Cerebellar synaptopathy in streptozotocin-induced diabetic rats / Sinaptopatía cerebelosa en ratas diabéticas inducidas por estreptozotocina

SUMMARY: There is an increasing amount of evidence that supports the diabetic complications of the central nervous system structure and function. The cerebellum, which is one of the primary structure derived from the hindbrain, plays an important role in motor control, motor coordination, and non-motor functions, such as cognitive processing. The synapse is a critical structure that regulates neuronal communication, and well-defined afferent and efferent fibre connections in the cerebellum help in maintaining the proper working order. Thus, the present study sought to investigate the long-term effects of diabetes-induced synaptopathy in the cerebellum, using both histological and ultrastructural studies. Twenty Sprague-Dawley male rats were divided randomly into control and diabetic groups, and diabetes was then induced through a single intraperitoneal injection of streptozotocin (60 mg/kg body weight). Six month later, the rats were sacrificed and the cerebellum was removed. Light and electron microscopic examinations showed a degeneration of Purkinje cells (Neuron purkinjense) with shrunken cells, pyknotic nuclei, and synaptopathy, including the reduction in synapse density, number of synaptic vesicles, and maturation of synapses in the molecular layer of diabetic cerebellum. The disruptions in synaptic profiles, which observed in the diabetic condition, could be related to cerebellar dysfunction, thus leading to the defects in coordinated movement, balance, as well as cognitive learning and memory.

RESUMEN: Actualmente existe una creciente evidencia que apoya las complicaciones diabéticas de la estructura y función del sistema nervioso central. El cerebelo, una de las estructuras primarias del cerebro posterior, desempeña un papel importante en el control motor, la coordinación motora y las funciones no motoras, tanto como en el procesamiento cognitivo. La sinapsis es una estructura crítica que regula la comunicación neuronal y las conexiones de fibras aferentes y eferentes bien definidas en el cerebelo, ayudan a mantener el funcionamiento correcto. Por lo tanto, en el presente estudio se investigaron los efectos a largo plazo de la sinaptopatía inducida por la diabetes en el cerebelo, utilizando estudios histológicos y ultraestructurales. Veinte ratas SpragueDawley macho se dividieron al azar en grupos de control y diabetes, se indujó la diabetes a través de una inyección intraperitoneal única de estreptozotocina (60 mg / kg de peso corporal). Seis meses después, se sacrificaron las ratas y se extrajo el cerebelo. Los exámenes de microscopías óptica y electrónica mostraron una degeneración de las neuronas purkinjenses (células de Purkinje), con células reducidas, núcleos picnóticos y sinaptopatía, como también la densidad reducida de sinapsis, el número de vesículas sinápticas y la maduración de las sinapsis en la capa molecular del cerebelo de las ratas diabéticas. Las interrupciones en los perfiles sinápticos, que se observaron en la condición diabética, podrían estar relacionadas con la disfunción cerebelosa, lo que lleva a defectos en el movimiento coordinado, el equilibrio, así como al aprendizaje cognitivo y la memoria.

Sodium-Glucose Linked Cotransporter-2 Inhibition Does Not Attenuate Disease Progression in the Rat Remnant Kidney Model of Chronic Kidney Disease.

Pharmacological inhibition of the proximal tubular sodium-glucose linked cotransporter-2 (SGLT2) leads to glycosuria in both diabetic and non-diabetic settings. As a consequence of their ability to modulate tubuloglomerular feedback, SGLT2 inhibitors, like agents that block the renin-angiotensin system, reduce intraglomerular pressure and single nephron GFR, potentially affording renoprotection. To examine this further we administered the SGLT2 inhibitor, dapagliflozin, to 5/6 (subtotally) nephrectomised rats, a model of progressive chronic kidney disease (CKD) that like CKD in humans is characterised by single nephron hyperfiltration and intraglomerular hypertension and where angiotensin converting enzyme inhibitors and angiotensin receptor blockers are demonstrably beneficial. When compared with untreated rats, both sham surgery and 5/6 nephrectomised rats that had received dapagliflozin experienced substantial glycosuria. Nephrectomised rats developed hypertension, heavy proteinuria and declining GFR that was unaffected by the administration of dapagliflozin. Similarly, SGLT2 inhibition did not attenuate the extent of glomerulosclerosis, tubulointerstitial fibrosis or overexpression of the profibrotic cytokine, transforming growth factor-ß1 mRNA in the kidneys of 5/6 nephrectomised rats. While not precluding beneficial effects in the diabetic setting, these findings indicate that SGLT2 inhibition does not have renoprotective effects in this classical model of progressive non-diabetic CKD.

