Growth Inhibitory Effect of (E)-2,4-bis(p-hydroxyphenyl)-2-Butenal Diacetate through Induction of Apoptotic Cell Death by Increasing DR3 Expression in Human Lung Cancer Cells

The Maillard Reaction Products (MRPs) are chemical compounds which have been known to be effective in chemoprevention. Death receptors (DR) play a central role in directing apoptosis in several cancer cells. In our previous study, we demonstrated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal, a MRP product, inhibited human colon cancer cell growth by inducing apoptosis via nuclear factor-κB (NF-κB) inactivation and G₂/M phase cell cycle arrest. In this study, (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate, a new (E)-2,4-bis(p-hydroxyphenyl)-2-butenal derivative, was synthesized to improve their solubility and stability in water and then evaluated against NCI-H460 and A549 human lung cancer cells. (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate reduced the viability in both cell lines in a time and dose-dependent manner. We also found that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate increased apoptotic cell death through the upregulation of the expression of death receptor (DR)-3 and DR6 in both lung cancer cell lines. In addition to this, the transfection of DR3 siRNA diminished the growth inhibitory and apoptosis inducing effect of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate on lung cancer cells, however these effects of (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate was not changed by DR6 siRNA. These results indicated that (E)-2,4-bis(p-hydroxyphenyl)-2-butenal diacetate inhibits human lung cancer cell growth via increasing apoptotic cell death by upregulation of the expression of DR3.     

Angiotensin II-dependent persistent podocyte loss from destabilized glomeruli causes progression of end stage kidney disease

Podocyte depletion is a major mechanism driving glomerulosclerosis. Progression is the process by which progressive glomerulosclerosis leads to end stage kidney disease (ESKD). In order to determine mechanisms contributing to persistent podocyte loss, we used a human diphtheria toxin transgenic rat model. After initial diphtheria toxin-induced podocyte injury (over 30% loss in 4 weeks), glomeruli became destabilized, resulting in continued autonomous podocyte loss causing global podocyte depletion (ESKD) by 13 weeks. This was monitored by urine mRNA analysis and by quantitating podocytes in glomeruli. Similar patterns of podocyte depletion were found in the puromycin aminonucleoside and 5/6 nephrectomy rat models of progressive end-stage disease. Angiotensin II blockade (combined enalapril and losartan) restabilized the glomeruli, and prevented continuous podocyte loss and progression to ESKD. Discontinuing angiotensin II blockade resulted in recurrent glomerular destabilization, podocyte loss, and progression to ESKD. Reduction in blood pressure alone did not reduce proteinuria or prevent podocyte loss from destabilized glomeruli. The protective effect of angiotensin II blockade was entirely accounted for by reduced podocyte loss. Thus, an initiating event resulting in a critical degree of podocyte depletion can destabilize glomeruli and initiate a superimposed angiotensin II-dependent podocyte loss process that accelerates progression resulting in eventual global podocyte depletion and ESKD. These events can be monitored noninvasively in real-time through urine mRNA assays.     

Hemoglobin conjugates with antioxidant enzymes (hemoglobin-superoxide dismutase-catalase) via poly(ethylene glycol) crosslinker for protection of pancreatic beta RINm5F cells in hypoxia

A low p50 hemoglobin (Hb) (p50 indicates O(2) tension at which Hb is half-saturated)-based oxygen carrier conjugated to antioxidant enzymes via dicarboxymethylated poly(ethylene glycol) (PEG) linker may have the beneficial effect in protecting pancreatic beta cells from severe hypoxia at transplantation sites. In this study, the oxygen dissociation curves, Hill plots, Bohr Effect, and oxygen content of Hb conjugates were measured. The protective effect due to incubation of Hb-conjugates (Hb/PEG molar ratio 1:10) with pancreatic beta cells (RINm5F) against hypoxia (6%, 3%, and 1% oxygen) was evaluated by an MTT assay and confocal microscopy. Quantitatively, Hb conjugates with antioxidant enzymes offered statistically significant protection (p<0.01, increased viability ～80%) from hypoxia compared to control cells in 1% oxygen environment. Confocal images also showed that the low p50?Hb conjugates with antioxidants protected RINm5F cells from hypoxia.     

Growth-dependent podocyte failure causes glomerulosclerosis

Podocyte depletion leads to glomerulosclerosis, but whether an impaired capacity of podocytes to respond to hypertrophic stress also causes glomerulosclerosis is unknown. We generated transgenic Fischer 344 rats that express a dominant negative AA-4E-BP1 transgene driven by the podocin promoter; a member of the mammalian target of rapamycin complex 1 (mTORC1) pathway, 4E-BP1 modulates cap-dependent translation, which is a key determinant of a cell's hypertrophic response to nutrients and growth factors. AA-4E-BP1 rat podocytes expressed the transgene and had normal kidney histology and protein excretion at 100 g of body weight but developed ESRD by 12 months. Proteinuria and glomerulosclerosis were linearly related to both increasing body weight and transgene dose. Uni-nephrectomy reduced the body weight at which proteinuria first developed by 40%-50%. The initial histologic manifestation of disease was the appearance of bare areas of glomerular basement membrane from the pulling apart of podocyte foot processes, followed by adhesions to the Bowman capsule. Morphometric analysis confirmed the mismatch between glomerular tuft volume and total podocyte volume (number × size) per tuft in relation to weight gain and nephrectomy. Proteinuria and glomerulosclerosis did not develop if dietary calorie restriction prevented weight gain and glomerular enlargement. In summary, failure of podocytes to match glomerular tuft growth in response to growth signaling through the mTORC1 pathway can trigger proteinuria, glomerulosclerosis, and progression to ESRD. Reducing body weight and glomerular growth may be useful adjunctive therapies to slow or prevent progression to ESRD.     

