sodium and water excretion in response to isotonic or hypertonic blood volume expansion.

Influence of renal denervation on blood pressure, sodium and water excretion in acute total obstructive apnea in rats

Obstructive apnea (OA) can exert significant effects on renal sympathetic nerve activity (RSNA) and hemodynamic parameters. The present study focuses on the modulatory actions of RSNA on OA-induced sodium and water retention. The experiments were performed in renal-denervated rats (D; N = 9), which were compared to sham (S; N = 9) rats. Mean arterial pressure (MAP) and heart rate (HR) were assessed via an intrafemoral catheter. A catheter was inserted into the bladder for urinary measurements. OA episodes were induced via occlusion of the catheter inserted into the trachea. After an equilibration period, OA was induced for 20 s every 2 min and the changes in urine, MAP, HR and RSNA were recorded. Renal denervation did not alter resting MAP (S: 113 ± 4 vs D: 115 ± 4 mmHg) or HR (S: 340 ± 12 vs D: 368 ± 11 bpm). An OA episode resulted in decreased HR and MAP in both groups, but D rats showed exacerbated hypotension and attenuated bradycardia (S: -12 ± 1 mmHg and -16 ± 2 bpm vs D: -16 ± 1 mmHg and 9 ± 2 bpm; P < 0.01). The basal urinary parameters did not change during or after OA in S rats. However, D rats showed significant increases both during and after OA. Renal sympathetic nerve activity in S rats increased (34 ± 9%) during apnea episodes. These results indicate that renal denervation induces elevations of sodium content and urine volume and alters bradycardia and hypotension patterns during total OA in unconscious rats.

Spot urine porphyrins/creatinine ratio profile of healthy Brazilian individuals adjusted for personal habits

Changes in urinary porphyrin excretion may be the result of hereditary causes and/or from environmental or occupational exposure. The objective of this study was to measure the amount of some porphyrins in spot urine samples obtained from volunteers randomly selected from a healthy adult population of São Paulo with a sensitive HPLC method and to estimate normal ranges for a non-exposed population. Spot urine samples were collected from 126 subjects (both genders, 18 to 65 years old) not occupationally exposed to porphyrinogenic agents. Porphyrin fractions were separated on RP-18 HPLC column eluted with a methanol/ammonium acetate buffer gradient, pH 4.0, and measured fluorometrically (excitation 405 nm/emission 620 nm). The amount of porphyrins was corrected for urinary creatinine excretion. Only 8-carboxyl (uro) and 4-carboxyl (copro) porphyrins were quantified as µg/g creatinine. Data regarding age, gender, occupational activities, smoking and drinking habits were analyzed by Mann-Whitney and Kruskal-Wallis tests. Uroporphyrin results did not differ significantly between the subgroups studied. Copro and uro + copro porphyrins were significantly different for smokers (P = 0.008) and occupational activities (P = 0.004). With respect to alcohol consumption, only men drinking >20 g/week showed significant differences in the levels of copro (P = 0.022) and uro + copro porphyrins (P = 0.012). The 2.5-97.5th percentile limit values, excluding those for subjects with an alcohol drinking habit >20 g/week, were 0-20.8, 11.7-93.1, and 15.9-102.9 µg/g creatinine for uro, copro and uro + copro porphyrins, respectively. These percentile limit values can be proposed as a first attempt to provide urinary porphyrin reference values for our population, serving for an early diagnosis of porphyrinopathies or as biomarkers of exposure to porphyrinogenic agents.

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.