N-linked glycoproteomic profiling in esophageal squamous cell carcinoma.

BACKGROUND: Mass spectrometry-based proteomics and glycomics reveal post-translational modifications providing significant biological insights beyond the scope of genomic sequencing. AIM: To characterize the N-linked glycoproteomic profile in esophageal squamous cell carcinoma (ESCC) via two complementary approaches. METHODS: Using tandem multilectin affinity chromatography for enrichment of N-linked glycoproteins, we performed N-linked glycoproteomic profiling in ESCC tissues by two-dimensional gel electrophoresis (2-DE)-based and isobaric tags for relative and absolute quantification (iTRAQ) labeling-based mass spectrometry quantitation in parallel, followed by validation of candidate glycoprotein biomarkers by Western blot. RESULTS: 2-DE-based and iTRAQ labeling-based quantitation identified 24 and 402 differentially expressed N-linked glycoproteins, respectively, with 15 in common, demonstrating the outperformance of iTRAQ labeling-based quantitation over 2-DE and complementarity of these two approaches. Proteomaps showed the distinct compositions of functional categories between proteins and glycoproteins with differential expression associated with ESCC. Western blot analysis validated the up-regulation of total procathepsin D and high-mannose procathepsin D, and the down-regulation of total haptoglobin, high-mannose clusterin, and GlcNAc/sialic acid-containing fraction of 14-3-3ζ in ESCC tissues. The serum levels of glycosylated fractions of clusterin, proline-arginine-rich end leucine-rich repeat protein, and haptoglobin in patients with ESCC were remarkably higher than those in healthy controls. CONCLUSION: Our study provides insights into the aberrant N-linked glycoproteome associated with ESCC, which will be a valuable resource for future investigations.

O-glycosylated clusterin as a sensitive marker for diagnosing early stages of prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) has been the most popular diagnostic marker for prostate cancer. The frequent occurrence of low PSA values (<10 ng/ml) in patients with highly suspicious prostate cancer, however, has undermined the accuracy of clinical examinations. The aim of this study was to develop a better resolution for diagnosing prostate cancer to overcome the disadvantage of PSA. METHODS: We focused on the glycosylation status of patients' serum proteins and conducted comprehensive lectin microarray analyses to characterize N- and O-glycans using sera from prostate cancer and benign prostatic diseases. Next, we retrieved candidate serum proteins with characteristic glycan structures using lectin-immobilized beads and identified them by quantitative mass spectrometry using a technique referred to as isobaric tag for relative and absolute quantitation (iTRAQ) labeling. Finally, we constructed a new assay to quantify a candidate glycoprotein with the newly identified glycans. RESULTS: Lectin microarray analyses revealed that sera from patients with prostate cancer had a higher affinity for Jacalin, Amaranthus caudatus (ACA) lectin, and Maclura pomifera (MPA) lectin, compared with that from patients with benign prostatic diseases and normal subjects, suggesting that O-glycosylated proteins are more abundant in sera from patients with prostate cancer. Then, serum glycoproteins preferentially adsorbed onto Jacalin-Agarose as well as biotin-ACA/and biotin-MPA/streptavidin-immobilized magnetic beads were isolated, labeled with iTRAQ, and identified using quantitative mass spectrometry. It was found that the ACA- and MPA-recognizable clusterin was more enriched in patients' sera from prostate cancer compared with those from benign prostatic diseases. Following this discovery, we constructed a Luminex-based assay to quantify O-glycosylated clusterin, in which total serum clusterin was first captured on anti-clusterin antibody-immobilized beads, and then clusterin-associated O-glycans were determined by the pair of biotin-MPA and streptavidin-phycoerythrin. When PSA values registered less than 10 ng/ml, the corresponding serum level of MPA-recognized clusterin determined by this assay was beneficial for distinguishing the patients with prostate cancer from the patients with benign prostatic disease. CONCLUSION: For PSA values that measure less than 10 ng/ml, the serum O-glycosylated clusterin level can be a complementary indicator for the malignancy of prostate cancer.

DNAJA4 deficiency augments hyperthermia-induced Clusterin and ERK activation: two critical protective factors of human keratinocytes from hyperthermia-induced injury.