Insulin resistance, steatohepatitis, and hepatocellular carcinoma in a new congenic strain of Fatty Liver Shionogi (FLS) mice with the Lep(ob) gene.

In order to examine the influence of obesity on metabolic disorder and liver pathogenesis of the Fatty Liver Shionogi (FLS) mouse, which develops hereditary fatty liver and spontaneous liver tumors, we established a new congenic strain named FLS-Lep(ob). The Lep(ob) gene of the C57BL/6JWakShi (B6)-Lep(ob)/Lep(ob) mouse was transferred into the genome of the FLS mouse, by backcross mating. FLS-Lep(ob)/Lep(ob) mice were maintained by intercrossing between Lep(ob)-heterozygous littermates. The FLS-Lep(ob)/Lep(ob) mice of both sexes developed remarkable hyperphagia, obesity and type 2 diabetes mellitus. At 12 weeks of age, glucosuria was detected in all male and female FLS-Lep(ob)/Lep(ob) mice. Biochemical examination demonstrated that the FLS-Lep(ob)/Lep(ob) mice have severe hyperlipidemia and hyperinsulinemia. The livers of FLS-Lep(ob)/Lep(ob) mice showed microvesicular steatosis and deposition of large lipid droplets in hepatocytes throughout the lobules. The steatohepatitis-like lesions including the multifocal mononuclear cell infiltration and clusters of foamy cells were observed earlier in FLS-Lep(ob)/ Lep(ob) mice than in FLS mice. B6-Lep(ob)/Lep(ob) mice did not show hepatic inflammatory change. Furthermore, FLS-Lep(ob)/Lep(ob) mice developed multiple hepatic tumors including hepatocellular adenomas and carcinomas following steatohepatitis. In conclusion, the FLS-Lep(ob)/Lep(ob) mice developed steatohepatitis and hepatic tumors following hepatic steatosis. The FLS-Lep(ob)/Lep(ob) mouse with obesity and type 2 diabetes mellitus might be a useful animal model for human non-alcoholic steatohepatitis (NASH).

Effects of the antifungal agent itraconazole on proliferative changes of the forestomach mucosa in alloxan-induced diabetic rats.

Alloxan-induced diabetic rats frequently exhibit proliferative lesions of squamous hyperplasia accompanied by chronic inflammation and Candida albicans infection in the forestomach, and some lesions progress to squamous cell carcinoma (SCC). Candida infection causes not only hyperplastic changes with inflammation but might also lead to SCC in human oral mucosa. Thus, the present study was conducted to examine the effects of the antifungal agent itraconazole (ITCZ) on proliferative and inflammatory changes of the forestomach in alloxan-induced diabetic WBN/Kob rats. Diabetes was induced by alloxan at fifteen weeks of age. Rats were allocated to three groups at forty-five weeks of age and were given ITCZ by gavage 0 (vehicle control), 5, and 10 mg/kg/day for four weeks, and they were sacrificed at the sixty-fifth week of age. Mucosal hyperplastic changes were consistently accompanied by inflammation and Candida infections in the 0 mg/kg group. These lesions were reduced by ITCZ (0 mg/kg; 100%, 5 mg/kg; 53.5%, 10 mg/kg; 61.5%). Squamous cell carcinoma was detected in three rats from the 0 mg/kg, but only one rat from the 10 mg/kg dose groups in this study. Itraconazole reduced the degree of mucosal hyperplasia, inflammatory changes, and Candida infection. Therefore, C. albicans infection was an important factor in pathogenesis of mucosal proliferation and inflammation.

Biotransformation, excretion, and nephrotoxicity of the hexachlorobutadiene metabolite (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide.

