RESUMEN

Polycystic kidney disease is a complex clinical entity which comprises a group of genetic diseases that leads to renal cyst development. We evaluated the most suitable housekeeping genes for microRNA expression by RT-qPCR analyses of kidney tissues in Pkd1-deficient mouse models from a panel of five candidates genes (miR-20a, miR-25, miR-26a, miR-191 and U6) and 3 target genes (miR-17, miR-21 and let-7a) using samples from kidneys of cystic mice (Pkd1flox/flox:Nestincre, CY), non-cystic controls (Pkd1flox/flox, NC), Pkd1-haploinsufficient (Pkd1+/-, HT), wild-type controls (Pkd1+/+, WT), severely cystic mice (Pkd1V/V, SC), wild-type controls (CO). The stability of the candidate genes was investigated using NormFinder, GeNorm, BestKeeper, DataAssist, and RefFinder software packages and the comparative ΔCt method. The analyses identified miR-26a as the most stable housekeeping gene for all kidney samples, miR-20a for CY and NC, miR-20a and miR-26a for HT and WT, and miR-25 and miR-26a for SC and CO. Expression of miR-21 was upregulated in SC compared to CO and trends of miR-21 upregulation and let-7a downregulation in CY and HT compared to its control kidneys, when normalized by different combinations of miR-20a, miR-25 and miR-26a. Our findings established miR-20a, miR-25, and miR-26a as the best housekeeping genes for miRNA expression analyses by RT-qPCR in kidney tissues of Pkd1-deficient mouse models.

Asunto(s)

Perfilación de la Expresión Génica , Genes Esenciales/genética , Riñón/metabolismo , MicroARNs/genética , Proteína Quinasa C/deficiencia , Animales , Haploinsuficiencia , Ratones , Proteína Quinasa C/genética

RESUMEN

BACKGROUND: Sodium reabsorption depends on the Na/K/ATPase activity coupled to basolateral K+ recycling through K+ channels. ATP depletion reduces pump activity and increases K+ leak resulting in transport dysfunction. Kir4.1 is a pH-sensitive K+ channel expressed in the basolateral membrane of distal tubules. In this study, we evaluated whether Kir4.1 is also expressed in proximal tubules (PTs) and whether renal ischemia alters Kir4.1 mRNA expression levels. METHODS: The presence of Kir4.1 mRNA was evaluated in PTs microdissected from collagenase-treated rat kidneys. Kir4.1 expression levels were estimated in the renal cortex by multiplex RT-PCR after 30 or 60 min of renal ischemia followed by 1, 24, 48 or 72 h of reperfusion. RESULTS: The PCR product obtained from isolated tubules was sequenced and showed approximately 98% homology with rat Kir4.1 cDNA. Ischemia/reperfusion for 30 min induced a time-dependent reduction in Kir4.1 mRNA expression in parallel with plasma creatinine, however recovery was delayed after 60 min of ischemia, remaining reduced after 72 h of reperfusion when plasma creatinine was already normalized. CONCLUSION: Kir4.1 mRNA expression was decreased by renal ischemia. The ischemia-induced cellular K+ loss may be minimized by Kir4.1 downregulation and may contribute to the mechanism by which cellular acidification induces cell protection against ATP depletion.

Asunto(s)

Túbulos Renales Proximales/metabolismo , Riñón/irrigación sanguínea , Riñón/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Potasio/metabolismo , Daño por Reperfusión/metabolismo , Animales , Expresión Génica , Concentración de Iones de Hidrógeno , Masculino , Ratas , Ratas Wistar , Distribución Tisular

RESUMEN

1. Activation of vascular ATP-sensitive K(+) (K(ATP)) channels has been implicated in vasodilator responses to pregnancy. 2. The effect of glibenclamide, a K(ATP) channel inhibitor, on systolic blood pressure (SBP) and renal function was evaluated in pregnant and non-pregnant spontaneously hypertensive rats, as well as in normotensive and hypertensive Wistar rats that had been made hypertensive by simultaneous treatment with N(G)-nitro-l-arginine methyl ester (0.4 mg/mL) and indomethacin (2 mg/kg, i.p.) from Day 1 of gestation. Pregnant animals received 10 mg/kg glibenclamide for 12 days starting at Day 7. In addition, the mRNA levels of the vascular K(ATP) channel (Kir6.2) were estimated in aorta and kidney using real-time reverse transcription-polymerase chain reaction on Day 19 of pregnancy. 3. The decreased SBP observed in pregnant Wistar rats was paralleled by an increase in Kir6.2 mRNA levels. Glibenclamide blunted systemic vasodilation and reduced the mRNA expression of Kir6.2. There was no pregnancy induced vasodilation and no change in Kir6.2 mRNA expression in SHR. Glibenclamide had no effect on pregnant SHR. Hypertensive Wistar rats exhibited high SBP, followed by increased Kir6.2 mRNA levels. The effects of glibenclamide were not evaluated in this group because glibenclamide induced intense vaginal bleeding. 4. The results of the present study suggest that K(ATP) channels may be involved in pregnancy induced vasodilation during normotensive pregnancy, but not in pregnant SHR. Glibenclamide may have an abortive effect if administered during the early phases of gestation or in association with nitric oxide and prostaglandin inhibitors.

