Abstract
BACKGROUND AND AIMS
Proteinuria is a major risk factor for the progression of chronic kidney disease. Protein overload in the proximal tubular epithelial cells causes oxidative stress, ...lysosomal dysfunction, inflammation and apoptosis, resulting in proximal tubule dysfunction. Recent studies have focussed on the association between proximal tubule injury and cellular senescence and the development of drugs targeting and removing senescent proximal tubular cells. Senescent cells show resistance to apoptosis and persistently secrete inflammatory cytokines, resulting in chronic inflammation. It has been suggested that excess proliferation induced by fatty acids causes senescence of proximal tubular cells. However, the association between protein overload, proliferation and cellular senescence in proximal tubular cells remains unclear. The present study aimed to clarify the effect of protein overload on cell proliferation and senescence in a proteinuric mouse model and in an immortalized proximal tubular epithelial cell line. Moreover, it was evaluated whether the endocytic receptors for protein uptake, megalin and cubilin, affect protein overload-induced proliferation and senescence using knockout (KO) of megalin and cubilin in the proteinuric mouse model.
METHOD
Experiments were performed using podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice. Kidneys from these mice were analysed using immunohistochemical and immunofluorescent staining. Immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were used for in vitro experiments, wherein RPTEC/TERT1 cells were incubated with 0.1–10 mg/mL of human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting, quantitative rt-PCR, senescence-associated beta-galactosidase (SA-β-gal) staining and immunofluorescence were performed to analyse cellular senescence. The effect of a PKC activator (phorbol 12-myristate 13-acetate) and an inhibitor (Go6983) on proliferation and senescence was also evaluated.
RESULTS
The proliferation markers EdU incorporation, PCNA (Figure 1) and Ki-67 were detected in proximal tubular cells of podocin-KO mice, but not in wild-type mice. In triple-KO mice, a decrease in PCNA-positive tubules was observed. This suggests that protein reabsorption via megalin and cubilin provoked cell proliferation. Light microscopy analysis and a proliferation assay with BrdU incorporation revealed that HSA overload-induced the proliferation of RPTEC/TERT1 cells, whereas fatty acid-free HSA or transferrin had no effect on the proliferation of RPTEC/TERT1 cells. Western blot analysis showed that HSA treatment, but not fatty acid-free HSA treatment, induced alterations in proliferation (PCNA and Ki-67), cell cycle (cyclin A, D, and Rb), cellular senescence (p21 and p16), and DNA injury (γ-H2AX). Using immunofluorescence, an increase in p21 and p16 expression was also observed in HSA-treated cells. Moreover, the HSA-treated cells showed positive staining for SA-β-gal. These results suggest that fatty acids bound to HSA induce cell proliferation and senescence. Furthermore, results showed that a PKC inhibitor suppressed HSA-induced proliferation and senescence, while a PKC activator accelerated these alterations without HSA treatment.
CONCLUSION
The present study showed that fatty acid-associated albumin induced proliferation and senescence of the proximal tubule cells, which were dependent on megalin/cubilin endocytosis of filtered protein. PKC activation is at least in part related to cell proliferation and senescence. It is unknown which molecular switch determine the cell cycle fate.
In order to investigate molecules related to insulin secretion, we conducted a practical method to imitate human insulin secretion through rats via organ baths of pancreatic preparations. Our ...previous study showed that insulin secretion from rat pancreas tissue was stimulated by glucagon-like peptide-1 and glimepiride. 1,5-anhydro-D-glucitol (1,5-AG) is a glucose analog and exists in humans. This study aims to assess the effects of short-term 1,5-AG stimulation of insulin secretion in rat pancreases to better understand the effects in humans. Rat pancreases were assigned to eight groups: two glucose concentrations (100 and 400 mg/dl) and pairs of varying 1,5-AG concentrations (0, 0.1, 1, and 10 mM). There was a significant increase in insulin outflow from low to high glucose concentrations. However, there was no significant enhancement of insulin secretion between the four groups with low and high 1,5-AG concentrations. This suggests that short-term exposure to 1,5-AG has no effect on insulin secretion in rat pancreas tissues. To justify whether the methods and techniques were useful as an experimental system, we isolated pancreases from another rodent species, the mouse, and similarly measured insulin outflow. In mouse pancreas preparations, stimulating with 400 mg/dl glucose significantly increased insulin outflow. Therefore, organ baths of isolated mouse pancreases are also considerably effective for assessing effects of novel molecules and/or therapeutics on insulin secretion.
