Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

Silencing HIF-1α aggravates growth inhibition and necrosis of proximal renal tubular epithelial cell under hypoxia.

The kidney is particularly susceptible to ischemia/hypoxia insult while dysfunction of proximal tubular epithelial cells (PTEC) is a primary pathologic hallmark in acute kidney injury. Hypoxia-inducible factor-1 (HIF-1) is a key regulator responsible for cellular hypoxic responses. Therefore, we investigated the effects of HIF-1 suppression, using small interference RNA (siRNA), upon the cell fate of PTEC under hypoxia, and explored the underlying possible molecular mechanism. Hypoxia was induced with hypoxia mimetic cobalt chloride. Our data showed that, in HIF-1α siRNA group, the HK-2 cells growth inhibition and necrosis became worse than those in hypoxia group. However, for apoptosis, no significant difference was observed between them. Consistent with the downregulation of HIF-1α in HIF-1α siRNA group, both mRNA and protein expression of glucose transporter-1 (Glut-1) and vascular endothelial growth factor (VEGF) also reduced more significantly than those in hypoxia group. In conclusion, silencing HIF-1α gene could aggravate growth inhibition and necrosis of PTEC under hypoxia. We provide evidence, from the opposite direction, that HIF-1 activation under hypoxia may facilitate adaptation and survival of proximal renal tubular cells, and the beneficial effects may be related to its downstream genes, such as Glut-1 and VEGF.

Prolyl hydroxylase 2 (PHD2) inhibition protects human renal epithelial cells and mice kidney from hypoxia injury.

Prolyl hydroxylase domain protein 2 (PHD2) is a key oxygen sensor, setting low steady-state level of hypoxia-inducible factor-α (HIF-α). Here, we showed that treatment of cobalt chloride (CoCl2), a hypoxia mimic, in HK-2 tubular epithelial cells induced PHD2 and HIF-1/2α expression as well as cell apoptosis and autophagy activation. Three methyladenine (3-MA), the autophagy inhibitor, blocked autophagy and protected HK-2 cells from CoCl2. Significantly, siRNA knockdown of PHD2 also protected HK-2 cells from CoCl2,possibly via increasing HIF-1α expression. Reversely, HIF-1α siRNA knockdown almost abolished cytoprotection by PHD2 siRNA in CoCl2-treated HK-2 cells. In vivo, pretreatment with a PHD inhibitor L-mimosine remarkably attenuated mice renal ischemia-reperfusion injuries. Molecularly, L-mimosine inhibited apoptosis and inflammatory responses in injured mice kidneys. Together, our results suggest that PHD2 silence or inhibition protects human renal epithelial cells and mice kidney from hypoxia injuries.

The protection of selenium on cadmium-induced inhibition of spermatogenesis via activating testosterone synthesis in mice.

Selenium (Se) is an essential trance element in testis. However, the potential protective effects of Se against cadmium (Cd)-induced reproductive toxicity remained to be elucidated. Male ICR mice were orally administered by gavage with Na2SeO3 (0.1, 0.2, 0.4 mg/kg BW) for 1h prior to CdCl2 (5 mg/kg BW) alone or in combination for 15, 25 or 35 days. Cd exposure caused a significant decrease in body weight, sperm concentration and motility as well as plasma testosterone level which was accompanied by decreased antioxidant enzymatic activity of SOD and GSH-Px and by increased lipid peroxidation (as malondialdehyde, MDA). Se pretreatment compensated deficits in the sperm parameters (concentration, motility and morphology) induced by Cd. Se (0.4 mg/kg BW) treatment significantly increased serum testosterone level that was reduced by Cd (on 15th, 25th and 35th day) (P<0.01). Se treatment ameliorated Cd-induced reduction in testicular steroidogenic acute regulatory (StAR) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) activities. The present study suggest that the protective potential of Se against Cd-induced reprotoxicity might be due to up-regulation StAR and testosterone synthetic enzyme activity, which could be useful for increasing testosterone synthesis for achieving optimum protection in sperm quality and spermatogenesis.

Transient hypoxia-inducible factor activation in rat renal ablation and reduced fibrosis with L-mimosine.

AIM: Hypoxia-inducible factor (HIF) activity during the course of chronic kidney disease (CKD) development is poorly defined, and the effect of HIF activation on CKD is still controversial. The purpose of the present study was to characterize HIF expression during the course of CKD development, and to investigate the effect of HIF activation on CKD by using prolyl hydroxylase (PHD) inhibitor L-mimosine. METHODS: Rats with remnant kidneys (RK) were killed at week 1, 2, 4, 6, 8, 12 after subtotal nephrectomy. An additional group of RK rats was treated with L-mimosine to study the effect of HIF-α activation. RESULTS: Tubulointerstitial hypoxia in the remnant kidney began at week 1 and continued, albeit attenuated, until week 12, the last time point examined. The nuclear expression of HIF-1α and HIF-2α, as well as typical HIF target genes VEGF (vascular endothelial growth factor), HO-1 (heme oxygenase-1), GLUT-1 (glucose transporter-1) and EPO (erythropoietin), were all upregulated in the early stage of RK when renal function was stable, and returned to the basal level later, accompanied by impaired renal function and interstitial fibrosis. L-mimosine administered from week 5 to week 12 led to accumulation of HIF-1α and HIF-2α proteins, increased expression of VEGF, HO-1 and GLUT-1, and improved renal function. Furthermore, fibrosis markers α-smooth muscle actin (α-SMA) and Collagen III, as well as peritubular capillary rarefaction index, were all significantly decreased after L-mimosine treatment. CONCLUSION: There was a transient HIF-α activation in the remnant kidney of rats at the early stage following subtotal nephrectomy. L-mimosine administered in later stages re-activated HIF-α and reduced tubulointerstitial fibrosis.