Asunto(s)

Adenosina Trifosfato/metabolismo , Hipertensión Inducida en el Embarazo/etiología , Canales de Potasio/fisiología , Preñez , Animales , Glucemia/análisis , Glucemia/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Femenino , Gliburida/farmacología , Canales KATP , Riñón/efectos de los fármacos , Riñón/metabolismo , Óxido Nítrico/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/metabolismo , Embarazo , Proteinuria/etiología , ARN Mensajero/metabolismo , Ratas , Ratas Wistar

RESUMEN

Phyllanthus niruri is a plant used for years in Brazil to treat urinary calculi. We prospectively evaluated the effect of P. niruri intake on 24 h urinary biochemical parameters in an attempt to assess its in vivo effect in calcium stone forming (CSF) patients. A total of 69 CSF patients (39 males and 30 females, 38+/-8 years old) were randomized to take either P. niruri ( n=33) (450 mg capsules, td) or placebo ( n=36) for 3 months. Blood calcium, uric acid, citrate, magnesium, oxalate, sodium and potassium were determined at baseline and at the end of the study. A subset analysis was made in patients classified according to the presence of metabolic abnormalities (hypercalciuria, hyperuricosuria, hyperoxaluria, hypocitraturia and hypomagnesiuria). Overall, there were no significant differences in the mean values of urinary parameters between the urine samples before and after P. niruri intake, except for a slight reduction in mean urinary magnesium after P. niruri, which was within the normal range. However, in the subset analysis, we observed that P. niruri induced a significant reduction in the mean urinary calcium in hypercalciuric patients (4.8+/-1.0 vs 3.4+/-1.1 mg/kg/24 h, P<0.05). In this short-term follow-up, no significant differences in calculi voiding and/or pain relief between the groups taking P. niruri or the placebo were detected. Our data suggest that P. niruri intake reduces urinary calcium based on the analysis of a subset of patients presenting with hypercalciuria. Larger trials including primary hypercalciuric stone formers should be performed in order to confirm these findings and to determine the possible clinical consequences of urinary calcium reduction during P. niruri administration.

Asunto(s)

Calcio/orina , Phyllanthus , Fitoterapia/métodos , Extractos Vegetales/uso terapéutico , Cálculos Urinarios/tratamiento farmacológico , Adulto , Brasil , Calcio/sangre , Calcio/metabolismo , Ácido Cítrico/orina , Femenino , Liofilización , Humanos , Magnesio/orina , Masculino , Persona de Mediana Edad , Oxalatos/orina , Potasio/orina , Estudios Prospectivos , Sodio/orina , Ácido Úrico/orina , Cálculos Urinarios/orina

RESUMEN

Increased intrarenal renin-angiotensin system activity contributes to diabetic nephropathy. ANG II generation in mesangial cells (MC) is increased by high-glucose (HG) exposure. This study assessed the mechanisms involved in the glucose-induced ANG II generation in rat MC. Under basal conditions, MC mainly secreted prorenin. HG decreased prorenin secretion and induced a striking 30-fold increase in intracellular renin activity. After 72 h of HG exposure, only the mRNA levels for angiotensinogen and angiotensin-converting enzyme (ACE) were significantly elevated. However, after shorter periods of 24 h of HG stimulation the mRNA levels of the enzymes prorenin and cathepsin B, besides that for ACE, were significantly increased. The results suggest that the HG-induced increase in ANG II generation in MC results from an increase in intracellular renin activity mediated by at least three factors: a time-dependent stimulation of (pro)renin gene transcription, a reduction in prorenin enzyme secretion, and an increased rate of conversion of prorenin to active renin, probably mediated by cathepsin B. The increase in angiotensinogen mRNA in parallel to increased renin activity indicates that HG also increased the availability of the renin substrate. The consistent upregulation of ACE mRNA suggests that, besides renin, ACE is directly involved in the increased mesangial ANG II generation induced by HG.

