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Acetaminophen (APAP) is a widely prescribed analgesic and antipyretic drug. In the present work, we studied the effects of glutamine (Gln) in an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. Renal function, histological characteristics, and Na+,K+-ATPase cortical abundance and distribution were analyzed. The appearance of HSP70 and actin in urine was also evaluated. Myeloperoxidase (MPO) activity in cortical tissue was measured as an index of the inflammatory response. Gln administration 30 min before APAP protected from the renal functional and histological damage promoted by APAP. Rats that received the dual treatment Gln and APAP (Gln/APAP) showed the same level of Na+,K+-ATPase cortical induction as APAP-treated animals, but the enzyme maintained its normal basolateral localization. HSP70 abundance was increased up to the same level in the Gln, APAP, and Gln/APAP groups. Urinary HSP70 and actin were detected only in the APAP-treated animals, reinforcing the protection of renal tubular integrity afforded by the Gln pretreatment. Gln pretreatment also protected from the increment in MPO activity promoted by APAP. Our results support the idea that Gln pretreatment could be a therapeutic option to prevent APAP-induced renal injury. Fil: Brovedan, Marco Alcides. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina Fil: Molinas, Sara Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina Fil: Pisani, Gerardo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina Fil: Monasterolo, Liliana Alicia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina Fil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Area Farmacología; Argentina. Universidad Nacional de Rosario. Centro de Investigaciones de la Universidad Nacional de Rosario; Argentina

Accumulating evidence indicates the association between changes in circulating sex steroid hormone levels and the development of diabetic nephropathy. However, the renal synthesis of steroid hormones during diabetes is essentially unknown. Male Wistar rats were injected with streptozotocin (STZ) or vehicle. After one week, no changes in functional or structural parameters related to kidney damage were observed in STZ group; however, a higher renal expression of proinflammatory cytokines and HSP70 was found. Expression of Steroidogenic Acute Regulatory protein (StAR) and P450scc (CYP11A1) was decreased in STZ kidneys. Incubation of isolated mitochondria with 22R-hydroxycholesterol revealed a marked inhibition in CYP11A1 function at the medullary level in STZ group. The inhibition of these first steps of renal steroidogenesis in early STZ-induced diabetes led to a decreased local synthesis of pregnenolone and progesterone. These findings stimulate investigation of the probable role of nephrosteroids in kidney damage associated with diabetes. Fil: Pagotto, Melina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Roldán, María Lorena. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Molinas, Sara Maria. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina Fil: Raices, Trinidad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Pisani, Gerardo Bruno. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Ciencias Fisiológicas. Área Morfología; Argentina Fil: Pignataro, Omar Pedro. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Monasterolo, Liliana Alicia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Ciencias Fisiológicas. Área Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina

Acetaminophen (APAP) is an analgesic-antipyretic drug widely used in children. In the present study, we used an in vivo model of APAP-induced nephrotoxicity in male Wistar rats. We analyzed whether toxic doses of APAP could induce heat shock protein 70 (HSP70) in the kidney and whether HSP70 could be detected in urine. Renal function and histological evaluation of the kidneys were performed at different times after APAP administration (1,000 mg/kg body weight i.p.). Cellular injury was assessed by Triton X-100 solubilization of Na(+)/K(+) ATPase. Renal and hepatic glutathione levels were also measured. Urinary N-acetyl-beta-D glucosaminidase (NAG) excretion increased 4 h after intoxication. At this time, urea and creatinine were at control levels and a slight degree of histological alteration was detected. Kidney microscopic evaluation, Na(+)/K(+) ATPase solubility, creatinine, and urea levels and NAG excretion did not differ from those of controls 48 h after APAP administration. HSP70 was detected in urine obtained from 4 to 24 h after APAP administration. HSP70 abundance in renal cortex was increased at early time points and 48 h after APAP administration. Urinary HSP70 excretion would be a marker of its renal induction combined with the loss of tubule integrity. NAG would be a suitable early biomarker of APAP-induced nephrotoxicity.

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.