Hexachlorobuta-1,3-diene (HCBD) is nephrotoxic in rodents. Its toxicity is based upon a multistep bioactivation pathway. Conjugation with glutathione by glutathione S-transferases to form (E)-S-(1,2,3,4,4-pentachlorobutadienyl)-L-glutathione (PCBG), further processing to the corresponding cysteine S-conjugate, and finally processing to a reactive thioketene are thought to be responsible for the observed nephrotoxic effects. A novel metabolite, identified as (E)-N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine sulfoxide (N-AcPCBC-SO), was described after administration of [14C]HCBD to male Wistar rats. This metabolite is formed by sulfoxidation of N-acetyl-S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine (N-AcPCBC) mediated by cytochrome P450 3A and has been found to be cytotoxic to proximal tubular cells in vitro without activation by beta-lyase. In rats, given HCBD in vivo, only one diastereomer of the sulfoxide is excreted; however, in rat hepatic microsomes two diastereomers, (R)- and (S)-N-AcPCBC-SO, are formed. This study focuses on the mechanisms responsible for this discrepancy and on a possible contribution of N-AcPCBC-SO to the nephrotoxicity of HCBD in vivo. (R,S)-N-AcPCBC-SO (1:1 mixture of both diastereomers) and N-acetyl-alpha-methyl-S-(1,2,3,4,4-pentachlorobutadienyl)-d, L-cysteine sulfoxide (alpha-Me-N-AcPCBC-SO) were administered iv to male and female Wistar rats (20, 40, and 80 micromol/kg of body weight). alpha-Me-N-AcPCBC-SO cannot be cleaved by cysteine conjugate beta-lyase even if alpha-Me-N-AcPCBC-SO is deacetylated by acylases. Excretion of gamma-glutamyltranspeptidase, protein, and glucose in the urine, indicative for kidney damage, and histopathological examination of the kidneys showed marked differences in the renal damage in male and female rats after application of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. Necroses of the kidney tubules were only found in male, but not female, rats. Major sex-specific differences were observed in the elimination of sulfoxides; the (R)-isomer was excreted in a 5-10-fold excess to the (S)-isomer after application of (R,S)-N-AcPCBC-SO. After purification, both isomers were administered to male rats resulting in the urinary excretion of (R)-N-AcPCBC-SO after giving the (R)-isomer; treatment with (S)-N-AcPCBC-SO, however, revealed the formation of (S)-N-acetyl-S-(2-glycinylcystein-S-yl-1,3,4, 4-tetrachlorobutadienyl)-L-cysteine. The results show major sex-specific differences in the nephrotoxic potency of N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO. However, both N-AcPCBC-SO and alpha-Me-N-AcPCBC-SO are nephrotoxic in males, suggesting the formation of a vinyl sulfoxide as an additional, beta-lyase-independent mechanism in HCBD-caused nephrotoxicity.

Glycosuria in glomerular diseases: histopathology and clinical correlations

There are doubts about the presence of glycosuria and the progress of glomerular disease. Some reports suggest that glycosuria could be an index of a more severe tubulointerstitial lesion. We investigated the presence of glycosuria in 60 patients with primary glomerular diseases: 17 patients (28 percent) and glycosuria and 43 patients (72 percent) were glycosuria free. The two groups were similar in age, arterial pressure and sex. Serum creatinine was higher in patients with glycosuria (2.0 + 1.7 vs 1.3 + 0.9 mg/dl, P<0.05). The protein excretion rate was 7.5 + 3.7 vs 5.3 + 4.2g/day (P>0.05) in patients with and without glycosuria, respectively, while serum albumin was lower in patients with glycosuria (1.7 + 0.6 vs 2.7 + 1.0 g/dl, P<0.05). Several histological forms were present in the group with glycosuria, with membranous glomerulonephritis being the most frequent. Histological evidence of tubular atrophy and interstitial fibrosis prevailed in patients with glycosuria, suggesting a poor prognosis for these patients. We may conclude that the presence of glycosuria in patients with glomerular disease is associated with more pronounced tubular atrophy and interstitial fibrosis and therefore imply a poorer prognosis.

Pancreatic islet cell tumors found in rats given alloxan and nicotinamide.

Morphological and biochemical studies were performed in pancreatic islet cell tumors found in rats given alloxan (40 mg/kg) and nicotinamide (305 mg/kg). Complete serial sections of the whole pancreas, combined with planimetric analysis, uncovered islet cell tumors in 5 of 7 rats which were killed 10 to 14 months after treatment. Hypoglycemia associated with hyperinsulinemia was found in a rat which developed a tumor which consisted of cells which reacted lightly with aldehyde fuchsin. Another rat developed a gross tumor which was composed of cells stained deeply with aldehyde fuchsin. However, flat insulin response to glucose associated with glucose intolerance was found in this rat. In addition to B cells, a few A and D cells were found within the two tumors. The present study suggests that pancreatic islet cell tumors found in rats given alloxan and nicotinamide are composed of at least three endocrine cell populations, although the majority of tumor cells are insulin-producing B cells.