BACKGROUND: Hyperthermia upregulates DNAJA4, a member of heat shock proteins (HSPs) 40 family, in human keratinocytes and HPV-infected tissue. DNAJA4 deficiency enhances growth arrest induced by hyperthermia. Clusterin (CLU) and phosphorylated ERK (p-ERK) play a role in regulating cell proliferation and apoptosis, under environmental stress. OBJECTIVES: To examine the downstream molecules and signalling pathways of DNAJA4 and assess their roles in cell cycle and apoptosis of keratinocytes in response to hyperthermia. METHODS: Wild-type and DNAJA4-knockout (KO) HaCaT cells were exposed to either 44 °C (hyperthermia) or 37 °C (control) for 30 min. The expression levels of CLU and p-ERK were determined by RT-PCR and Western blotting. RNAi and PD98059 were used to inhibit the expression of CLU and p-ERK, respectively. Cell viability, cell cycle and apoptosis were analysed by MTS assay and flow cytometry. Fresh biopsy samples of human normal foreskin or condyloma acuminatum (CA) were utilized to examine the expression of CLU and p-ERK after ex vivo culture at 44 °C. RESULTS: The expression of CLU and p-ERK was significantly increased by hyperthermia treatment at 44 °C in HaCaT cells, foreskin and HPV-infected tissues. In HaCaT cells subjected to hyperthermia, DNAJA4 deficiency further augmented the expression of CLU and p-ERK. CLU deficiency enhanced the p-ERK expression. Hyperthermia-induced CLU and p-ERK exerted protective roles mainly through inhibiting apoptosis and maintaining cell cycle, respectively. CONCLUSIONS: In keratinocytes, CLU and p-ERK are induced by hyperthermia, an effect which can be further enhanced by DNAJA4 deficiency. CLU deficiency also increases p-ERK expression. Both CLU and p-ERK are critical protective factors of human keratinocytes from hyperthermia-induced injury.

Dynamics of clusterin protein expression in the brain and plasma following experimental traumatic brain injury.

Progress in the preclinical and clinical development of neuroprotective and antiepileptogenic treatments for traumatic brain injury (TBI) necessitates the discovery of prognostic biomarkers for post-injury outcome. Our previous mRNA-seq data revealed a 1.8-2.5 fold increase in clusterin mRNA expression in lesioned brain areas in rats with lateral fluid-percussion injury (FPI)-induced TBI. On this basis, we hypothesized that TBI leads to increases in the brain levels of clusterin protein, and consequently, increased plasma clusterin levels. For evaluation, we induced TBI in adult male Sprague-Dawley rats (n = 80) by lateral FPI. We validated our mRNA-seq findings with RT-qPCR, confirming increased clusterin mRNA levels in the perilesional cortex (FC 3.3, p < 0.01) and ipsilateral thalamus (FC 2.4, p < 0.05) at 3 months post-TBI. Immunohistochemistry revealed a marked increase in extracellular clusterin protein expression in the perilesional cortex and ipsilateral hippocampus (7d to 1 month post-TBI), and ipsilateral thalamus (14d to 12 months post-TBI). In the thalamus, punctate immunoreactivity was most intense around activated microglia and mitochondria. Enzyme-linked immunoassays indicated that an acute 15% reduction, rather than an increase in plasma clusterin levels differentiated animals with TBI from sham-operated controls (AUC 0.851, p < 0.05). Our findings suggest that plasma clusterin is a candidate biomarker for acute TBI diagnosis.

Verteporfin blocks Clusterin which is required for survival of gastric cancer stem cell by modulating HSP90 function.

Gastric cancer stem cell (GCSC) is implicated in gastric cancer relapse, metastasis and drug resistance. However, the key molecule(s) involved in GCSC survival and the targeting drugs are poorly understood. We discovered increased secreted clusterin (S-Clu) protein expression during the sphere-forming growth of GCSC via mass spectrometry. Overexpression of clusterin was detected in 69/90 (77%) of primary GC tissues and significantly associated with T stage, lymph node metastasis and TNM stage. Depletion of clusterin (Clu, the full-length intracellular clusterin) led to the declustering of GCSC tumorspheres and apoptosis of GCSC. Subsequently, we found clusterin was in complex with heat shock protein 90 beta (HSP90) and involved in regulating the cellular level of HSP90 client proteins. Furthermore, by screening a collection of drugs/inhibitors, we found that verteporfin (VP), a phototherapy drug, blocked clusterin gene expression, decreased the HSP90 client proteins and caused cell death of GCSC. VP treatment is more effective in eradicating GCSCs than in killing GC cells. Both clusterin silencing or VP treatment deterred tumor growth in human GCSC xenografts. These findings collectively suggest that GC patients can promptly benefit from clusterin-targeted therapy as well as VP treatment in combination with or subsequent to conventional chemotherapy for reducing mortality of GC.

