Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat

Prolonged ischemia amplified iscehemia/reperfusion (IR) induced renal apoptosis and autophagy. We hypothesize that ischemic conditioning (IC) by a briefly intermittent reperfusion during a prolonged ischemic phase may ameliorate IR induced renal dysfunction. We evaluated the antioxidant/oxidant mechanism, autophagy and apoptosis in the uninephrectomized Wistar rats subjected to sham control, 4 stages of 15-min IC (I15 × 4), 2 stages of 30-min IC (I30 × 2), and total 60-min ischema (I60) in the kidney followed by 4 or 24 hours of reperfusion. By use of ATP assay, monitoring O2-. amounts, autophagy and apoptosis analysis of rat kidneys, I60 followed by 4 hours of reperfusion decreased renal ATP and enhanced reactive oxygen species (ROS) level and proapoptotic and autophagic mechanisms, including enhanced Bax/Bcl-2 ratio, cytochrome C release, active caspase 3, poly-(ADP-ribose)-polymerase (PARP) degradation fragments, microtubule-associated protein light chain 3 (LC3) and Beclin-1 expression and subsequently tubular apoptosis and autophagy associated with elevated blood urea nitrogen and creatinine level. I30 × 2, not I15 × 4 decreased ROS production and cytochrome C release, increased Manganese superoxide dismutase (MnSOD), Copper-Zn superoxide dismutase (CuZnSOD) and catalase expression and provided a more efficient protection than I60 against IR induced tubular apoptosis and autophagy and blood urea nitrogen and creatinine level. We conclude that 60-min renal ischemia enhanced renal tubular oxidative stress, proapoptosis and autophagy in the rat kidneys. Two stages of 30-min ischemia with 3-min reperfusion significantly preserved renal ATP content, increased antioxidant defense mechanisms and decreased ischemia/reperfusion enhanced renal tubular oxidative stress, cytosolic cytochrome C release, proapoptosis and autophagy in rat kidneys

The role of TonEBP in regulation of AAD expression and dopamine production in renal proximal tubule cells upon hypertonic challenge

a b s t r a c t Renal proximal tubule cells overexpress aromatic L-amino acid decarboxylase (AAD) to produce dopamine, which inhibits salt absorption in the hypertonic environment. We examined the effect of TonEBP on AAD expression in human proximal tubule epithelial cells, HK-2 cell line. Confocal microscopy showed that after 2 h of exposure to the hypertonic medium, TonEBP accumulation in nuclei increased as compared to the isotonic control. The activated TonEBP enhanced the mRNA expression of the representative downstream genes (i.e., SMIT and TauT). Meanwhile, AAD protein abundance also increased with TonEBP activation. EMSA and luciferase reporter assay showed that TonEBP was involved in transcriptional regulation of AAD upon hypertonic stress. Inactivation of TonEBP by the p38 inhibitor SB203580, or TonEBP shRNA significantly reduced AAD expression, which was rescued by re-expressing Myc-tagged TonEBP. Up-regulation of AAD increased dopamine synthesis, and dopamine inhibited NKA activity in hypertonic condition. These results suggested that TonEBP played an important role in the epithelial cells of renal proximal tubule upon hypertonic stress by enhancing AAD expression, which could promote dopamine secretion to negative regulate NKA activity. The elucidation of a new mechanism described in this study combined with previous findings provides more insights into this issue

Trypsin-induced proteome alteration during cell subculture in mammalian cells

<p>Abstract</p> <p>Background</p> <p>It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.</p> <p>Methods</p> <p>In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.</p> <p>Results</p> <p>36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.</p> <p>Conclusions</p> <p>In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.</p

BPR1K653, a Novel Aurora Kinase Inhibitor, Exhibits Potent Anti-Proliferative Activity in MDR1 (P-gp170)-Mediated Multidrug-Resistant Cancer Cells

Over-expression of Aurora kinases promotes the tumorigenesis of cells. The aim of this study was to determine the preclinical profile of a novel pan-Aurora kinase inhibitor, BPR1K653, as a candidate for anti-cancer therapy. Since expression of the drug efflux pump, MDR1, reduces the effectiveness of various chemotherapeutic compounds in human cancers, this study also aimed to determine whether the potency of BPR1K653 could be affected by the expression of MDR1 in cancer cells.BPR1K653 specifically inhibited the activity of Aurora-A and Aurora-B kinase at low nano-molar concentrations in vitro. Anti-proliferative activity of BPR1K653 was evaluated in various human cancer cell lines. Results of the clonogenic assay showed that BPR1K653 was potent in targeting a variety of cancer cell lines regardless of the tissue origin, p53 status, or expression of MDR1. At the cellular level, BPR1K653 induced endo-replication and subsequent apoptosis in both MDR1-negative and MDR1-positive cancer cells. Importantly, it showed potent activity against the growth of xenograft tumors of the human cervical carcinoma KB and KB-derived MDR1-positive KB-VIN10 cells in nude mice. Finally, BPR1K653 also exhibited favorable pharmacokinetic properties in rats.BPR1K653 is a novel potent anti-cancer compound, and its potency is not affected by the expression of the multiple drug resistant protein, MDR1, in cancer cells. Therefore, BPR1K653 is a promising anti-cancer compound that has potential for the management of various malignancies, particularly for patients with MDR1-related drug resistance after prolonged chemotherapeutic treatments

