Relationship between serum lithium concentration and kidney damage in a preclinical model.

OBJECTIVES: The aim of the present study was to assess whether there is a relationship between serum lithium concentrations and the magnitude of kidney damage in a preclinical model. METHODS: Thirty Wistar male rats were randomized into three groups: control group fed ad libitum powered standard diet for 3 months; and experimental groups fed ad libitum the same diet supplemented with 30 or 60 mmol/kg diet for 3 months (LowLi and HighLi groups respectively). Laboratory parameters were assessed at months 1 and 3 and histopathological changes were evaluated after 3 months. RESULTS: Serum lithium levels in experimental rats were within therapeutic range used in humans throughout the entire experiment. After 3 months of treatment, lithium levels were statistically higher in HighLi group. Rats of the LowLi group showed dilation of cortical tubules although with similar clearance of creatinine. Rats from the HighLi group had greater histopathological damage in addition to lower creatinine clearance than the other two groups. CONCLUSIONS: Our study suggests that during long-term treatments, even with serum lithium levels within the therapeutic range used in humans, the risk of kidney damage could increase proportionally to the serum lithium concentration.

Changes in CNS cells in hyperammonemic portal hypertensive rats.

Rats with pre-hepatic portal hypertension because of partial portal vein ligation develop minimal hepatic encephalopathy (MHE) with hyperammonemia, impaired blood-brain barrier, mild brain edema, and severe mitochondrial changes in the hippocampus. The aim of this study was to evaluate changes of different neural cells in the cerebral cortex and the hippocampus. Animals were divided into two groups, MHE and sham. Astrocytes were studied by immunostaining with glial fibrillary acidic protein and S100ß protein; neurons were immunostained with neuronal nuclear marker, microtubule associated protein-2, and NF-200 and capillaries with Nestin. The hypoxia-inducible factor 1α (HIF-1α) and its downstream proteins, P-glycoprotein (P-gp) and erythropoietin receptor (Epo-R), were also evaluated. Astrocytes were increased in area and number only in the hippocampus, while S100ß increased in both brain areas in MHE animals. Microtubule associated protein-2 and NF-200 immunoreactivities (-ir) were significantly reduced in both areas. Hippocampal Nestin-ir was increased in MHE animals. These cellular changes were similar to those described in ischemic conditions, thus HIF-1α, P-gp, and Epo-R were also evaluated. A high expression of HIF-1α in cortical neurons was observed in the MHE group. It is likely that this hypoxia-like state is triggered via ammonia occupying the binding domain of HIF-1α and thereby preventing its degradation and inducing its stabilization, leading to the over-expression of P-gp and the Epo-R.

High fat diet-induced liver steatosis promotes an increase in liver mitochondrial biogenesis in response to hypoxia.

Mitochondrial DNA (mtDNA) copy number plays a key role in the pathophysiology of metabolic syndrome-related phenotypes, but its role in non-alcoholic fatty liver disease (NAFLD) is not well understood. We evaluated the molecular mechanisms that may be involved in the regulation of liver mtDNA content in a high-fat-induced rat model of NAFLD. In particular, we tested the hypothesis that liver mtDNA copy number is associated with liver expression of HIF-1α. Rats were given either standard chow diet (SCD, n = 10) or high-fat diet (HFD, n = 15) for 20 weeks. Subsequently, mtDNA quantification using nuclear DNA (nDNA) as a reference was carried out using real time quantitative PCR. HFD induced a significant increase in liver mtDNA/nDNA ratio, which significantly correlated with the liver triglyceride content (R: 0.29, P < 0.05). The liver mtDNA/nDNA ratio significantly correlated with the hepatic expression of HIF-1α mRNA (R: 0.37, P < 0.001); liver HIF-1α mRNA was significantly higher in the HFD group. In addition, liver cytochrome c oxidase subunit IV isoform 1 (COX4I1) mRNA expression was also positively correlated with liver mtDNA content. The hepatic expression of mRNA of transcriptional factors that regulate mitochondrial biogenesis, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and PGC-1ß, nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor δ and Tfam, was not associated with the liver mtDNA content. Neither hepatocyte apoptosis nor oxidative stress was involved in the HIF-1α-mediated increase in mtDNA copy number. In conclusion, we found that HFD promotes an increase in liver mitochondrial biogenesis in response to hypoxia via HIF-1α, probably to enhance the mitochondrial function as well as to accommodate the metabolic load.