Novel and classical renal biomarkers as evidence for the nephroprotective effect of Carica papaya leaf extract.

The present study is aimed at utilization of novel and classical kidney function biomarkers to evaluate the nephroprotective potential of Carica papaya leaf extract (CPLE) in gentamicin nephrotoxicity model in albino rats. The used classical biomarkers were urea and creatinine; while the new biomarkers were Kidney injury molecule-1 (KIM-1) and Clusterin. Forty-five male albino rats were assigned into five groups and subjected to different treatments for nine consecutive days (vehicles; gentamicin, 100 mg/kg, subcutaneously; ascorbic acid, 200 mg/kg, orally; CPLE, 150 and 300 mg/kg b wt., orally). Three rats/group were killed on days 3, 6, and 9 for blood and tissue samples for renal and oxidation markers. Gentamicin resulted in significant increase in urea and creatinine only by the end of the experimental course; while the novel biomarkers were evident as early as 3 days upon gentamicin injection. When concurrently administered with gentamicin, CPLE significantly protected kidney tissues against gentamicin nephrotoxic effects indicated by decrement of both the novel and the classical standard biomarkers, in a dose-dependent manner. CPLE-mediated protection was attributed to its antioxidant potential indicated by significant inhibition of malondialdehyde (MDA) levels in both serum and kidney homogenates. The results were further supported by histopathological examination that revealed considerable amelioration of the pathological microscopic alterations induced by repeated gentamicin injection. Phytochemical analysis of CPLE indicated presence of tannins and flavonoids. These data may suggest CPLE, based on improvement of both classical and novel renal markers, as a highly potent nephroprotective and antioxidant from natural source that could be a good remedy in conditions associated with renal disorders.

Melittin inhibits tumor growth and decreases resistance to gemcitabine by downregulating cholesterol pathway gene CLU in pancreatic ductal adenocarcinoma.

Melittin is a Chinese traditional medicine for treating chronic inflammation, immunological diseases and cancers, however, the efficacy of melittin and its mechanism for treating pancreatic ductal adenocarcinoma (PDAC) are still unknown. Here we investigated the anti-cancer activity of melittin and its regulated mechanism(s) in the PDAC models. Melittin was found to suppress tumor growth by promoting cell apoptosis and cell-cycle arrest. Interestingly, the microarray analyses demonstrated that melittin significantly regulated cholesterol biosynthesis pathway during treatment. For instance, the cholesterol pathway gene clusterin (CLU) was highly downregulated by melittin which also enhanced gemcitabine sensitivity in PDAC cells by inhibiting CLU expression. In contrast, overexpression of CLU significantly diminished melittin mediated tumor suppression and gemcitabine sensitization, suggesting that CLU is the target of melittin. Furthermore, in the xenograft mouse model, the combination therapy of melittin and gemcitabine is more efficacious for inhibiting PDAC tumor growth than either single regimen. Taken together, our study has indicated that melittin is capable of suppressing tumor growth and promoting gemcitabine sensitivity in PDAC by downregulating cholesterol pathway.

Clusterin and Pycr1 alterations associate with strain and model differences in susceptibility to experimental pancreatitis.