Asunto(s)

Angiotensina II/biosíntesis , Mesangio Glomerular/metabolismo , Glucosa/farmacología , Renina/metabolismo , Animales , Células Cultivadas , Medios de Cultivo , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Masculino , Peptidil-Dipeptidasa A/biosíntesis , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptor de Angiotensina Tipo 1/biosíntesis , Receptor de Angiotensina Tipo 2/biosíntesis , Sistema Renina-Angiotensina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Phyllanthus niruri is a plant used in Brazilian folk medicine for the treatment of urolithiasis. It was previously observed that P. niruri shows no toxicity, potentially increases calculus voiding by stone forming patients and inhibits the endocytosis of calcium oxalate (CaOx) crystals by MDCK cells. In addition, in a rat model of urolithiasis it reduced calculus growth. In the present study, we evaluated the effect of an aqueous extract of P. niruri on CaOx crystallization in vitro. CaOx precipitation was induced by the addition of 0.1 M sodium oxalate to unfiltered urine samples from Wistar rats (n=14) and normal humans (n=18) in the presence or absence of P. niruri extract (0.25 mg/ml of urine). The presence of CaOx crystals was evaluated immediately and 24 h later. In vitro crystallization of human urine produced typical mono- and dihydrated CaOx crystals, but only a few typical CaOx crystals were found in rat urine. The presence of P. niruri extract did not inhibit CaOx precipitation and even more crystals were obtained, although they were significantly smaller than those in the control urine. Crystal aggregation observed 24 h after crystallization was also inhibited by P. niruri extract. The results showed an inhibitory effect of P. niruri extract on CaOx crystal growth and aggregation in human urine, suggesting that it may interfere with the early stages of stone formation and may represent an alternative form of treatment and/or prevention of urolithiasis

Asunto(s)

Oxalato de Calcio/química , Cálculos Renales/tratamiento farmacológico , Phyllanthus , Fitoterapia , Extractos Vegetales/farmacología , Animales , Cristalización , Femenino , Humanos , Técnicas In Vitro , Cálculos Renales/química , Masculino , Ratas , Ratas Wistar , Orina/química

RESUMEN

OBJECTIVE: To evaluate the effect of an aqueous extract of Phyllanthus niruri (Pn), a plant used in folk medicine to treat lithiasis, on the urinary excretion of endogenous inhibitors of lithogenesis, citrate, magnesium and glycosaminoglycans (GAGs). MATERIALS AND METHODS: The effect of chronic (42 days) administration of Pn (1.25 mg/mL/day, orally) was evaluated in a rat model of urolithiasis induced by the introduction of a calcium oxalate (CaOx) seed into the bladder of adult male Wistar rats. The animals were divided into four groups: a sham control (16 rats); a control+Pn (six); CaOx+water instead of Pn (14); and CaOx+Pn (22). Plasma and urine were collected after 42 days of treatment for biochemical analysis and the determination of urinary excretion of citrate, magnesium and GAGs. The animals were then killed and the calculi analysed. RESULTS: The creatinine clearance or urinary and plasma concentrations of Na+, K+, Ca2+, oxalate, phosphate and uric acid were unaffected by Pn or the induction of lithiasis. Treatment with Pn strongly inhibited the growth of the matrix calculus and reduced the number of stone satellites compared with the group receiving water. The calculi were eliminated or dissolved in some treated animals (three of 22). The urinary excretion of citrate and magnesium was unaffected by Pn treatment. However, the mean (sd) urinary concentration of GAGs was significantly lower in rats treated with CaOx+Pn, at 5.64 (0.86) mg/g creatinine, than when treated with CaOx + water, at 11.78 (2.21) mg/g creatinine. In contrast, the content of GAGs in the calculi was higher in the CaOx + Pn rats, at 48.0 (10.4) g/g calculus, than in the CaOx + water group, at 16.6 (9.6) g/g calculus. CONCLUSION: These results show that Pn has an inhibitory effect on crystal growth, which is independent of changes in the urinary excretion of citrate and Mg, but might be related to the higher incorporation of GAGs into the calculi.

Asunto(s)

Oxalato de Calcio/antagonistas & inhibidores , Cálculos Renales/tratamiento farmacológico , Phyllanthus , Fitoterapia/métodos , Extractos Vegetales/uso terapéutico , Cálculos de la Vejiga Urinaria/tratamiento farmacológico , Animales , Ácido Cítrico/antagonistas & inhibidores , Cristalización , Glicosaminoglicanos/orina , Cálculos Renales/orina , Masculino , Compuestos Organometálicos/antagonistas & inhibidores , Fosfatos/sangre , Fosfatos/orina , Potasio/sangre , Potasio/orina , Ratas , Ratas Wistar , Sodio/sangre , Sodio/orina , Ácido Úrico/orina , Cálculos de la Vejiga Urinaria/orina

RESUMEN

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 +/- 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 +/- 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 +/- 3.1 and 3.9 +/- 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II