Renal Damage During Continuous Versus Intermittent Treatment with Lithium.

The aim of this study was to evaluate renal damage in animals treated with lithium continuously versus intermittently. Rats were randomized into three groups: control group fed ad libitum powered standard diet for 3 months and two experimental groups, one of them fed ad libitum the same diet or the same diet supplemented with 60 mmol of lithium/kg diet every alternate week, for 3 months and the other fed ad libitum powered standard diet for one and a half month and the same diet supplemented with 60 mmol of lithium/kg diet for the last month and a half. Lithemias in experimental groups were within therapeutic range used in humans. At the end of the protocol, diuresis was higher in experimental groups compared to control group. There was no difference in serum creatinine and creatinine clearance. Both experimental groups showed hypertrophy, hyperplasia, and dilatation of cortical collecting tubules although dilatation was greater in continuous group. Longer studies are necessary to clarify the evolution of renal damage. Our preliminary study shows that histopathological damage associated with the use of lithium occurs during both continuous and intermittent treatment, but it seems to be somewhat greater in the continuous group.

Chronic progressive nephropathy: functional, morphological, and morphometrical studies.

Some aspects of the functional, morphological, and morphometrical characteristics of chronic progressive nephropathy occurring in 18- to 26-month-old male rats and in 3-month-old control rats were studied. Rats with chronic progressive nephropathy were proteinuric and showed a slight increase in serum creatinine and no changes in blood pressure. The morphological changes were studied by light microscopy, high-resolution light microscopy, and electron microscopy. They showed focal and segmental or global glomerulosclerosis, the three types of atrophic tubules ("classic," "thyroid-like," and "endocrine") described by Nadasdy et al, as well as interstitial fibrosis with mononuclear cell infiltrates. On certain occasions, small vessels showed hyalinosis. Glomerular morphometrical studies showed a biphasic pattern in the glomeruli progressing toward obsolescence. Vascular morphometrical studies showed significant increase in media wall thickness and media cross-sectional area in the 18- to 26-month-old rats. These results support the hypothesis that changes in the vascular system are not of utmost importance in the pathogenesis of chronic progressive nephropathy, and that glomerular sequential changes seem to be of paramount significance in the progression of the disease.

Toxicological study of bee venom (Apis mellifera mellifera) from different regions of the province of Buenos Aires, Argentina.

Samples of Apis mellifera mellifera venom from different hives in two regions of the Buenos Aires province and its pool were analyzed for their lethal potency, myotoxic, defibrinogenating, hemolytic and inflammatory-edematizing activity and for the histological alterations they produce in the heart, lungs, kidneys, skeletal muscle and liver of mice. In vitro studies focused on the venom's hemolytic activity in different systems and species (horse, man, sheep and rabbit), the cytotoxicity in cellular lines, and on the proteolytic and coagulant activity in plasma and fibrinogen. Hemolytic activity, either observed in vitro or in vivo, showed similar toxicity levels for all samples. Erythrocytes of different species varied in their sensitivity to the venom pool, equines being the most sensitive and sheep the most resistant to direct hemolytic action. Local and systemic myotoxicity was evidenced by either the elevation of serum creatine kinase and/or histopathological lesions, observed in different muscles. All samples caused significant pathological alterations; pulmonary, cardiac, renal and skeletal muscle lesions were substantive and can be related to the pathophysiological mechanisms of envenomation. The venoms from different apiaries and regions of the Buenos Aires province showed very similar toxicological characteristics. These results suggest that severity of envenomation in case of a swarming could therefore be more related to the number of bees than to the differential toxicity of the venom from different regions of the province. This is the first study on the toxicity and toxicological characteristics of Apis mellifera venom in Argentina.

A new venomous scorpion responsible for severe envenomation in Argentina: Tityus confluens.