Maternal diabetes and risk of attention-deficit/hyperactivity disorder in offspring in a multinational cohort of 3.6 million mother-child pairs

Previous studies report an association between maternal diabetes mellitus (MDM) and attention-deficit/hyperactivity disorder (ADHD), often overlooking unmeasured confounders such as shared genetics and environmental factors. We therefore conducted a multinational cohort study with linked mother-child pairs data in Hong Kong, New Zealand, Taiwan, Finland, Iceland, Norway and Sweden to evaluate associations between different MDM (any MDM, gestational diabetes mellitus (GDM) and pregestational diabetes mellitus (PGDM)) and ADHD using Cox proportional hazards regression. We included over 3.6 million mother-child pairs between 2001 and 2014 with follow-up until 2020. Children who were born to mothers with any type of diabetes during pregnancy had a higher risk of ADHD than unexposed children (pooled hazard ratio (HR) = 1.16, 95% confidence interval (CI) = 1.08-1.24). Higher risks of ADHD were also observed for both GDM (pooled HR = 1.10, 95% CI = 1.04-1.17) and PGDM (pooled HR = 1.39, 95% CI = 1.25-1.55). However, siblings with discordant exposure to GDM in pregnancy had similar risks of ADHD (pooled HR = 1.05, 95% CI = 0.94-1.17), suggesting potential confounding by unmeasured, shared familial factors. Our findings indicate that there is a small-to-moderate association between MDM and ADHD, whereas the association between GDM and ADHD is unlikely to be causal. This finding contrast with previous studies, which reported substantially higher risk estimates, and underscores the need to reevaluate the precise roles of hyperglycemia and genetic factors in the relationship between MDM and ADHD

The potential role of FXYD2 isoform 3 in regulation of Na+, K+-ATPase activity in HK-2 cells upon hypertonic challenge