Asunto(s)

Animales , Masculino , Ratones , Ratas , Mesangio Glomerular , Renina , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Mesangio Glomerular , Técnica del ADN Polimorfo Amplificado Aleatorio , Ratas Wistar , Renina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ARN Mensajero

RESUMEN

Renin is an enzyme involved in the stepwise generation of angiotensin II. Juxtaglomerular cells are the main source of plasma renin, but renin activity has been detected in other cell types. In the present study we evaluated the presence of renin mRNA in adult male Wistar rat and mouse (C-57 Black/6) mesangial cells (MC) and their ability to process, store and release both the active and inactive forms of the enzyme. Active renin and total renin content obtained after trypsin treatment were estimated by angiotensinogen consumption analyzed by SDS-PAGE electrophoresis and quantified by angiotensin I generation by HPLC. Renin mRNA, detected by RT-PCR, was present in both rat and mouse MC under basal conditions. Active renin was significantly higher (P<0.05) in the cell lysate (43.5 +/- 5.7 ng h-1 10(6) cells) than in the culture medium (12.5 +/- 2.5 ng h-1 10(6) cells). Inactive prorenin content was similar for the intra- and extracellular compartments (9.7 +/- 3.1 and 3.9 +/- 0.9 ng h-1 10(6) cells). Free active renin was the predominant form found in both cell compartments. These results indicate that MC in culture are able to synthesize and translate renin mRNA probably as inactive prorenin which is mostly processed to active renin inside the cell. MC secrete both forms of the enzyme but at a lower level compared with intracellular content, suggesting that the main role of renin synthesized by MC may be the intracellular generation of angiotensin II.

Asunto(s)

Mesangio Glomerular/enzimología , Renina/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Mesangio Glomerular/citología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Renina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

In order to evaluate the progression of renal disease, Munich-Wistar rats were submitted to 5/6 nephrectomy and given whole-body x- or gamma-irradiation with or without remnant kidney protection or were submitted only to remnant kidney irradiation. All groups received a single 6-Gy dose immediately after surgery. Whole-kidney function, glomerular hemodynamics, 24-hour proteinuria and histopathology were assessed 60 days after surgery and irradiation. The irradiated nephrectomized animals presented whole-kidney function parameters comparable to those of normal rats. In addition, they were less hypertensive and had higher hematocrit. They showed glomerular hyperfiltration and hypertension even greater than their respective nephrectomized controls. However, the interrelations among the glomerular filtration determinants were somewhat different in irradiated animals. Their 24-hour proteinuria was significantly lower and the sclerosis index and tubulointerstitial injury score were markedly smaller. Among irradiated animals, the worst sclerosis index was observed in those with a shielded remnant kidney and the best in those without protection of the remnant kidney. This led us to speculate about a possible influence of resident mesangial cells on the early events following renal mass ablation and on the maintenance of subsequent physiopathologic changes. Therefore, radiation undoubtedly provoked a beneficial change in the course of renal disease when the renal mass ablation model was employed. Many factors could have contributed to this favorable feature including lower levels of systemic arterial pressure, less increment in DeltaP, diminished proteinuria, and maintenance of tubulointerstitial space integrity. Our data also suggest that development of glomerulosclerosis seems to be determined by events occurring immediately after injury.

Asunto(s)

Fallo Renal Crónico/fisiopatología , Fallo Renal Crónico/radioterapia , Glomérulos Renales/fisiopatología , Glomérulos Renales/efectos de la radiación , Circulación Renal/efectos de la radiación , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Glomérulos Renales/irrigación sanguínea , Masculino , Nefrectomía , Proteinuria/fisiopatología , Proteinuria/radioterapia , Ratas , Ratas Wistar , Irradiación Corporal Total