In Argentina the scorpions of medical importance belong to the genus Tityus (T.), particularly the species T. trivittatus, the only scorpion whose sting is recognized to be associated with severe human envenoming and death. This genus is distributed from the north of the Patagonian region to the center and some provinces in the north of the country. During the period 2003-2006 four children died following scorpion stings, of which one was certainly and three were probably by T. confluens. In 2006, in the province of Tucumán, a girl died by scorpion envenoming and the scorpion responsible for the death, found in her shoe, was T. confluens. We thus studied the toxicity of venom gland homogenates from T. confluens from the provinces of Jujuy and Catamarca, and of crude venom from specimens from Catamarca and the province of La Rioja. The lethal potencies of the telson homogenates were 7.0 and 18.6microg/g for Jujuy and Catamarca, respectively, while the lethal potency of the crude venom was 0.7microg/g. Injected mice showed generalized congestion and hepatic lesions. Pancreatic damage was observed in some animals. Lungs showed congestion and foci of hemorrhage and mild edema. The heart showed injury in the muscular fibers. The venom showed high reactivity against anti-T. trivittatus antivenom and against two anti-T. serrulatus antivenoms. The anti-T. trivittatus antivenom neutralized the lethal activity of T. confluens venom. In addition, the venom reacted very slightly against an anti-Centruroides antivenom. Therefore, the stings of this scorpion must be considered of risk for humans to the same degree as the stings of T. trivittatus.

Role of Oxidative Stress in Lithium-Induced Nephropathy.

Long-term lithium treatment was associated with chronic kidney disease and renal failure although the underlying pathogenic mechanisms are not certainty known. The aim of this study was to evaluate changes in oxidative stress measures as well as renal functional and structural alterations associated with chronic use of lithium in rats. Forty Wistar male rats were randomized into four groups: control groups fed ad libitum powered standard diet for 1 and 3 months and experimental groups fed ad libitum the same diet supplemented with 60 mmol/kg diet for 1 and 3 months. Histopathological changes, laboratory parameters, and oxidative stress measurements were assessed at months 1 and 3. The experimental animals showed alteration of the cortical tubules from the first month of lithium-treatment and a decrease in the glomerular filtration rate and in the glomerular area at the third month. There was an increase in thiobarbituric acid reactive substances and carbonyls, as well as an increase in reduced glutathione, in the kidney of rats exposed to lithium. These changes were evident from the first month of treatment and remained throughout the experiment. Our results suggest that, oxidative stress could be one of the pathogenic mechanisms involved in the structural and functional alterations of the kidney associated with prolonged use of lithium. The study of the pathogenic mechanisms involved in lithium-induced nephropathy is a critical issue for the development of new strategies for prevention and/or early detection.

Menhaden Oil Rich Diet and Experimental Renal Damage Due to Ischemia Reperfusion.

Renal necrosis can be induced in weanling rats due to choline deficient diet. Menhaden oil has a protective effect against the development of renal necrosis in choline deficient weanling rats. The aim of this work was to determine the effects of menhaden oil in a model of acute kidney injury due to ischemia reperfusion. Wistar rats were divided into two groups and fed vegetable oils or menhaden oil as lipids. Unilateral renal ischemia was performed for 30 minutes and animals were sacrificed 48 hours later. Histopathological examination showed no significant differences between groups. Menhaden oil did not prevent histopathological lesions.

Ocular lesions and experimental choline deficiency.

Previous studies have shown ocular haemorrhages in choline-deficient rats. The aim of this paper is to study further the relationship between ocular and renal lesions and biochemical alterations in rats fed a choline-deficient diet. Fifty one weanling male Wistar rats, were divided into two groups. Thirty one of them were fed a choline-deficient diet and the rest was fed a choline-supplemented diet ad libitum. Animals from both groups were killed between the fifth and the eighth day. Urea, creatinine and homocysteine concentrations in blood were determined. Eyes were used for light microscopy study; high resolution light microscopy and the study of the retina as "rétine a plat". Kidneys were studied by light microscopy. Choline-supplemented rats did not show ocular or renal lesion. Choline-deficient rats that showed renal lesions, tubular or cortical necrosis, did not always have ocular changes. There were no ocular changes in the only choline-deficient rat without renal lesion. The ocular changes consisted mainly in haemorrhage in both cameras and ciliary and vitreous bodies. Correlations between ocular and renal lesion (r = 0.72, p < 0.0001, CI 95%: 0.48-0.86); ocular lesion and creatinine (r = 0.86, p < 0.0001, Cl 95%: 0.72-0.93) and ocular lesion and urea (r = 0.70, p < 0.0001, Cl 95%: 0.44-0.85) were positive. Choline-deficiency induces ocular haemorrhagic lesions after the development of renal necrosis. The ocular pathology could be due to the immaturity of the ocular vasculature at this age. The hyaloid, choroid and retinal system are involved.