Na+, K+-ATPase (NKA)為廣泛存在細胞內且對於滲透壓調節極具重要性的運輸蛋白，其利用水解ATP排出三個鈉離子和吸收兩個鉀離子製造出離子濃度梯度，藉此造成其他二級主動運輸蛋白的驅動力。細胞利用調節NKA的功能來達到細胞對外界環境滲透壓變化的適應。哺乳類腎臟為扮演滲透壓平衡的調節器官，其中近曲小管為主要部位之一。因此本研究利用人類腎臟近曲小管表皮細胞(HK-2)給予高滲透壓刺激再搭配microarray的分析，篩選出與調節NKA功能相關且具高度表現之基因。其中FXYD2，一個具腎臟組織特異性且可影響NKA對鈉鉀親和力的蛋白質即為首選。在驗證microarray的結果時發現，FXYD2具有三個isoforms，其中isoform 3具有相對的顯著性表現。此外，isoform 3至今未有文獻探討其在滲透壓調節的功能，因此FXYD2 isoform 3為主要探討目標。實驗結果發現FXYD2 isoform 3和NKA α1 subunit的mRNA及蛋白表現量在高滲透壓下都有顯著性上升。另外也觀察到FXYD2 isoform 3的表現位置位於細胞膜上，而前人的研究也指出NKA α1 subunit表現在細胞膜上，所以更進一步去檢視FXYD2 isoform 3是否有跟NKA α1 subunit有交互作用，結果使用共同免疫沉澱得到他們確實有關係。為更深入探討FXYD2 isoform 3的角色，本研究使用了RNAi-A (knockdown isoform 1, 2, and 3)、RNAi-B (knockdown isoform 1 and 2)之細胞。FXYD2 isoforms 的knockdowm並未影響NKA α1 subunit的蛋白質表現，但NKA活性卻受到改變，NKA活性的排序由高至低為RNAi-B > RNAi-A > wild type。經由互相比較發現FXYD2 isoform 1, 2扮演著抑制NKA活性的角色；而FXYD2 isoform 3則是扮演促進NKA活性之角色。另一方面，在高滲透壓環境下，有文獻指出TonEBP為大量表現的轉錄因子，其主要活化其下游基因表現以保護細胞能適應此逆境並得以生存，所以TonEBP的調節機制對於細胞適應高滲透壓有舉足輕重的角色。經TonEBP knockdown實驗發現， TonEBP會影響FXYD2 isoform 3的mRNA和蛋白質表現，間接影響NKA的活性。綜合以上的結果本篇研究首先發現，在高滲透壓環境下HK-2細胞藉由TonEBP增加 FXYD2 isoform 3的表現，進而調節NKA之活性。Na+,K+-ATPase (NKA) is a widely existence and important transporter in animal cells. NKA uptake two of K+ and extrusion of Na+ in providing the driving force to trigger the secondary active transporters by using the energy of the hydrolysis of ATP. Cells regulated NAK function to adapt the environmental osmotic changes. The mammalian kidney was the key organ in osmotic homeostasis and proximal tubule was the one of major tissue. This study used human renal proximal tubule cells HK-2 and microarray screened the up-regulated genes which correlate to NKA function upon hypertonic challenge. FXYD2 protein was specificity in kidney, and it could effect on sodium/potassium affinity of NKA. To confirm the microarray data, FXYD2 isoform 3 was markedly expressed. There were still no reporters to discuss detail function of isoform 3 in osmotic regulation. Therefore, FXYD2 isoform 3 was the target of this study with functional assay. In this study, the mRNA and protein abundance of FXYD2 isoform 3 were significantly increased upon hypertonic challenge. In addition, the localization of FXYD2 isoform 3 was distributed on cell membrane as NKA α1 subunit. Moreover, we obtained FXYD2 isoform 3 indeed had interaction with NKA α1 subunit. Accordingly, it was suggested that FXYD2 isoforms3 played the role in regulation of NKA activity. Therefore, the potential role of FXYD2 isoform 3 will be illustrated during hypertonic challenge by using the RNAi-A (knockdown isoform 1, 2 and 3) and RNAi-B (knockdown isoform 1 and 2) knockdown cells. FXYD2 knockdown cells did not affect NKA protein expression level, but NKA activity was affected upon hypertonic stress. NKA activity high to low was RNAi-B > RNAi-A > wild type. FXYD2 isoform 1and 2 played the repressor role, and isoform 3 played an enhancer role in NKA activity regulation. On the other hand, some studies indicated TonEBP was a transcription factor and it was higher expression upon hypertonic challenge. TonEBP modulated its downstream genes to protect cells could adapt the effects of hypertonic challenge. Hence, modulation mechanism of TonEBP was important when cells were faced hypertonic environment. In TonEBP knockdown cells, FXYD2 isoform 3 expression was regulated by TonEBP and result in affecting NKA activity. Take together, this study was the first reporter to demonstrate that hypertonicity promoted FXYD2 isoform 3 expression through TonEBP-dependent regulation, and then enhanced NKA activity in HK-2 cells.Contents......i Tables and figures contents......ii 中文摘要......1 Abstract......3 Introduction......5 Materials and methods......11 Results......21 Discussion......30 References......37 Tables......47 Figures......5

Temporal Trends of Severe Hypoglycemia and Subsequent Mortality in Patients with Advanced Diabetic Kidney Diseases Transitioning to Dialysis

Background: Patients with diabetic kidney disease (DKD) are at higher risk of hypoglycemia than diabetic patients without DKD. We aimed to investigate the temporal trends of severe hypoglycemia in advanced DKD patients transitioning to dialysis and examine risk factors associated with severe hypoglycemia. We also investigated the association of severe hypoglycemia episodes with one-year mortality after initiation of dialysis in patients with advanced DKD. Methods: Using the Taiwan National Health Insurance Research Database, 46,779 advanced DKD patients transitioning to dialysis (Peritoneal dialysis 4216, hemodialysis 42,563) between 1997 and 2011 were enrolled. We calculated the rates of severe hypoglycemia from 5 years before dialysis until 10 years after dialysis. Cox proportional hazard model was used to examine the risk factors of post end stage renal disease (ESRD) one-year hypoglycemia and post ESRD one-year mortality in advanced DKD patients transitioning to dialysis. Results: We found that 11.5% of advanced DKD patients had at least one episode of severe hypoglycemia the year leading up to dialysis initiation. Multivariate analysis revealed hemodialysis compared with peritoneal dialysis, stroke, use of sulfonylurea, glinide, and insulin were associated with higher risk of severe hypoglycemia one year after transitioning to dialysis. Increased frequency of severe hypoglycemia-related hospitalizations was associated with incrementally higher mortality risk one year after transitioning to dialysis (Pre-ESRD hypoglycemia: Hazard ratios: 1.28 (1.18&#8211;1.38, p &lt; 0.001), 1.64 (1.49&#8211;1.81, p &lt; 0.001) for one, two hypoglycemia-related hospitalizations, respectively; post-ESRD hypoglycemia: HRs of 1.56 (1.40&#8211;1.73, p &lt; 0.001), 1.72 (1.39&#8211;2.12, p &lt; 0.001) for one, two hypoglycemia-related hospitalizations, respectively (reference group: no hypoglycemia related hospitalization)). Conclusions: Among advanced DKD patients, we observed a progressive elevated risk of hypoglycemia during the critical dialysis transition period. Increased frequency of severe hypoglycemia-related hospitalizations was associated with higher mortality risk one year after transitioning to dialysis. Further study of glycemic management strategies which prevent hypoglycemia during the critical transition period are warranted