RESUMEN

The determination of urinary excretion of lithogenic elements in healthy newborns involves factors ranging from urine collection and data handling to maternal influences, which can cause difficulties in analyzing the results. The objective of this study was to determine normal values of parameters related to lithogenesis, such as calcium, uric acid, citrate, and oxalate, in urine of healthy newborns using isolated samples, focusing on variations according to gender, weight, milk ingestion, and family history of lithiasis. Parameters measured in isolated urine samples from 104 healthy newborns (77 males and 27 females) were corrected by creatinine. The ratios were expressed as milligram/milligram of creatinine: calcium/creatinine of 0.10+/-0.01 (X+/-SE), uric acid/ creatinine of 1.10+/-0.10, citrate/creatinine of 0.56+/-0.04, and oxalate/creatinine of 0.07+/-0.01. Differences were observed between males and females, in terms of uric acid (0.80+/-0.07 vs. 1.10+/-0.10 mg/mg, P<0.05), citrate (0.05+/-0.06 vs. 0.17+/-0.05, P<0.05), sodium (0.17+/- 0.01 vs. 0.05+/-0.01, P<0.001), and potassium (0.05+/- 0.01 vs. 0.20+/-0.02, P<0.001). Interestingly, the urinary concentration of protector factors such citrate and potassium was higher in females than in males with low sodium excretion. Artificial milk feeding leads to higher calcium (0.10+/-0.06 vs. 0.06+/-0.01), uric acid (1.40+/-0.20 vs. 0.90+/-0.09, P<0.05), citrate (0.90+/-0.09 vs. 0.50+/-0.04, P<0.001), and oxalate (0.17+/-0.03 vs. 0.05+/-0.01, P<0.001) excretion when compared with breast feeding. There was higher excretion of calcium and sodium in patients under 3 kg. Children with familial antecedents presented some differences in urinary excretion, with higher uric acid (1.50+/-0.30 vs. 0.80+/-0.08, P<0.05) but lower calcium (0.05+/-0.02 vs. 0.10+/-0.01, P<0.05) and sodium (0.15+/-0.02 vs. 0.20+/-0.02, P<0.05) excretion, respectively. This report provides urinary parameters obtained in healthy newborns and correlates them with factors that could be involved in the genesis of osteopenia, renal stones, and/or nephrocalcinosis.

Asunto(s)

Recién Nacido/orina , Cálculos Renales/etiología , Lactancia Materna , Calcio/orina , Citratos/orina , Creatinina/orina , Femenino , Humanos , Alimentos Infantiles , Masculino , Oxalatos/orina , Valores de Referencia , Caracteres Sexuales , Ácido Úrico/orina

RESUMEN

The activities of serine endopeptidase, prolyl endopeptidase and neutral endopeptidase were determined in tubular fluid collected from several portions of the rat nephron as well as in urine. The enzyme activities were measured by HPLC using bradykinin (BK) as substrate. Free residual peptides of BK obtained by the action of these enzymes on the locally produced BK were also determined. The endopeptidase activities were found to be present throughout the nephron. Equimolar fragments of BK were detected in the early proximal tubule (Arg(1)-Pro(7), Phe(8)-Arg(9), Arg(1)-Gly(4), Phe(5)-Arg(9), and BK), late proximal tubule (Arg(1)-Phe(5), Arg(1)-Pro(7), Gly(4)-Pro(7), Gly(4)-Arg(9), and BK), late distal tubule (Arg(1)-Gly(4), Phe(5)-Arg(9), Arg(1)-Phe(5), Ser(6)-Arg(9), Gly(4)-Arg(9), BK, and [des-Arg(9)]BK) and urine (Phe(8)-Arg(9), Phe(5)-Arg(9), Arg(1)-Phe(5), Ser(6)-Arg(9), Arg(1)-Pro(7), Gly(4)-Pro(7), Gly(4)-Arg(9), BK, and [des-Arg(9)]BK). Our data suggest that the endopeptidases and exopeptidases are secreted by the nephron. Early proximal tubules secrete angiotensin converting enzyme and neutral endopeptidase, differing from late distal tubules that produce prolyl endopeptidase, serine endopeptidase, carboxypeptidase, and also neutral endopeptidase. All enzymes detected along the rat nephron were found in the urine. The existence of endopeptidases and carboxypeptidase in the distal nephron may have a potential physiological role in the inactivation of the kinins formed by kallikrein in the kidney and also in the inactivation of additional peptides other than BK.

Asunto(s)

Líquidos Corporales/enzimología , Endopeptidasas/metabolismo , Cininas/metabolismo , Nefronas/enzimología , Aminoácidos/análisis , Animales , Líquidos Corporales/química , Bradiquinina/metabolismo , Cromatografía Líquida de Alta Presión , Hidrólisis , Masculino , Ratas , Ratas Wistar

RESUMEN

The effects of recombinant human erythropoietin (rHuEPO)-induced polycythemia on renal function and glomerular hemodynamics were evaluated in Munich-Wistar rats (MW+EPO) before and after infusion of indomethacin; the rHuEPO effects on total renal function were also evaluated in 5/6 nephrectomized (CRF) MW and spontaneously hypertensive rats (MW-CRF+EPO and SHR-CRF+EPO, respectively). In normal MW rats, rHuEPO (300 IU/kg BW, 3 x /week, during 2 weeks) induced elevation in MAP, with maintenance of GFR, paralleled by superficial vasodilatation and elevation in SNGFR, suggesting cortical blood redistribution. These hemodynamic alterations induced by rHuEPO were blunted by indomethacin, suggesting a participation of the vasodilator prostaglandins in the renal compensatory mechanism of polycythemia. Elevation in MAP and reduction in GFR occurred in the MW-CRF+EPO group compared with the group receiving vehicle. In contrast, the SHR-CRF+EPO presented a reduction in MAP and maintenance of GFR, suggesting different rHuEPO effects depending on previous renal function and/or hypertensive state.