The purpose of this study was to assess the effects of the cyclooxygenase inhibitor, indomethacin, and some vasoactive agents on the renal functional parameters during the early liver injury induced by four days bile duct ligation (BDL). Wistar rats with four days-BDL and control-sham operated were used. Renal function was measured in anesthetized rat treated with a single dose of indomethacin (control, 0.3, 1.0, 3.0 mg/kg b.w.; i.v.) one hour before clearance studies. Sulindac effects were also evaluated (5 mg/kg b.w., i.p). Isolated rat kidney preparations from control and BDL donor rats were used to study renal vascular response to noradrenaline, carbachol or sodium nitroprusside. The bile duct ligation promoted a diminished renal cortical plasma flow (RCPF) on the fourth day post surgery accompanied with a diminution in the glomerular filtration rate (GFR), increased filtration fraction and increased fractional excretion of water and sodium. Indomethacin 0.3 mg/kg induced an increase in GFR and RCPF, maintaining the high filtration fraction in BDL rats. The other doses did not alter these parameters as compared with bile duct ligated rats without treatment, but indomethacin 3 mg/kg caused a significant increase in filtration fraction. Indomethacin induced dose-dependent diminution in natriuresis in sham and BDL groups. Sulindac did not modify hemodynamic parameters, but induced antinatriuresis and antidiuresis in both experimental groups. Maximal vascular responses to noradrenaline measured in isolated rat kidneys were statistically diminished in BDL-rats as compared with controls (C, n=7; 35.0+/-2.3 mmHg ml(-1) min; BDL-rats, n=5; 23.8+/-0.7 mmHg ml(-1) min; p0.02), without changes in EC15. Maximal relaxation induced by sodium nitroprusside in the phenylephrine (PHE)-pre-constricted renal vasculature in control preparations did not differ from that observed in BDL group (C: n=6; 49.5+/-2.3%). Values of EC50 were 1.26+/-0.07 microM (n=6) in control preparations and 0.34+/-0.03 microM (n=4) in kidneys from BDL-rats (p0.001). Carbachol induced a biphasic relaxation of PHE-pre-constricted renal vasculature. No differences in maximal responses were found. EC50 value of the second phase in BDL group was significantly decreased compared to control preparations (C: n=6, 0.47+/-0.05 microM; BDL: n=6, 0.22+/-0.03 microM p0.001). The present results show that the altered renal function after a short time post bile duct ligation is determined, at least in part, by increased release of arachidonic derivatives in vascular bed and tubular cells. At this stage of liver injury, the alteration in the renal vascular response to different vasoactive agents is remarkable.

The effects of diazepam were studied in the isolated rat kidney under conditions of constant flow. Kidneys were perfused with modified Ringer-Krebs solution. Diazepam produced a raised fractional excretion of water and sodium without hemodynamic changes, suggesting a direct effect on tubular transport mechanisms. Diazepam decreased renal perfusion pressure in a concentration-dependent fashion when kidneys were pretreated with either noradrenaline or potassium chloride. Similar responses were observed when 7-chloro-5-[4-chloro-phenyl]-1, 3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro 5-4864) or clonazepam was used. These data provide evidence for a relaxant effect of benzodiazepines on preconstricted renal vasculature.

The effects of different acetaminophen (APAP) concentrations (1,5 or 10 mM) on renal function were investigated in the isolated perfused rat kidney (IPK). APAP was added to the perfusion media as a single dose after a equilibration time and control periods. Changes in fractional excretion of sodium (FENa), water \(\left( {FE_{H_2 O} } \right)\), glucose (FEglu) and in glomerular filtration rate (GFR) were measured. The lower concentration used only modified the \(FE_{H_2 O} \). APAP 10 mM induced an increment in \(FE_{H_2 O} \) (72% higher than control preparation), FENa (79% higher than control preparation) and an elevation in glucose excretion (55% higher than control preparation), associated with a decrease in GFR (23% lower than control preparation). The influence of PGE2 on the effects of APAP was also investigated. PGE2 prevented the APAP-induced decrease in GFR and in glucose reabsorption, but did not change the pattern of sodium and water handling. The effects of another vasodilator, verapamil, on APAP-induced renal effects were also tested. Verapamil prevented the glomerular but not the tubular effects of APAP. Urinary APAP excretion data showed a similar availability of APAP to the tubular cells in all the groups. Our data suggest that APAP exerts a direct action in the IPK, affecting hemodynamic and tubular functions, and that the latter are not a consequence of hemodynamic alterations.