Acute pancreatitis has several underlying etiologies, and results in consequences ranging from mild to complex multi-organ failure. The wide range of pathology suggests a genetic predisposition for progression. We compared the susceptibility to acute pancreatitis in BALB/c and FVB/N mice, coupled with proteomic analysis, in order to identify potential protein associations with pancreatitis progression. METHODS: Pancreatitis was induced in BALB/c and FVB/N mice by administration of cerulein or feeding a choline-deficient, ethionine-supplemented (CDE) diet. Histology and changes in serum amylase were examined. Proteome profiling in cerulein-treated mice was performed using 2-dimensional differential in gel electrophoresis (2D-DIGE) followed by mass spectrometry analysis and biochemical validation. RESULTS: Male and female FVB/N mice manifested more severe cerulein-induced pancreatitis as compared with BALB/c mice, but both strains were similarly susceptible to CDE-induced pancreatitis. Few of the 2D-DIGE alterations were validated by immunoblotting. Clusterin was markedly up-regulated after cerulein-induced pancreatitis in FVB/N but less-so in BALB/c mice. Pyrroline-5-carboxylate reductase (Pycr1), an enzyme involved in proline biosynthesis, had higher basal levels in FVB/N male and female mouse pancreata compared with BALB/c pancreata, and was relatively more resistant to degradation in FVB/N pancreata. However, serum and pancreas tissue proline levels were similar in the two strains. CONCLUSION: FVB/N is more susceptible than BALB/c mice to cerulein-induced but not CDE-induced pancreatitis. Most of the 2D-DIGE alterations in the two strains likely relate to posttranslational modifications rather than protein level differences. Clusterin levels increase dramatically in association with pancreatitis severity, while Pycr1 is higher in FVB/N versus BALB/c pancreata basally and after induction of pancreatitis. Changes in proline metabolism may represent a novel potential genetic modifier in the context of pancreatitis.

Therapeutic targeting of myeloid-derived suppressor cells involves a novel mechanism mediated by clusterin.

Myeloid-derived suppressor cells (MDSCs) constitute a key checkpoint that impedes tumor immunity against cancer. Chemotherapeutic intervention of MDSCs has gained ground as a strategy for cancer therapy but its mechanism remains obscure.We report here a unique mechanism by which monocytic (M)-MDSCs are spared, allowing them to polarize towards M1 macrophages for reactivation of immunity against breast cancer. We first demonstrated that curcumin, like docetaxel (DTX), can selectively target CD11b(+)Ly6G(+)Ly6C(low) granulocytic (G)-MDSCs, sparing CD11b(+)Ly6G(-)Ly6C(high) M-MDSCs, with reduced tumor burden in 4T1-Neu tumor-bearing mice. Curcumin treatment polarized surviving M-MDSCs toward CCR7(+) Dectin-1(-)M1 cells, accompanied by IFN-γ production and cytolytic function in T cells. Selective M-MDSC chemoresistence to curcumin and DTX was mediated by secretory/cytoplasmic clusterin (sCLU). sCLU functions by trapping Bax from mitochondrial translocation, preventing the apoptotic cascade. Importantly, sCLU was only found in M-MDSCs but not in G-MDSCs. Knockdown of sCLU in M-MDSCs and RAW264.7 macrophages was found to reverse their natural chemoresistance. Clinically, breast cancer patients possess sCLU expression only in mature CD68(+) macrophages but not in immature CD33(+) immunosuppressive myeloid cells infiltrating the tumors. We thus made the seminal discovery that sCLU expression in M-MDSCs accounts for positive immunomodulation by chemotherapeutic agents.

[Evaluation of nephrotoxicity induced by total terpenoids from Alismatis Rhizoma on HK-2 cells in vitro and its induction of apoptosis].

To evaluate the nephrotoxicity of total terpenoids from Alismatis Rhizoma on human kidney proximal tubular cells (HK-2), explore the iraction in inducing apoptosis of HK-2 cells, and provide reference for the research of controversial nephrotoxicity of total terpenoids from Alismatis Rhizoma, HK-2 cells were used and cells viability was measured by MTT colorimetric method. An assessment of cells apoptosis was also conducted by using flow cytometry. Meanwhile western blot assay was used to detect the protein expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. At last, q-PCR was used to detect the mRNA expressions of caspase-3, Bcl-2, Bcl-xl, Kim-1, clusterin and TFF-3. The flow cytometry results showed that cells apoptosis rate was (37.48±1.76)%, (26.91±1.91)% and (25.61±2.05)% respectively after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results showed that Bcl-2 and Bcl-xl protein levels were significantly decreased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹), while the protein expression of caspase-3 was significantly increased. q-PCR results were the same with western blot results, that mRNA expressions of Bcl-2 and Bcl-xl were significantly decreased while mRNA expression of caspase-3 was significantly increased after treating with total terpenoids (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). Western blot results and q-PCR results showed that both mRNA and protein expressions of Kim-1, clusterin and TFF-3 were significantly increased after treating with total terpenoids from Alismatis Rhizoma (6.25×10-5, 3.125×10-5, 1.562 5×10-5 g•mL⁻¹). HK-2 cells in vitro evaluation results showed that, total terpenoids from Alismatis Rhizoma may have nephrotoxicity effect, but further study is still needed for verification; meanwhile, they could induce HK-2 cells apoptosis, providing basis for nephrotoxicity study and safe application of Alismatis Rhizoma.