An integrated analysis was performed with data from 4 phase 2 and phase 3 studies of tofogliflozin in which patients with type 2 diabetes mellitus received the sodium‐glucose cotransporter 2 ...inhibitor tofogliflozin for up to 24 weeks. Sex differences, baseline haemoglobin A1c (HbA1c) and serum uric acid (UA) levels, and log10‐transformed urinary N‐acetyl‐β‐D‐glucosaminidase ratio were significantly correlated with the reduction in serum UA levels at both 4 and 24 weeks in multivariate analysis (respectively, P < .0001). The decrease in HbA1c levels was greatest in the group with the highest baseline HbA1c level (quartile 4; HbA1c > 8.6%) and lowest in the group with the lowest baseline HbA1c level (quartile 1; HbA1c ≤ 7.4%). The decrease in serum UA levels was greatest in the quartile 1 group and lowest in the quartile 4 group. In most groups, the maximum decrease in serum UA levels was seen in the first 4 weeks, while the maximum decrease in HbA1c was seen at week 24. Thus, serum UA levels were significantly decreased in patients with moderate HbA1c levels.
Due to the coronavirus pandemic situation, the pharmacological role-playing was conducted by online in 2020 and 2021. We reported consideration of pros and cons of the online pharmacology ...role-playing from results of a two-year student questionnaire survey. Two hundred twenty five 3rd-grade medical students joined the role-playing at Dokkyo Medical University. Twenty-four students were assigned as physicians or patients and played the informed consent in two cases; hypertension accompanying diabetes and dementia with polypharmacy. The remaining students were observers. All students answered the questionnaire with a score of one to five, regarding the usefulness of study on disease and pharmacotherapy, understanding the patients' feeling, improvement of motivation to become a doctor, and change of attitude to studying. The percentage of students who scored five or four was 63 to 83% for the players and 71 to 80% for the observers. The frequent answers regarding necessary points of the study were "quality of the study" and "communication ability" and "perspective from the patients" for the players, and "communication ability", "quality of the study" and "perspective from the patients" for the observers in order of frequency. Most students described positive impression in the free entry field that was considered as pons. A description of the difficulty of adjusting the online settings was considered as cons. Online pharmacological role-playing may be a useful approach for the medical students to learn the pharmacotherapy and doctor-patient relationship.
Abstract Aims Spontaneously diabetic Torii (SDT) rats exhibit vascular abnormalities in pancreatic islets as the initial changes at pre-diabetes stage (8 weeks old), which is followed by β cell ...deterioration. In the present study, we investigated pathophysiological interactions between β cells and intra-islet microvasculature of SDT rats at pre- and peri-onset of diabetes. Methods SDT rats were treated with Habu snake venom (HSV) to assess its hemorrhagic effects in glomeruli and pancreatic islets. SDT rats were treated with streptozotocin (STZ) to assess acute β cell fragility toward cytotoxic insult and the late-stage consequence of β cell ablation in neighboring structures. The receptor tyrosine kinase inhibitor sunitinib was administered to SDT rats to examine its therapeutic effect. Results HSV administration at 5 weeks old induced severe hemorrhage in and around islets in SDT rats. By contrast, precedent β cell depletion using STZ ameliorated hemorrhage, inflammation, and fibrosis around the islets at 13 weeks old, which is normally seen in SDT rats of this age. Blockade of vascular endothelial growth factor (VEGF)-like activity attenuated HSV-induced hemorrhage in SDT islets. VEGF release from SDT islets was increased at 13 weeks old but not at 5 weeks old, while interleukin-1β release was increased as early as 5 weeks old. Sunitinib treatment started at 5 weeks of age inhibited the onset of intra-islet hemorrhage, β cell loss, and hyperglycemia in SDT rats. Conclusions Enhanced VEGF signaling in islets contributes to β cell injury, microvascular failure, and consequential diabetes in SDT rats.