A Patient Friendly Corifollitropin Alfa Protocol without Routine Pituitary Suppression in Normal Responders.

The release of corifollitropin alfa simplifies daily injections of short-acting recombinant follicular stimulating hormone (rFSH), and its widely-used protocol involves short-acting gonadotropins supplements and a fixed GnRH antagonist regimen, largely based on follicle size. In this study, the feasibility of corifollitropin alfa without routine pituitary suppression was evaluated. A total of 288 patients were stimulated by corifollitropin alfa on cycle day 3 following with routine serum hormone monitoring and follicle scanning every other day after 5 days of initial stimulation, and a GnRH antagonist (0.25 mg) was only used prophylactically when the luteinizing hormone (LH) was ≧ 6 IU/L (over half of the definitive LH surge). The incidence of premature LH surge (≧ 10 IU/L) was 2.4% (7/288) before the timely injection of a single GnRH antagonist, and the elevated LH level was dropped down from 11.9 IU/L to 2.2 IU/L after the suppression. Two hundred fifty-one patients did not need any antagonist (87.2% [251/288]) throughout the whole stimulation. No adverse effects were observed regarding oocyte competency (fertilization rate: 78%; blastocyst formation rate: 64%). The live birth rate per OPU cycle after the first cryotransfer was 56.3% (161/286), and the cumulative live birth rate per OPU cycle after cyrotransfers was 69.6% (199/286). Of patients who did and did not receive GnRH antagonist during stimulation, no significant difference existed in the cumulative live birth rates (78.4% vs. 68.3%, p = 0.25). The results demonstrated that the routine GnRH antagonist administration is not required in the corifollitropin-alfa cycles using a flexible and hormone-depended antagonist regimen, while the clinical outcome is not compromised. This finding reveals that the use of a GnRH antagonist only occasionally may be needed

Identification of mosaic and segmental aneuploidies by next-generation sequencing in preimplantation genetic screening can improve clinical outcomes compared to array-comparative genomic hybridization

Abstract Background Chromosomal mosaicism is observed as the presence of both euploid and aneuploid cells in a particular blastocyst. Recent studies have reported that the implantation rate of mosaic embryo transfer is remarkably lower than the euploid embryos. The superior capability of next-generation sequencing (NGS) to detect chromosomal mosaicism in preimplantation genetic screening (PGS) remains controversial, and several data displayed similar implantation and pregnancy rates using NGS or array comparative genomic hybridization (aCGH). Results In this study, the main inconsistency of aneuploidy detection and clinical performance between the NGS and aCGH were assessed. The phase I consisted of a parallel comparison in 182 blastocysts from 45 selected PGS patients for both the NGS and aCGH platforms. The phase II retrospectively compared the clinical outcomes of 90 patients with NGS-screened euploid embryo transfer to that of 129 patients with aCGH-screened euploid embryo transfer. The parallel comparison showed that the inconsistency of embryo euploidy was 11.8% (p = 0.01). Chromosomal mosaicism (10.7% with NGS vs. 3.9% with aCGH) and segmental aneuploidy (10.7% with NGS vs. 6.7% with aCGH) contributed to the discrepancy mainly. The chromosomally mosaic embryos (20%–50% of aneuploidy) and several embryos with segmental aneuploidy (≥10 Mbp) were hard to distinguish using the aCGH platform, but could be clearly identified using the NGS platform. After the first euploid embryo cryotransfer, the β-HCG(+) rate and implantation rate significantly increased in the PGS/NGS patients (HCG[+] rate: 73.3% in PGS/NGS vs. 60.5% in PGS/aCGH, p = 0.048; implantation rate: 53.2% in PGS/NGS vs. 45.0% in PGS/aCGH, p = 0.043). The clinical and ongoing pregnancy rates appeared higher in the NGS group, but did not reached statistical significance. Conclusions The results demonstrated that the NGS platform can identify embryos with chromosomal mosaicism and segmental aneuploidy more precisely than the aCGH platform, and the following clinical performance of NGS was more favorable