Microalbuminuria and early renal response to lethal dose Shiga toxin type 2 in rats.

In Argentina, hemolytic uremic syndrome (HUS) constitutes the most frequent cause of acute renal failure in children. Approximately 2%-4% of patients die during the acute phase, and one-third of the 96% who survive are at risk of chronic renal sequelae. Little information is available about the direct effect of Shiga toxin type 2 (Stx2) on the onset of proteinuria and the evolution of toxin-mediated glomerular or tubular injury. In this work, rats were injected intraperitoneally with recombinant Escherichia coli culture supernatant containing Stx2 (sStx2; 20 µg/kg body weight) to induce HUS. Functional, immunoblotting, and immunohistochemistry studies were carried out to determine alterations in slit diaphragm proteins and the proximal tubule endocytic system at 48 hours post-inoculation. We detected a significant increase in microalbuminuria, without changes in the proteinuria values compared to the control rats. In immunoperoxidase studies, the renal tubules and glomerular mesangium showed an increased expression of transforming growth factor ß(1)(TGF-ß(1)). The expression of megalin was decreased by immunoperoxidase and the cytoplasm showed a granular pattern of megalin expression by immunofluorescence techniques. Western blot analysis performed in the renal cortex from sStx2-treated and control rats using anti-nephrin and anti-podocalyxin antibodies showed a decreased expression of these proteins. We suggest that the alterations in slit diaphragm proteins and megalin expression could be related to the development of microalbuminuria in response to lethal doses of Stx2.

Mn-loaded apoferritin: a highly sensitive MRI imaging probe for the detection and characterization of hepatocarcinoma lesions in a transgenic mouse model.

Mn-Apo is a highly sensitive MRI contrast agent consisting of ca. 1000 manganese atoms entrapped in the inner cavity of apoferritin. Part of the metallic payload is in the form of Mn(2+) ions that endow the nano-sized system with a very high relaxivity that can be exploited to detect hepatocellular carcinoma in mice. Cellular studies showed that Mn-Apo is readily taken up by normal hepatocytes via the ferritin transporting route. Conversely, hepatoma cells (HTC) displayed a markedly reduced ability to entrap Mn-Apo from the culture medium. The i.v. administration of Mn-Apo into C57BL/6 J mice resulted in a marked liver tissue hyperintensity in T(1)-weighted MR image 20 min after injection. When injected into HBV-tg transgenic mice that spontaneously develop hepatocellular carcinoma (HCC), Mn-Apo allowed a clear delineation of healthy liver tissue and tumor lesions as hyperintense and hypointense T(1)-weighted MR images, respectively. Immunohistochemistry analysis correlated Mn-Apo cellular uptake to SCARA5 receptor expression. When the MRI contrast induced by Mn-Apo was compared with that induced by Gd-BOPTA (a commercial contrast agent known to enter mouse hepatocytes through organic anion transporters) it was found that only some of the lesions were detected by both agents while others could only be visualized by one of the two. These results suggest that Mn-Apo may be useful to detect otherwise invisible lesions and that the extent of its uptake directly reports the expression/regulation of SCARA5 receptors. Mn-Apo contrast-enhanced MR images may therefore contribute to improving HCC lesion detection and characterization.

Isolation, amino acid sequence and biological characterization of an "aspartic-49" phospholipase A2 from Bothrops (Rhinocerophis) ammodytoides venom.