To investigate substances related to insulin secretion, we reported a convenient experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath. While ...the method has experimental utility for investigating insulin secretion, optimization of the experimental design is still needed. The level of insulin outflow in the control decreased over time in our previous study. Decreasing serum 1,5-anhydroglucitol (1,5-AG) levels is also known to be shown in patients with worsening glycemic control. There is one in vitro report demonstrated that 1,5-AG induced insulin release. It appears that discussion needs to be deepened further on it. In this study, we investigated the effect of 1,5-AG on insulin secretion through to optimize the condition of endocrine function using the ex vivo organ bath technique. The level of insulin outflow in the control and 1,5-AG groups decreased over time in the organ bath experiment. To analyze the effect of trypsin on reduced insulin secretion, pancreas preparation was treated with soybean trypsin inhibitor (TI). Insulin outflow levels of the TI group were significantly higher than the control group. An enzyme indicator of tissue damage tended to be lower in the TI group. There was no significant enhancement of insulin secretion by 1,5-AG. The present study demonstrated the utility of TI application for the organ bath technique. This finding supported the development of an organ bath technique for the assessment of the effects of novel therapeutics on insulin secretion.
Recently, anserine (beta-alanyl-3-methyl-L histidine), one of the dipeptides, was reported to lower the serum uric acid level in humans. This level in humans is regulated by the balance between ...urinary excretion mediated by renal tubular transporters in the kidney and enzymatic production mainly in the liver xanthine oxidase (XO). The mechanism of anserine lowering the serum uric acid level is still unknown, so we investigated it by examining whether anserine inhibits urate excretion or production. To analyze the uricosuric action, we performed a 14Curate uptake experiment in human embryonic kidney cells stably expressing human urate transporter 1 (HEK293- URAT1) or mock (HEK293-mock) cells with 10 mM of the following substrates: beta-alanine, 1-methylL-histidine, 3-methyl-L-histidine, carnosine (betaalanyl-L-histidine), and anserine nitrate. Because only anserine nitrate was commercially available and there was a report that sodium nitrate injected intracellularly increased urate uptake mediated by URAT1, we also examined the effect of sodium nitrate on urate uptake via URAT1. Next, we carried out a XO activity assay to analyze the inhibitory effect on urate production with 10 mM of substrates, as previously mentioned. In the uptake experiment, anserine nitrate and sodium nitrate markedly inhibited urate uptake mediated by URAT1. 3-Methyl-L-histidine and carnosine showed weak inhibitory effects. Beta-alanine and 1-methylL-histidine did not show inhibitory effects. In the XO activity assay, anserine nitrate markedly inhibited XO activity and its IC50 was 6.45 ± 1.57 mM. 3-MethylL-histidine and carnosine showed weak inhibitory effects. Beta-alanine, 1-methyl-L-histidine, and sodium nitrate did not show inhibitory effects. Although the effect of anserine nitrate on URAT1 was unsettled, it showed a marked inhibitory effect on XO activity. This suggests that the effect of anserine to lower the serum urate level in humans is partly due to the inhibition of urate production by XO activity.
Aims/Introduction: Oral ingestion of carbohydrate triggers secretion of glucagon‐like peptide (GLP)‐1, which inhibits the postprandial rise in blood glucose levels. However, the mechanism of ...carbohydrate‐induced GLP‐1 secretion from enteroendocrine L cells remains unclear. In the present study, GLP‐1 secretion was examined by meal tolerance tests of healthy Japanese volunteers.