Asunto(s)

Eritropoyetina/farmacología , Nefrectomía , Policitemia/inducido químicamente , Circulación Renal/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Indometacina/farmacología , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/fisiología , Masculino , Policitemia/fisiopatología , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Proteínas Recombinantes , Circulación Renal/efectos de los fármacos , Vasodilatación/fisiología

RESUMEN

OBJECTIVE: Previous analysis of the angiotensin I-converting enzyme (ACE) gene in this laboratory showed that primary mesangial cells in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on glomerular microcirculation. The aim of this study was to purify and characterize the secreted and intracellular ACE forms from mesangial cells in culture. METHODS AND RESULTS: Medium from Wistar rats mesangial cells was collected (third passage), incubated for 20 h with RPMI without fetal bovine serum and concentrated 29 times in an Amicon concentrator. The concentrated medium was submitted to gel filtration on an AcA-34 column and two peaks (ACE1, mol. wt 130 000 and ACE2, 60000) with ACE on activity Hippuryl-His-Leu and Z-Phe-His-Leu were separated. The mesangial cells were collected and ACE enzyme was extracted using Triton X-114, followed by centrifugation and concentration. The supernatant was submitted to the same chromatography as described above and two peaks with ACE activity (ACEInt1, mol. wt 130000 and ACEInt2, 68000) were separated. The purified ACE were inhibited by enalaprilat and captopril, two potent competitive inhibitors of ACE and by EDTA, using Hippuryl-His-Leu as a substrate. The Km values were 2 mM for ACE1 and ACE2 and 3 mM for ACEInt1 and ACEInt2. The enzymes ACE1 and ACE2 presented an optimum pH of 8.0 and ACEInt1 and ACEInt2 an optimum pH of 7.5. CONCLUSION: The activities of full-length wild-type and N-domain ACE were characterized by the ratio of the hydrolysis of Z-Phe-His-Leu/Hippuryl-His-Leu, which was 1 and 4, respectively. The ratios found for ACE1, ACE2, ACEInt1 and ACEInt2 in the present study were similar to those described above, suggesting that mesangial cells, besides showing the presence of intracellular ACE, are able to secret both full-length wild-type ACE and N-domain ACE. Thus, they may potentially have an effect, not only on bradykinin and angiotensin I (ACE wild-type), but also on substance P, luteinizing hormone-releasing hormone and Met-enkephalin to interfere with glomerular haemodynamics and with the renal microcirculation.

Asunto(s)

Mesangio Glomerular/enzimología , Peptidil-Dipeptidasa A/aislamiento & purificación , Angiotensina I/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Bradiquinina/metabolismo , Captopril/farmacología , Bovinos , Células Cultivadas , Cloruros/farmacología , Cromatografía en Gel , Enalaprilato/farmacología , Mesangio Glomerular/citología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Peso Molecular , Oligopéptidos , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Ratas , Especificidad por Sustrato , Temperatura

RESUMEN

The role of catecholamines in the distribution of intrarenal blood flow and in single-nephron glomerular filtration rate (SNGFR) was evaluated in anesthetized Wistar rats by the Hanssen technique. Epinephrine (EPI) and norepinephrine (NOR) were infused to produce elevations of 20-30 mmHg in mean arterial pressure. Superficial and juxtamedullary nephron perfusion and filtration were determined by the presence of Prussian blue dye. In the control group, 100% of the nephrons presented a homogeneous pattern of perfusion and filtration. In contrast, a heterogeneous distribution of the dye was found even in the larger arteries (arciform and radial), indicating variable perfusion and filtration in both superficial and juxtamedullary nephrons. The effects of EPI and NOR were also evaluated in the superficial cortex by the micropuncture technique in two additional groups of Munich-Wistar rats. Mean SNGFR was 27% and 54% lower in the EPI- and NOR-treated groups, respectively. No change in mean intraglomerular hydraulic pressure was observed after EPI or NOR infusion in spite of a highly scattered pattern, indicating an important variability in perfusion along the superficial cortex, and/or different sensitivity of the pre- and post-glomerular arterioles. The present data suggest that EPI and NOR may affect intrarenal hemodynamics by modifying perfusion and filtration in both superficial and juxtamedullary glomeruli and not by shifting blood flow from superficial to juxtamedullary nephrons. The heterogeneous pattern of perfusion was a consequence of differential vasoconstriction along the intrarenal arteries, probably due to different density and/or sensitivity of the adrenergic receptor subtypes present in the intrarenal vascular tree.