A phospholipase enzyme was separated by chromatography from the venom of the snake Bothrops (Rhinocerophis) ammodytoides and characterized. The experimentally determined molecular weight was 13,853.65 Da, and the full primary structure was determined by Edman degradation and mass spectrometry analysis. The enzyme contains 122 amino acids residues closely stabilized by 7 disulfide bridges with an isoelectric point of 6.13. Sequence comparison with other known secretory PLA2 shows that the enzyme isolated belongs to the group II, presenting an aspartic acid residue at position 48 (numbered by convention as Asp49) of the active site, and accordingly displaying enzymatic activity. The enzyme corresponds to 3% of the total mass of the venom. The enzyme is mildly toxic to mice. The intravenous LD50 of this phospholipase in CD-1 mice was around 6 µg/g of mouse body weight (more exactly 117 µg/mouse of 20 g) and the minimal mortal dose (MMD) was estimated to be close to 10 µg/g. In contrast, the LD50 of the venom was circa 2 µg/g mouse body weight. Toxicological analyses of the purified enzyme were performed in vitro and in vivo using experimental animals (mice and rats). The enzyme at high doses caused pulmonary congestion, intraperitoneal bleeding, inhibition of clot retraction and muscle tissue alterations with increasing of creatine kinase levels.

[Obtention of an antivenom against Phoneutria nigriventer (arachnida; ctenidae) venom]. / Obtención de un antiveneno contra el veneno de Phoneutria nigriventer (arachnida; ctenidae).

Envenomation by spiders of the genus Phoneutria ("banana spider") may be lethal, especially in children. The only available specific treatment is the use of antivenom, which is produced by only one laboratory in the world. In this study we report the development of an equine F (ab')2 experimental antivenom raised against the venom of Phoneutria nigriventer. The antivenom neutralized the venom of spiders from different regions of Argentina (Misiones and Jujuy), the development of envenomation symptoms in experimental animals was totally inhibited. These results show that local production of this type of antivenom is possible. Independence of production is important since international acquisition is always conditioned by the availability of stock surplus from the sole producer.

Obtención de un antiveneno contra el veneno de Phoneutriab nigriventer (arachnida; ctenidae) / Obtention of an antivenom against Phoneutria nigriventer (arachnida; ctenidae) venom

Los envenenamientos por la picadura de arañas del género Phoneutria (“araña de los bananeros”) pueden causar la muerte, especialmente en niños. El único tratamiento específico es la aplicación del antiveneno, del cual hay un solo productor mundial. En este trabajo se comunica el desarrollo de un antivenenoexperimental en equinos, a fragmentos F(ab´)2, contra el veneno de Phoneutria nigriventer. El antiveneno obtenidoneutralizó el veneno de arañas de esta especie provenientes de distintas zonas de la Argentina (Misiones y Jujuy). La sinología del envenenamiento en los animales de experimentación fuetotalmente inhibida. La experiencia desarrollada muestra que la producción local de este antiveneno es viable, lo que equivaldría a no depender de la adquisición de este tipo de productosdel extranjero, lo que en ocasiones es imposible, pues está supeditada al excedente de stock del productor.

Lesión destructiva de la línea media: granulomatosis de Wegener / Medline destructive lesion: Wegener’s granulomatosis

Un paciente de 38 años de edad fue derivado con diagnóstico de leishmaniasis mucocutánea por un proceso destructivo de la nariz y los senos paranasales, con severo compromiso oftalmológico. Este trabajo discute los diagnósticos diferenciales de las lesiones destructivas de la línea media. El estudio de estas lesiones es dificultoso, pero debe efectuarse con una aproximación sistemática y múltiples técnicas diagnósticas. El diagnóstico final fue de Granulomatosisde Wegener.

Ocular lesions and experimental choline deficiency

Previous studies have shown ocular haemorrhages in choline-deficient rats. The aim of this paper is to study further the relationship between ocular and renal lesions and biochemical alterations in rats fed a choline-deficient diet. Fifty one weanling male Wistar rats, were divided into two groups. Thirty one ofthem were fed a choline-deficient diet and the rest was fed a choline- supplemented diet ad libitum. Animalsfrom both groups were killed between the fifth and the eighth day. Urea, creatinine and homocysteine concentrations in blood were determined. Eyes were used for light microscopy study; high resolution lightmicroscopy and the study of the retina as “rétine a plat”. Kidneys were studied by light microscopy. Cholinesupplementedrats did not show ocular or renal lesion. Choline-deficient rats that showed renal lesions, tubular or cortical necrosis, did not always have ocular changes. There were no ocular changes in the only cholinedeficient rat without renal lesion. The ocular changes consisted mainly in haemorrhage in both cameras andciliary and vitreous bodies. Correlations between ocular and renal lesion (r=0.72, p<0.0001, CI 95%: 0.48-0.86); ocular lesion and creatinine (r=0.86, p<0.0001, CI 95%: 0.72-0.93) and ocular lesion and urea (r=0.70, p<0.0001, CI 95%: 0.44-0.85) were positive. Choline-deficiency induces ocular haemorrhagic lesions after the development of renal necrosis. The ocular pathology could be due to the immaturity of the ocular vasculature at this age. The hyaloid, choroid and retinal system are involved