Intake of cooked tomato sauce preserves coronary endothelial function and improves apolipoprotein A-I and apolipoprotein J protein profile in high-density lipoproteins.

Intake of tomatoes has been linked with healthy diets (eg, Mediterranean diet). However, it remains unknown whether tomato intake exerts protective effects on the vasculature. The aim of this study was to determine whether medium-term supplementation with cooked tomato sauce (CTS) Mediterranean style (sofrito) attenuates diet-induced coronary endothelial dysfunction in an animal model with clinical impact and explore the mechanisms behind the effects. Pigs (N = 18) were fed a 10-day hypercholesterolemic diet. Half of the animals were given a supplement of 100 g/d of CTS (21.5 mg lycopene per day). Coronary responses to escalating doses of vasoactive drugs (acetylcholine, calcium ionophore, and sodium nitroprusside) and L-NG-monomethylarginine (endothelial nitric oxide synthase [eNOS] inhibitor) were measured using flow Doppler. In the coronary arteries, we investigated eNOS gene expression and activation, monocyte chemoattractant protein 1 (MCP-1) expression, and oxidative DNA damage. In the circulation, we investigated lipoprotein resistance to oxidation and the differential proteomic protein profile. In dyslipidemic animals, CTS intake prevented diet-induced impairment of receptor-operated and nonreceptor-operated endothelial-dependent coronary vasodilation. These beneficial effects were associated with enhanced eNOS transcription and activation and diminished DNA damage in the coronary arteries. CTS-fed animals showed lower lipid peroxidation, higher high-density lipoprotein (HDL) antioxidant potential and plasma lycopene levels of 0.16 mg/L. Interestingly, improved HDL functionality was associated with protein profile changes in apolipoprotein A-I and apolipoprotein J. Lipids levels and MCP-1 expression were not affected by CTS. We report that CTS intake protects against low-density lipoprotein-induced coronary endothelial dysfunction by reducing oxidative damage, enhancing eNOS expression and activity, and improving HDL functionality.

Se-methylselenocysteine inhibits apoptosis induced by clusterin knockdown in neuroblastoma N2a and SH-SY5Y cell lines.

Apoptosis, as a programmed cell death process, is essential for the maintenance of tissue function in organisms. Alteration of this process is linked to many diseases. Over-expression of clusterin (Clu) can antagonize apoptosis in various cells. Selenium (Se) is an essential trace element for human health. Its biological function is also associated with cell apoptosis. To explore the function of Clu and the impact of Se in the process of apoptosis, several short-hairpin RNAs (shRNA) were designed for the construction of two sets of recombinant plasmids: one set for plasmid-transfection of mouse neuroblastoma N2a cells (N2a cells); and the other set for lentiviral infection of human neuroblastoma SH-SY5Y cells (SH-SY5Y cells). These shRNAs specifically and efficiently interfered with the intracellular expression of Clu at both the mRNA and protein levels. The Clu-knockdown cells showed apoptosis-related features, including down-regulation of antioxidative capacity and the Bcl-2/Bax ratio and up-regulation of caspase-8 activity. Se-methylselenocysteine (MSC) at an optimum concentration of 1 µM could reverse the alteration in antioxidative capacity, Bcl2/Bax ratio and caspase-8 activity caused by Clu-knockdown, thus inhibiting apoptosis and maintaining cell viability. The results hereby imply the potentiality of Clu and Se in neuroprotection.

Ochratoxin A induces rat renal carcinogenicity with limited induction of oxidative stress responses.