Asunto(s)

Epinefrina/farmacología , Tasa de Filtración Glomerular/efectos de los fármacos , Norepinefrina/farmacología , Animales , Catecolaminas/farmacología , Masculino , Ratas , Ratas Wistar , Flujo Plasmático Renal/efectos de los fármacos

RESUMEN

The role of catecholamines in the distribution of intrarenal blood flow and in single-nephron glomerular filtration rate (SNGFR) was evaluated in anesthetized Wistar rats by the Hanssen technique. Epinephrine (EPI) and norepinephrine (NOR) were infused to produce elevations of 20-30 mmHg in mean arterial pressure. Superficial and juxtamedullary nephron perfusion and filtration were determined by the presence of Prussian blue dye. In the control group, 100 per cent of the nephrons presented a homogeneous pattern of perfusion and filtration. In contrast, a heterogenous distribution of the dye was found even in the larger arteries (arciform and radial), indicating variable perfusion and filtration in both superficial and juxtamedullary nephrons. The effects of EPI and NOR were also evaluated in the superficial cortex by the micropuncture technique in two additional groups of Munich-Wistar rats. Mean SNGFR was 27 per cent and 54 per cent lower in the EPI-and NOR-treated groups, respectively. No change in mean intraglomerular hydraulic pressure was observed after EPI or NOR infusion in spite of a highly scattered pattern, indicating an important variability in perfusion along the superficial cortex, and/or different sensitivity of the pre-and post-glomerular arterioles. The present data suggest that EPI and NOR may affect intrarenal hemodynamics by modifying perfusion and filtration in both superficial and juxtamedullary glomeruli and not by shifting blood folow from superficial to juxtamedullary nephrons. The heterogenous pattern of perfusion was a consequence of differential vasoconstriction along the intrarenal arteries, probably due to different density and/or sensitivity of the adrenergic receptor subtypes present in the intrarenal vascular tree.

Asunto(s)

Ratas , Animales , Epinefrina/farmacología , Tasa de Filtración Glomerular/efectos de los fármacos , Norepinefrina/farmacología , Catecolaminas/farmacología , Ratas Wistar , Flujo Plasmático Renal/efectos de los fármacos

RESUMEN

Endogenous glucocorticoid (GC) has been proposed to play a role in the adaptive functions of remnant nephron and participates in the progression of renal disease. The effect of GC blockade by RU-486 (20 mg/kg), an anti-GC agent, on the progression of chronic renal failure (CRF) was evaluated in Munich-Wistar rats. CRF was induced by 5/6 nephrectomy. Global renal function, glomerular hemodynamics, proteinuria and renal histopathology studies were performed after 60 days of CRF induction. RU administration in control or CRF groups did not induce significant changes in total renal function, mean arterial or intraglomerular hydraulic pressures, 24-hour proteinuria or sclerosis index. However, RU induced a significant reduction in single-nephron glomerular filtration rate in the superficial nephrons in both groups' control (decreases 20%) and CRF (decreases 57%), without changing total glomerular filtration rate, when compared with vehicle administration. These reductions were due to a decline in glomerular plasma flow rate (QA) and in glomerular ultrafiltration coefficient (Kf). These data suggest that GC played a role in the adaptive hyperfiltration associated with the compensatory mechanism but did not participate in the genesis of proteinuria or glomerulosclerosis in this experimental model.

Asunto(s)

Glucocorticoides/antagonistas & inhibidores , Antagonistas de Hormonas/farmacología , Glomérulos Renales/irrigación sanguínea , Glomérulos Renales/efectos de los fármacos , Mifepristona/farmacología , Nefronas/irrigación sanguínea , Nefronas/efectos de los fármacos , Animales , Tasa de Filtración Glomerular/efectos de los fármacos , Glucocorticoides/fisiología , Hemodinámica/efectos de los fármacos , Fallo Renal Crónico/etiología , Fallo Renal Crónico/fisiopatología , Glomérulos Renales/fisiología , Masculino , Nefrectomía , Nefronas/fisiología , Ratas , Ratas Wistar

Asunto(s)

Lesión Renal Aguda/fisiopatología , Glomérulos Renales/fisiopatología , Flujo Sanguíneo Renal Efectivo/fisiología , Lesión Renal Aguda/etiología , Animales , Ciclosporina/efectos adversos , Ciclosporina/farmacología , Tasa de Filtración Glomerular , Hemodinámica/fisiología , Humanos , Inmunosupresores/efectos adversos , Inmunosupresores/farmacología , Isquemia/fisiopatología , Glomérulos Renales/efectos de los fármacos , Flujo Sanguíneo Renal Efectivo/efectos de los fármacos