Estudios previos han demostradohemorragia ocular en ratas deficientes en colina. El objetivo de este trabajo es profundizar en la relación entre las alteraciones oculares, renales y bioquímicas en ratas deficientes en colina. Cincuenta y una ratas Wistar macho recién destetadas fueron divididas en dos grupos: treinta y una fueron alimentadas con una dieta colino deficiente y el resto con colina suplementada ad-libitum. Los animales de ambos grupos fueron sacrificados entre el quinto y el octavo día. Se midió la concentración de urea, creatinina y homocisteína en sangre. Los ojos fueron estudiados por microscopía de luz, microscopía óptica de alta resolución y para el estudio de la retina como retina plana. Los riñones fueron estudiados por microscopía de luz. Las ratas suplementadas con colina no mostraron lesiones oculares o renales. Las colino deficientes que mostraron lesiones renales, necrosis tubular o cortical, no siempre tuvieron cambios oculares. No se encontraron cambios oculares en la única rata deficiente en colina sin lesión renal. Los cambios oculares consistieron principalmente en hemorragia enambas cámaras, cuerpo ciliar y vítreo. La correlación entre la lesión ocular y renal (r=0.72, p<0.0001, CI 95%:0.48-0.86), lesión ocular y creatinina (r=0.86, p<0.0001, CI 95%: 0.72-0.93) y lesión ocular y urea (r=0.70,p<0.0001, CI 95%: 0.44-0.85) fue positiva. La deficiencia de colina induce lesiones oculares luego del desarrollode la necrosis renal. La patología ocular podría ser debida a la inmadurez de los vasos oculares. El sistemahialoide, coroideo y retinal están involucrados

Nefritis colagena / Collagen nephritis

En los últimos años se ha descripto un nuevo tipo de glomerulopatía primaria, denomminada nefritis colágena, caracterizada por la acumulación masiva de colágeno fibrilar en mesangio y membrana basal glomerular sin asociación con patología osteoarticular. Se comunica aquí el primer caso en nuestro medio. Se trata de una paciente de 26 años con síndrome nefrótico y progressiva caída del filtrado cuya biopsia renal mostró una esclerosis focal y segmentaria en la microscopia óptica. La evaluación ultraestructural reveló acúmulos en membrana basal y mesangio de colágeno fibrilar que permitió el diagnóstico de esta entidad.

Immunohistochemical studies on the small intestine of immunodeficient rats

Se estudió la histoarquitectura de la mucosa intestinal en un modelo experimental de inmunodeficiencia utilizando ratas Wistar que al destete recibieron una dieta libre de protéinas (13-17 días) a las que posteriormente se les adminsitró, por vía oral, caseína al 20% durante 39 días (grupo A) y en controles que recibieron desde el destete dieta comercial (grupo B). En el estudio del intestino delgado no se observaron diferencias significativas en los siguientes parámetros: altura de la mucosa, altura de las vellosidades, relación cripta/vellosidad, número de células epiteliales y número decélulas caliciformes. Se observó diferencia en el número de células del corion: (p < 0.05). A partir de estos dados resultó de interés investigar el número de células plasmáticas IgA, IgM y IgE presentes en la lámina propia del intestino delgado. Los resultados indican disminución estadísticamente significativa en el número de células plasmáticas IgA (p < 0.002), IgM (p < 0.002) e IgE (p < 0.02) en las ratas que después de la deficiencia proteica recibieron caseína al 20% durante 40 días. El daño provocado en la diferenciación terminal de los precursores de células plasmáticas IgA, Por la deficiencia proteica, no se revieste con la administración de caseína al 20% durante 39 días. La deficiencia de IgA provocada por la carencia de proteínas no es compensada con un aumento del número de células plamáticas IgM o IgE