Ochratoxin A (OTA) has displayed nephrotoxicity and renal carcinogenicity in mammals, however, no clear mechanisms have been identified detailing the relationship between oxidative stress and these toxicities. This study was performed to clarify the relationship between oxidative stress and the renal carcinogenicity induced by OTA. Rats were treated with 70 or 210 µg/kg b.w. OTA for 4 or 13 weeks. In the rats administrated with OTA for 13 weeks, the kidney was damaged seriously. Cytoplasmic vacuolization was observed in the outer stripe of the outer medulla. Karyomegaly was prominent in the tubular epithelium. Kidney injury molecule-1 (Kim-1) was detected in the outer stripe of the outer medulla in both low- and high-dose groups. OTA increased the mRNA levels of clusterin in rat kidneys. Interestingly, OTA did not significantly alter the oxidative stress level in rat liver and kidney. Yet, some indications related to proliferation and carcinogenicity were observed. A dose-related increase in proliferating cell nuclear antigen (PCNA) was observed at 4 weeks in both liver and kidney, but at 13 weeks, only in the kidney. OTA down-regulated reactive oxygen species (ROS) and up-regulated vimentin and lipocalin 2 in rat kidney at 13 weeks. The p53 gene was decreased in both liver and kidney at 13 weeks. These results suggest that OTA caused apparent kidney damage within 13 weeks but exerted limited effect on oxidative stress parameters. It implies that cell proliferation is the proposed mode of action for OTA-induced renal carcinogenicity.

Evaluation of the usefulness of novel biomarkers for drug-induced acute kidney injury in beagle dogs.

As kidney is a major target organ affected by drug toxicity, early detection of renal injury is critical in preclinical drug development. In past decades, a series of novel biomarkers of drug-induced nephrotoxicity were discovered and verified in rats. However, limited data regarding the performance of novel biomarkers in non-rodent species are publicly available. To increase the applicability of these biomarkers, we evaluated the performance of 4 urinary biomarkers including neutrophil gelatinase-associated lipocalin (NGAL), clusterin, total protein, and N-acetyl-ß-D-glucosaminidase (NAG), relative to histopathology and traditional clinical chemistry in beagle dogs with acute kidney injury (AKI) induced by gentamicin. The results showed that urinary NGAL and clusterin levels were significantly elevated in dogs on days 1 and 3 after administration of gentamicin, respectively. Gene expression analysis further provided mechanistic evidence to support that NGAL and clusterin are potential biomarkers for the early assessment of drug-induced renal damage. Furthermore, the high area (both AUCs=1.000) under receiver operator characteristics (ROC) curve also indicated that NGAL and clusterin were the most sensitive biomarkers for detection of gentamicin-induced renal proximal tubular toxicity. Our results also suggested that NAG may be used in routine toxicity testing due to its sensitivity and robustness for detection of tissue injury. The present data will provide insights into the preclinical use of these biomarkers for detection of drug-induced AKI in non-rodent species.

Clusterin/ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis.

Hypothalamic leptin signaling plays a central role in maintaining body weight homeostasis. Here, we show that clusterin/ApoJ, recently identified as an anorexigenic neuropeptide, is an important regulator in the hypothalamic leptin signaling pathway. Coadministration of clusterin potentiates the anorexigenic effect of leptin and boosts leptin-induced hypothalamic Stat3 activation. In cultured neurons, clusterin enhances receptor binding and subsequent endocytosis of leptin. These effects are mainly mediated through the LDL receptor-related protein-2 (Lrp2). Notably, inhibition of hypothalamic clusterin, Lrp2 or endocytosis abrogates anorexia and hypothalamic Stat3 activation caused by leptin. These findings propose a novel regulatory mechanism in central leptin signaling pathways.

Ligustrazine attenuates elevated levels of indoxyl sulfate, kidney injury molecule-1 and clusterin in rats exposed to cadmium.

In this study, we aimed at evaluating the effect of ligustrazine, a major constituent of Ligusticum wallichii from traditional Chinese medicine, on Cd-induced changes in nephrotoxicity indices. Rats were divided into four experimental groups: control; ligustrazine; Cd and ligustrazine+Cd. Cd treated alone group showed significant decreases (P<0.05) in body weight, renal levels of superoxide dismutase (SOD) and glutathione reductase (GR); and significant increases (P<0.05) in urine volume (24h), pH values, serum blood urea nitrogen (BUN), serum uric acid, kidney malondialdehyde (MDA), urinary total protein, urinary glucose, urinary lactate dehydrogenase (LDH) and urinary alkaline phosphatase (ALP). Apart from indoxyl sulfate (a uremic toxin), two newly accepted nephrotoxicity biomarkers including kidney injury molecule-1 (kim-1) and clusterin were also found to be increased. Nonetheless, all these effects induced by Cd were reversed upon treatment by ligustrazine although it failed in decreasing the concentrations of Cd in kidney and urine. Histopathological studies in Cd-treated rats exhibited renal tubule damage, which was also ameliorated by ligustrazine pretreatment. These results suggest that ligustrazine exhibits protective effects on Cd-induced nephrotoxicity. Additionally, this study also demonstrates Cd exposure induces elevated levels of indoxyl sulfate in serum and kidney, and clusterin in urine.