Cumulative evidence demonstrated effective downstream metabolism of pregnenolone in renal tissue. The aim of this study was to evaluate the expression and functional activity of cytochrome P450 side chain cleavage enzyme (CYP11A1), which converts cholesterol into pregnenolone, in adult rat kidney. Immunohistochemical labeling for CYP11A1 was observed in renal cortex and medulla, on structures identified as distal convoluted tubule and thick ascending limb of Henle's loop, respectively. Immunoblotting analysis corroborated the renal expression of the protein in inner mitochondrial membrane fractions. The incubation of isolated mitochondria with the membrane-permeant cholesterol analogue 22R-hydroxycholesterol resulted in efficient formation of pregnenolone, the immediate precursor for the synthesis of all the steroid hormones. The low progesterone production rate observed in these experiments suggested a poor activity of 3β-hydroxysteroid dehydrogenase enzyme in renal mitochondria. The steroidogenic acute regulatory protein (StAR), involved in the mitochondrial import of cholesterol, was detected in renal tissue at both mRNA and protein level. Immunostaining for StAR showed similar distribution to that observed for CYP11A1. The expression of StAR and CYP11A1 was found to be higher in medulla than in cortex. This enhanced expression of steroidogenesis-related proteins correlated with a greater pregnenolone synthesis rate and higher steroid hormones tissular content measured in medulla. In conclusion, we have established the expression and localization of StAR and CYP11A1 protein, the ability of synthesizing pregnenolone and a region-specific content of sex hormones in the adult rat kidney. These data clearly show that the kidney is a steroid hormones synthesizing organ. It is proposed that the existence in the kidney of complete steroidogenic machinery would respond to a physiological significance. © 2010 Elsevier Ireland Ltd. Fil: Pagotto, Melina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Roldán, María Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pagotto, Romina María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Lugano, Maria C.. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pisani, Gerardo. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Rogic, Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Molinas, Sara Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Fisiología Experimental. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Fisiología Experimental; Argentina Fil: Trumper, Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina Fil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Monasterolo, Liliana Alicia. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Background/Aims: Contributions to the understanding of acute renal failure (ARF) pathogenesis have not been translated into an effective clinical therapy. We studied the effects of pretreatment with the angiotensin II type 1 (AT1) receptor blocker, losartan, on renal function, tissue injury, inflammatory response and serum aldosterone levels in a model of ischemic ARF. Methods: Rats underwent unilateral renal ischemia followed by 24 h of reperfusion (IR), and were pretreated or not with 8 (IRL8) or 80 (IRL80) mg/kg/day of losartan for 3 days. Results: IR kidneys showed marked renal dysfunction, epithelial damage, capillary congestion, increased myeloperoxidase (MPO) activity and increased TNF-α, IL1-β and IL-6 mRNA levels. IRL80 kidneys showed protection against dysfunction and tissue injury, associated with normal MPO activity and cytokine mRNA levels. The lower dose was not able to achieve the same degree of functional renoprotection and could not prevent an increase of MPO or proinflammatory cytokine mRNA levels. The high losartan dose completely prevented an increase of serum aldosterone levels induced by IR. Conclusion: Renoprotection of the high losartan dose would be mainly mediated by its anti-inflammatory actions. Our results show a potential pathophysiological role of AT1 activation in promoting renal dysfunction, structural injury, inflammation and aldosterone elevation after IR injury.

The effects of the central-type benzodiazepine receptor antagonist flumazenil on renal vascular tone and its ability to reverse the benzodiazepine-induced vasodilation were investigated. The isolated and perfused rat kidney model was used. Flumazenil was unable to modify renal vascular resistance under basal conditions and in noradrenaline-pretreated kidneys. Relaxation induced by diazepam or clonazepam of noradrenaline-preconstricted renal vasculature was blunted by 10 microM flumazenil. These results suggest that central-type benzodiazepine receptors could be involved in benzodiazepine-induced renal vasodilation.

Glutamic acid decarboxylase (GAD) activity was measured in the oviduct of normal rats in diestrous and in rats ovariectomized (OVX) seven days before. OVX induced a significant decrease of GAD activity in the Fallopian tube. This effect was completely reversed by coadministration of estradiol benzoate + progesterone (E + P). Simultaneous injection of atropine, but not of α-methyl-para-tyrosine or labetalol, completely prevented the activation of GAD induced by ovarian sterois. Moreover. prostigmin significantly potentiated the action of E + P on GAD activity in the rat oviduct. These data clearly suggest the participation of acetylcholine in the mecanisms whereby ovarian steroids regulate GAD activity in the rat Fallopian tube.