Lysosomal enzymes are decreased in the kidney of diabetic rats

The objective of the present study was to investigate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in diabetic rat kidney. Cathepsins, glycosidases and sulfatases were studied on the 10th (DM-10) and on the 30th (DM-30) day of streptozotocin-induced diabetes mellitus (DM). The activity of cathepsin B, the main kidney cysteine protease, was decreased both in DM-10 and DM-30. Gel filtration chromatography of urinary proteins has shown the prevalence of low molecular weight peptides in normal and DM-10 urine, in contrast to the prevalence of high molecular weight peptides and intact proteins in DM-30. These results show that the decrease in lysosomal proteases could explain, at least in part, the increased albuminuria detected by radial immunodiffusion (RID), due to the excretion of less degraded or intact albumin. Concerning sulfated polysaccharides, the activities of β-glucuronidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase were also decreased in DM-30, while aryl sulfatases did not vary. Increased toluidine blue metachromatic staining of the tissue suggests that the lower activities of glycosidases could lead to intracellular deposition of partially digested molecules, and this could explain the decreased urinary excretion and increased tissue buildup of these molecules. The main morphological changes observed in kidney were proximal convoluted tubules with thinner walls and thinner brush border. Immunohistochemistry revealed that most of cathepsin B was located in the brush border of proximal tubular cells, highlighting the involvement of proximal convoluted tubules in diabetic nephropathy.     

Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized femal Wistar rats

Aging seems to be due to the accumulation of oxidative damage in cells and molecules. On the other hand, menopause and ovariectomy induce deleterious effects on different organs and systems that have been shown to be counteracted by estrogens and in a not so evident form also with phytoestrogens. The present study has investigated whether the administration of a commercial soy extract that contains approximately 10% isoflavones was able to modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female Wistar rats. Eighteen 22-month-old animals that had been previously ovariectomized at 12 months of age were divided into four groups: ovariectomized control rats, estradiol-treated ovariectomized females and ovariectomized rats treated with isoflavones. Six intact female rats of 2 months of age were used as reference group. Hepatocytes were isolated and cultured, and carbon monoxide (CO) and nitric oxide (NO) release, as well as adenosyl triphosphate (ATP), cyclic guanosyl monophosphate (cGMP), phosphatidylcholine (PC) and lipid peroxide (LPO) content of cells were evaluated. Uterus was also removed and weighed. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content as compared to young animals. Age also induced an increase in LPO cell content. NO, CO and cGMP were augmented with age, and PC synthesis showed a dramatic reduction. Treatment with either estradiol or isoflavones were able to improve all the mentioned parameters altered in hepatocytes isolated from old ovariectomized rats, and the magnitude of the improvement was similar for both treatments. Ovariectomy induced a significant reduction in uterine weight, which was significantly counteracted by estradiol treatment but not by isoflavone administration. In conclusion, the administration of a soy extract containing isoflavones seems to prevent oxidative changes in hepatocytes isolated from old ovariectomized female rats, without modifying uterus weight.     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

Over-expression of Zip-13 mRNA in kidney and lung during dietary zinc deficiency in Wistar rats

Zinc is an essential nutrient for all organisms, which is involved in the function of numerous key enzymes in metabolism. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while Zip transporters increase intracellular zinc. Previous studies in our laboratory have shown that Zip-1, ZnT-1, Zip-2 and LIV-1 mRNA are associated with zinc level in established human breast cancer in nude mice model. In this study, six zinc transporters: ZnT-1, ZnT-2, ZnT-4, Zip-1, Zip-8 and Zip-13 were chosen. We aim to determine the relation between zinc transporters and zinc level in kidney and lung of Wistar rats. Eighteen Wistar rats were randomly divided into three groups: normal group, zinc-deficiency group and pair-fed group. After 22 days, the rats were killed and organs samples were taken, then zinc transporters mRNA were detected by RT-PCR. Compared with the normal group, Zip-13 shows an up-regulation (P < 0.05) in zinc-deficiency group both in kidney and lung, and Zip-8 was significantly lower (P < 0.05) in zinc-deficiency group in kidney.     

Auricular chondritis in Wistar rats

The macroscopic and microscopic features of auricular chondritis in Wistar rats are described. Histologic lesions are characterized by granulomatous inflammation, chondrolysis, and multinodular proliferative foci. The condition in Wistar rats is compared to auricular chondritis in other strains of rats.     