BMI, spKt/V, and SBP but not DBP are related to LVH in Chinese maintenance hemodialysis patients.

OBJECTIVE: Left ventricular hypertrophy (LVH) is the strongest predictor of cardiovascular mortality, the leading cause of death in hemodialysis (HD) patients. This study aims to identify the potential risk factors for LVH in HD patients. METHODS: Exactly, 164 patients (84 men and 80 women) who had been on HD treatment for at least 6 months were enrolled. Clinical data were collected. Anthropometric measurements, biochemical analyses, and echocardiography were performed. The risk factors were determined by multivariate linear and logistic regression. RESULTS: In all the patients, the prevalence of LVH was 66.5%. The patients with LVH had higher body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure, and lower single-pool kt/V (spKt/V) compared with those without LVH. Multivariate linear regression showed that BMI (ß = 7.608, p = 0.014), SBP (ß = 9.462, p = 0.001), and spKt/V (ß = -14.226, p = 0.024) were independently correlated with left ventricular mass index (LVMI). Multivariate logistic regression showed the same results that BMI (ß = 7.193, p = 0.032), SBP (ß = 9.382, p = 0.02), and spKt/V (ß = -12.535, p = 0.001) were independently correlated with LVH. CONCLUSIONS: In Chinese maintenance hemodialysis patients, BMI, single-pool Kt/V (spKt/V), and SBP were independently correlated with left ventricular mass index and were independent risk factors for LVH.

Acute kidney injury in a Chinese hospitalized population.

OBJECTIVES: This study's objective was to determine the incidence and mortality rate of acute kidney injury (AKI) among hospitalized adult patients in a tertiary metropolitan hospital of China, and to evaluate the impact of AKI on in-hospital mortality, cost and length of stay (LOS). METHODS: Patients who were admitted to Zhongshan Hospital, Fudan University, Shanghai, China between September 1st, 2004 and June 30th, 2008 were involved. The presence and severity of AKI were assessed using absolute and relative increases from baseline to peak serum creatinine concentration during hospitalization. AKI was defined as a relative 50% increase or an absolute increment of 0.3 mg/dl (26.5 µmol/l) in serum creatinine within 48 h. After screening the computer-based data on kidney function, patients with AKI were identified and further history reviews were performed to obtain information regarding patients' demography, prognosis, severity of kidney injury and causes of AKI. RESULTS: There were 176,155 admissions during the study period and 5,619 met the diagnostic criteria of AKI. The overall incidence rate of AKI was 3.19%. Cardiovascular diseases followed by urogenital diseases and malignancy were the most common admission diagnoses. In-hospital mortality rate was 2.84% in all discharges and 19.68% in patients with AKI. Of AKI patients, old age, intensive care unit admission, Acute Kidney Injury Network score, need for renal replacement therapy and organ system failure number were independent predictors of hospital mortality according to forward conditional logistic regression. CONCLUSIONS: AKI is prevalent in the Chinese hospitalized patients. Slight elevations of serum creatinine are associated with significantly increased mortality, LOS and hospital cost. Moreover, outcomes are related directly to the severity of AKI characterized by percent changes in serum creatinine.

Bladder epithelial cell proliferation of rats induced by terephthalic acid-calculi.

OBJECTIVE: Urinary bladder hyperplasia associated with terephthalic acid (TPA) treatment was examined with concomitant use of sodium bicarbonate (NaHCO3) or hydrochlorothiazide to allow assessment of the relationship among bladder stones, epithelial hyperplasia, and corresponding cell cycle checkpoint gene expression in Sprague-Dawley (SD) rat. METHODS: A total of 112 weanling male SD rats that divided between six groups were given basal diet (control), diets containing 5% TPA or in combination with either 4% sodium NaHCO3 or 0.02% hydrochlorothiazide. After 90-day feeding, bladder samples were collected for histopathological diagnoses, and immunohistochemical method was used to characterize the expression of p16Ink4a cyclin D1, CDK4, EGFr and cyclin E in relation to that of proliferating cell nuclear antigen (PCNA). RESULTS: In TPA treatment groups, bladder stone incidence was 40% (21/52) with 14 cases of proliferative bladder. In control and other groups, neither stone nor epithelial cell proliferation was diagnosed. PCNA-positive focal hyperplasic lesions involved all epithelial layers. Overexpressions of cyclin D1, CDK4, EGFr are found in the corresponding lesion. p16Ink4a nuclear staining reduced in proliferative bladders especially with a great quantity of stone. In addition, no positive expression was detected on cyclin E. CONCLUSION: The present study provides a strong evidence of a link between induction of bladder hyperplasia, deregulation of the p16Ink4a-cyclin D1/CDK4 pathway, and abnormal EGFr mediated signal transduction pathway.