Hypothalamic and pituitary clusterin modulates neurohormonal responses to stress.

Clusterin is a sulfated glycoprotein abundantly expressed in the pituitary gland and hypothalamus of mammals. However, its physiological role in neuroendocrine function is largely unknown. In the present study, we investigated the effects of intracerebroventricular (ICV) administration of clusterin on plasma pituitary hormone levels in normal rats. Single ICV injection of clusterin provoked neurohormonal changes seen under acute stress condition: increased plasma adrenocorticotropic hormone (ACTH), corticosterone, GH and prolactin levels and decreased LH and FSH levels. Consistently, hypothalamic and pituitary clusterin expression levels were upregulated following a restraint stress, suggesting an involvement of endogenous clusterin in stress-induced neurohormonal changes. In the pituitary intermediate lobe, clusterin was coexpressed with proopiomelanocortin (POMC), a precursor of ACTH. Treatment of clusterin in POMC expressing AtT-20 pituitary cells increased basal and corticotropin-releasing hormone (CRH)-stimulated POMC promoter activities and intracellular cAMP levels. Furthermore, clusterin treatment triggered ACTH secretion from AtT-20 cells in a CRH-dependent manner, indicating that increased clusterin under stressful conditions may augment CRH-stimulated ACTH production and release. In summary, hypothalamic and pituitary clusterin may function as a modulator of neurohormonal responses under stressful conditions.

Clusterin and LRP2 are critical components of the hypothalamic feeding regulatory pathway.

Hypothalamic feeding circuits are essential for the maintenance of energy balance. There have been intensive efforts to discover new biological molecules involved in these pathways. Here we report that central administration of clusterin, also called apolipoprotein J, causes anorexia, weight loss and activation of hypothalamic signal transduction-activated transcript-3 in mice. In contrast, inhibition of hypothalamic clusterin action results in increased food intake and body weight, leading to adiposity. These effects are likely mediated through the mutual actions of the low-density lipoprotein receptor-related protein-2, a potential receptor for clusterin, and the long-form leptin receptor. In response to clusterin, the low-density lipoprotein receptor-related protein-2 binding to long-form leptin receptor is greatly enhanced in cultured neuronal cells. Furthermore, long-form leptin receptor deficiency or hypothalamic low-density lipoprotein receptor-related protein-2 suppression in mice leads to impaired hypothalamic clusterin signalling and actions. Our study identifies the hypothalamic clusterin-low-density lipoprotein receptor-related protein-2 axis as a novel anorexigenic signalling pathway that is tightly coupled with long-form leptin receptor-mediated signalling.

From genetics to dietetics: the contribution of epidemiology to understanding Alzheimer's disease.

Late-life dementia results from non-modifiable risk factors such as age and genetics, modulated by deleterious and protective environmental factors among which nutrition may play a major role. This paper highlights five major recent contributions of the French Three-City (3C) and PAQUID epidemiological studies to Alzheimer's disease (AD) knowledge, targeting genetic and dietary risk factors, and the impact of cognitive decline in daily living. The 3C study contributed to a large genome-wide association study to identify new genetic risk factors for AD. In addition to apolipoprotein E (APOE), two loci gave replicated evidence of association: one within CLU, encoding clusterin or apolipoprotein J, and the other within CR1, encoding the complement component receptor 1. Although the attributable fraction of risk for these polymorphisms is moderate, genetic studies provide significant insights into the molecular bases of AD. Regarding dietary data, findings from 3C suggest that healthy diets associating sources of both omega 3 fatty acids (fish) and antioxidants (fruits and vegetables) such as the Mediterranean diet, and caffeine could be associated with decreased risk for AD. However, the protective effect of omega3 fatty acids might be limited to APOE4 non-carriers. Future research should focus on gene-nutrient interactions. Regarding the functional impact of prodromal AD, the PAQUID study showed that taking into account mild functional limitations considerably increases the predictive value of neuropsychological tests for conversion to dementia. Research should focus on sensitive instruments to capture early functional decline to improve the identification of elderly patients at high risk of conversion to dementia.