Frequency of hydronephrosis in Wistar rats

Kidneys from 1806 Wistar rats were examined grossly for hydronephrosis and ureteral dilation. Hydronephrosis was seen more often on the right side (11%) than the left (0.3%). Overall frequency of hydronephrosis in males (181/1305) was greater than in females (23/501), and the frequency was statistically greater in male rats aged 5,6,8 and 9 weeks than in age-matched females.     

Lysosomal enzymes in pregnant and steroid treated rats

The contents of three lysosomal enzymes (beta-hexosaminidase, beta-glucuronidase and alpha-fucosidase) were studied in plasma and different tissues of pregnant and steroid treated rats. All these enzymes were found to be increased in plasma from pregnant rats in analogy with the findings in pregnant women. In liver tissue only beta-hexosaminidase and alpha-fucosidase were significantly increased. In rats with diethylstilbestrol (DES) and a combination of DES and progesterone, there was an increase of alpha-fucosidase in plasma and liver. No significant changes were observed for the other two enzymes. Thus, steroid treatment did not fully reproduce the enzyme changes seen in pregnancy, which may indicate that these are not solely due to a hormone effect.     

Lysosomal degradation of bile deposits in cholestasis. An ultrastructural and cytochemical study.

Deposits of bile in the cytoplasm of hepatocytes from six patients with cholestasis were studied by electron microscopy and ultrastructural cytochemistry. The deposits were identified in vacuoles of varying size and complexity. The demonstration of acid phosphatase activity in these vacuoles suggests that lysosomes participate in the elimination of bile deposits retained in hepatocytes.     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

Differential activation of mitogen-activated protein kinases by methyl methanesulfonate in the kidney of young and old rats

Mitogen-activated protein kinases (MAPKs) play a critical role in the regulation of cell proliferation, differentiation and apoptosis. We evaluated MAPKs, extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs) and p38 MAPKs in the kidney of young and old rats in response to a direct-acting alkylating agent, methyl methanesulfonate (MMS). It is shown that the basal activity of ERKs was strongly down-regulated in the kidney of old rats compared to their young counterparts without a significant difference in the basal expression of ERKs. Upon treatment with MMS, ERKs were deactivated about 5-fold (P<0.05) in the kidney of young rats, whereas they were activated about 4-fold (P<0.01) in old rats. Strikingly, expression of JNKs was not detected in old animals, whereas it was clearly present and strongly activated after MMS treatment in the kidney of young animals. The basal activity of p38 significantly increased in the kidney of old rats as compared to young animals, whereas no difference in the basal expression of p38 was detected. After treatment with MMS, p38 was activated in the kidney of both young and old rats, where activation was dramatically stronger than in young animals. Taken together, these results demonstrate age-specific MAPKs signaling pathways in the rat kidney. The implications in age-related changes in susceptibility of the kidney to MMS-induced carcinogenesis are discussed.     

Lysosomal dysfunction–induced autophagic stress in diabetic kidney disease

The catabolic process that delivers cytoplasmic constituents to the lysosome for degradation, known as autophagy, is thought to act as a cytoprotective mechanism in response to stress or as a pathogenic process contributing towards cell death. Animal and human studies have shown that autophagy is substantially dysregulated in renal cells in diabetes, suggesting that activating autophagy could be a therapeutic intervention. However, under prolonged hyperglycaemia with impaired lysosome function, increased autophagy induction that exceeds the degradative capacity in cells could contribute toward autophagic stress or even the stagnation of autophagy, leading to renal cytotoxicity. Since lysosomal function is likely key to linking the dual cytoprotective and cytotoxic actions of autophagy, it is important to develop novel pharmacological agents that improve lysosomal function and restore autophagic flux. In this review, we first provide an overview of the autophagic‐lysosomal pathway, particularly focusing on stages of lysosomal degradation during autophagy. Then, we discuss the role of adaptive autophagy and autophagic stress based on lysosomal function. More importantly, we focus on the role of autophagic stress induced by lysosomal dysfunction according to the pathogenic factors (including high glucose, advanced glycation end products (AGEs), urinary protein, excessive reactive oxygen species (ROS) and lipid overload) in diabetic kidney disease (DKD), respectively. Finally, therapeutic possibilities aimed at lysosomal restoration in DKD are introduced.