Materials and Methods: Twenty‐one healthy Japanese men participated in the study. The meal tolerance test was performed with modified nutrient compositions, with or without pretreatment with the α‐glucosidase inhibitor acarbose, or with substitution of sucrose with an equivalent dose of sweeteners in the meal. Blood concentrations of glucose, insulin, GLP‐1, and apolipoprotein (Apo) B‐48 were measured.
Results: GLP‐1 secretion started concomitant with the increase in blood glucose levels 10 min after meal ingestion. Insulin secretion started at 5 min, before the increase in blood glucose levels, reflecting the contribution of direct nutrient stimulation on the former parameter and neural regulation in the latter. Carbohydrate retention in the gut lumen induced by acarbose pretreatment extended postprandial GLP‐1 secretion and negated the increase in serum ApoB‐48 levels. GLP‐1 secretion was markedly decreased by a reduction in the amount of sucrose in the meal and was not restored by an equivalent dose of sweeteners used to compensate for the sweet taste.
Conclusions: The results indicate that direct stimulation of L cells with sugar, but not sweetener, is required for carbohydrate‐induced GLP‐1 secretion. In addition, inhibition of digestion of dietary carbohydrate by α‐glucosidase inhibitors may prevent postprandial hyperglycemia by increasing GLP‐1 secretion and by inhibiting glucose absorption. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00163.x, 2011)
Mammalian sperm, including human, must undergo several physiological and biochemical changes, collectively called capacitation, to be fertilization-competent. Capacitated sperm actively generate ...reactive oxygen species (ROS). A low level of ROS facilitates capacitation whereas an excessive ROS impairs capacitation. Hypotaurine (HT) is a precursor of taurine, and is abundant in the oviduct. HT is known to mitigate oxidative stress in hamster sperm, and is transported by taurine transporter (TauT) in a Na+- and Cl-- dependent manner. However, how HT protect sperm from oxidative stress remains unknown. This study aimed to elucidate the antioxidant mechanisms by HT in hamster sperm, focusing on the involvement of TauT. We first examined the effects of HT on sperm motility, intra- and extra- cellular ROS levels. HT was shown to be necessary to maintain motility. HT lowered intracellular ROS levels, but had no effect on extracellular ROS levels at the concentration tested, although HT itself has an antioxidative capacity at higher concentrations. Incorporation and enrichment of HT in sperm were confirmed by LC-MS/MS analysis. Next, the involvement of TauT was investigated. TauT was present in hamster sperm. β-alanine, a blocker of TauT, inhibited HT transport, increased intracellular ROS levels and impaired sperm motility. Moreover, the elimination of Na+ and Cl- inhibited HT transport, and increased intracellular ROS levels. In conclusions, the results indicate that hamster sperm incorporate and concentrate HT via TauT to protect themselves from ROS.
It is well known that glucagon-like peptide 1 (GLP-1) can bind to the GLP-1 receptor of pancreatic islet to enhance insulin secretion through a cAMP-dependent pathway. However, little is known about ...the effects of GLP-1 on the pancreatic exocrine gland. In the gland, a signal transduction of amylase release is evoked mainly by an increase in intracellular Ca2+ levels and activation of PKC. Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major substrate for PKC. We previously demonstrated that MARCKS is involved in pancreatic amylase release through the Ca2+-dependent pathway. Here, we studied the effects of GLP-1 on MARCKS phosphorylation and amylase release through the cAMP-dependent pathway in rat pancreatic acini. By the organ bath technique, GLP-1 did not induce amylase release in the intact pancreas. In contrast, it induced amylase release and MARCKS phosphorylation in isolated pancreatic acini. An inhibitor of PKA suppressed those effects. Furthermore, a MARCKS-related peptide inhibited the GLP-1-induced amylase release. These findings suggest that GLP-1 induces amylase release through MARCKS phosphorylation via activation of PKA in the isolated acini, but not in the intact pancreas.
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