RESUMEN

Whole-kidney function and glomerular hemodynamics were evaluated after acute (50 mg/kg, iv, in bolus) and short-term chronic (50 mg mg/kg, ip, 5 days) acyclovir (ACV) and short-term chronic ganciclovir (Gan; 30 mg/kg, ip, 5 days) treatment in envolemic Munich-Wistar rats. The evaluation of whole-kidney function of the ACV groups showed a significant reduction in total GFR (0.96 +/- 0.10 to 0.28 +/- 0.02 mL/min in the acute group, P < 0.05, and 1.04 +/- 0.09 to 0.33 +/- 0.04 mL/min in the chronic group, P < 0.05) with a marked increase in total renal vascular resistance (TRVR) (33 +/- 5 to 122 +/- 26 mm Hg.min/mL in the acute group and 28 +/- 3 to 74 +/- 18 mm Hg.min/mL in the chronic group, P < 0.05) and a reduction in RPF (2.29 +/- 0.25 to 0.81 +/- 0.15 mL/min in the acute group and 2.57 +/- 0.36 to 1.30 +/- 0.40 mL/min in the chronic group, P < 0.05). Conversely, urinary flow (V') was unchanged (3.6 +/- 0.4 to 3.6 +/- 0.2 microL/min in the acute group) or elevated (3.7 +/- 0.6 to 6.6 +/- 1.4 microL/min in the chronic group, P < 0.05). The evaluation of glomerular hemodynamics after ACV treatment showed a reduction in single-nephron GFR (SNGFR) (46.4 +/- 5.3 to 26.2 +/- 3.4 nL/min in the acute group and 38.7 +/- 5.7 to 21.1 +/- 5.7 nL/min in the chronic group, P < 0.05), a significant elevation in total arteriolar resistance (RT) (2.90 +/- 0.44 to 4.94 +/- 0.77 x 10(10) dyn.s.cm-5 in the acute group and 3.72 +/- 0.45 to 9.00 +/- 2.40 x 10(10) dyn.s.cm-5 in the chronic group, P < 0.05) and a severe reduction in glomerular plasma flow rate (QA) (152.6 +/- 29.5 to 103.8 +/- 27.8 nL/min in the acute group and 149.1 +/- 29.8 to 68.5 +/- 10.0 nL/min in the chronic group, P < 0.05). However, the glomerular ultrafiltration coefficient, Kf, was changed only in the chronic group (0.1002 +/- 0.0165 to 0.0499 +/- 0.0090 nL/(s.mm Hg), P < 0.05). After Gan treatment, no changes were observed in GFR (1.04 +/- 0.09 to 0.96 +/- 0.08 mL/min, with the maintenance of RPF (2.57 +/- 0.36 to 2.66 +/- 0.34 mL/min) and a nonsignificant reduction in TRVR (28 +/- 3 to 20 +/- 3 mm Hg.min/mL. The short-term Gan treatment also showed a different pattern in glomerular hemodynamics by inducing an elevation in SNGFR (38.7 +/- 5.7 to 50.3 +/- 2.8 nL/min, P < 0.05) with no changes in QA (150 +/- 30 to 135 +/- 22 nL/min) and a mild vasodilation, RT (3.7 +/- 0.5 to 2.7 +/- 0.3 x 10(10) dyn.s.cm-5, P < 0.05) associated with an increment in Kf (0.1002 +/- 0.0165 to 0.2400 +/- 0.0700 nL/(s.mm Hg), P < 0.05). Thus, ACV induced acute renal failure by reducing GFR and SNGFR by an increase in TRVR and RT with a reduction in RPF and QA. Also, after short-term treatment with ACV, a reduction in Kf led to a reduction of SNGFR. On the other hand, Gan treatment did not induce acute renal failure by the adopted techniques.

Asunto(s)

Aciclovir/envenenamiento , Antivirales/envenenamiento , Ganciclovir/envenenamiento , Glomérulos Renales/irrigación sanguínea , Riñón/efectos de los fármacos , Lesión Renal Aguda/inducido químicamente , Animales , Tasa de Filtración Glomerular/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Riñón/fisiopatología , Masculino , Ratas , Ratas Wistar , Circulación Renal/efectos de los fármacos , Resistencia Vascular/efectos de los fármacos

RESUMEN

The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.

Asunto(s)

Mesangio Glomerular/enzimología , Túbulos Renales/enzimología , Nefronas/enzimología , Peptidil-Dipeptidasa A/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Mesangio Glomerular/citología , Túbulos Renales Colectores/enzimología , Túbulos Renales Proximales/enzimología , Masculino , Oligopéptidos , Peptidil-Dipeptidasa A/orina , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Especificidad por Sustrato , Transcripción Genética