Estimating Podocyte Number and Density Using a Single Histologic Section

The reduction in podocyte density to levels below a threshold value drives glomerulosclerosis and progression to ESRD. However, technical demands prohibit high-throughput application of conventional morphometry for estimating podocyte density. We evaluated a method for estimating podocyte density using single paraffin-embedded formalin-fixed sections. Podocyte nuclei were imaged using indirect immunofluorescence detection of antibodies against Wilms’ tumor-1 or transducin-like enhancer of split 4. To account for the large size of podocyte nuclei in relation to section thickness, we derived a correction factor given by the equation CF=1/(D/T+1), where T is the tissue section thickness and D is the mean caliper diameter of podocyte nuclei. Normal values for D were directly measured in thick tissue sections and in 3- to 5-μm sections using calibrated imaging software. D values were larger for human podocyte nuclei than for rat or mouse nuclei (P<0.01). In addition, D did not vary significantly between human kidney biopsies at the time of transplantation, 3–6 months after transplantation, or with podocyte depletion associated with transplant glomerulopathy. In rat models, D values also did not vary with podocyte depletion, but increased approximately 10% with old age and in postnephrectomy kidney hypertrophy. A spreadsheet with embedded formulas was created to facilitate individualized podocyte density estimation upon input of measured values. The correction factor method was validated by comparison with other methods, and provided data comparable with prior data for normal human kidney transplant donors. This method for estimating podocyte density is applicable to high-throughput laboratory and clinical use.Pagtalunan et al. used the term podocyte density to describe the key relationship between podocyte number and glomerular tuft volume.¹ Model systems have proven the causative relationship between podocyte depletion (resulting from reduced podocyte number or dysfunction and/or glomerular enlargement) and glomerulosclerosis and progression to ESRD.^2–9 Groundbreaking kidney morphometric biopsy reports from type 1 and 2 diabetes, IgA nephropathy, and hypertensive kidney biopsies in humans support the concept that reduced podocyte number and density is associated with development of glomerulosclerosis and progression,^1,10–15 and strongly imply that podocyte density estimation could help guide clinical decision making.The importance of avoiding simplistic podocyte counting strategies and using appropriate stereologic considerations for estimating podocyte number and density have recently been re-emphasized.^16–20 Optimal research methods for estimating podocyte density, such as the disector/fractionator approach, are too technically demanding for high-throughput use in laboratory work, drug testing by pharmaceutical companies, routine clinical biopsy readout, and automated biopsy analysis. We therefore assessed whether it might be feasible to use single formalin-fixed paraffin-embedded histologic sections to estimate podocyte density in biopsy samples with adequate accuracy and reproducibility. A similar approach for counting nuclei in tissue sections was suggested by Abercrombie in 1946.²¹     

Evaluation of nose-only aerosol inhalation chamber and comparison of experimental results with mathematical simulation of aerosol deposition in mouse lungs

In vivo small rodent efficacy testing of new synthetic and biological molecules for the pulmonary route requires an efficient delivery device. For this purpose, a nose-only inhalation chamber was used to deliver aerosolized aqueous compounds to the respiratory tract of mice. The aim of the study was to determine the efficiency of dose delivery and deposition in the lungs of the mice using this chamber. A secondary goal was to compare the experimental lung deposition results with values predicted from mathematical simulation. Experimental tests were conducted by generating aerosols of a radiolabeled formulation of human serum albumin (HSA) with a mass median aerodynamic diameter (MMAD) of 3.9 +/- 0.5 microm and a geometric standard deviation (GSD) of 1.43 +/- 0.05 using PARI LC STAR jet nebulizers. Based on the total activity placed in the nebulizer, the chamber delivered 0.108 +/- 0.027% to the mice and 0.0087 +/- 0.0021% to the lungs of the mice. In vivo lung deposition was found to be 8.19 +/- 3.56% of total activity deposited in the mouse. Mathematical simulation predictions ranged between 5.89 and 4.40% for various breathing patterns, and did not differ significantly from the in vivo results (p > 0.10). These results provide important quantitative information relevant to aerosol delivery experiments in mouse models. Our results also suggest that the nose-only inhalation chamber would benefit from significant changes to increase the efficiency of deposition in mice such that it can be used for nebulization of expensive therapeutic drugs.     

Imoxin attenuates high fructose‐induced oxidative stress and apoptosis in renal epithelial cells via downregulation of protein kinase R pathway

Double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR), a ubiquitously expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance, and glucose homeostasis in obesity. Recent studies have demonstrated that PKR can respond to metabolic stress in mice as well as in humans. However, the underlying molecular mechanism is not fully understood. The aim of this study was to examine the effect of high fructose (HF) in cultured renal tubular epithelial cells (NRK‐52E) derived from rat kidney and to investigate whether inhibition of PKR could prevent any deleterious effects of HF in these cells. PKR expression was determined by immunofluorescence staining and Western blotting. Oxidative damage and apoptosis were measured by flow cytometry. HF‐treated renal cells developed a significant increase in PKR expression. A significant increase in reactive oxygen species generation and apoptosis was also observed in HF‐treated cultured renal epithelial cells. All these effects of HF were attenuated by a selective PKR inhibitor, imoxin (C16). In conclusion, our study demonstrates PKR induces oxidative stress and apoptosis, is a significant contributor involved in vascular complications and is a possible mediator of HF‐induced hypertension. Inhibition of PKR pathway can be used as a therapeutic strategy for the treatment of cardiovascular and metabolic disorders.