[show abstract][hide abstract] ABSTRACT: Melamine was recently identified as a risk factor for renal calculi following the milk powder contamination in China. However, the long-term natural history of melamine exposure and its renal effects remain unknown. We evaluated renal function and other adverse health effects using a rat model administered melamine and cyanuric aid, considering age and sex.
Twelve male F334/N rats each of ages 6, 10, and 26 weeks (N = 36) were equally assigned to Group M + C or controls. Group M + C rats were administered 12 mg . kg-1 . day-1 of melamine and cyanuric acid for 28 days. Serum and urine samples and kidney sections were evaluated on day 28. Six-week-old male and female F344/N rats were administered 12 mg of melamine and cyanuric acid for 28 days. Body weights were measured weekly; on days 0, 28, 90, and 180 after the 28-day period of melamine and cyanuric acid administration, serum samples and kidney sections were obtained.
Although the control group had no crystals, 6-week-old Group M + C rats had more crystals compared to the 10- and 26-week old Group M + C rats. Male rats also had significantly more crystals than females of the same age. Male rats were affected to a greater extent than females.
Younger rats experienced more severe renal failure and greater renal crystal deposition following melamine and cyanuric acid administration. However, after melamine and cyanuric acid administration cessation, crystal deposition and renal failure improved and did not cause growth arrest. Therefore, early diagnosis of melamine-associated calculi is critical.
[show abstract][hide abstract] ABSTRACT: 3β-Hydroxysteroid-Δ24 reductase (DHCR24) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. Accumulating evidence suggests that ER stress is involved in the pathogenesis of neurodegenerative disease. In this study, we investigated whether DHCR24 may function as a neuroprotective protein under ER stress. Neuroblastoma N2A cells were infected with adenovirus expressing myc-tagged DHCR24 (Ad-DHCR24) or lacZ (Ad-lacZ, serving as a control) and subjected to ER-stress, induced with Tunicamycin (TM). Cells infected with Ad-DHCR24-myc were resistant to TM-induced apoptosis, and showed weaker level of caspase-12 activity. These cells also exhibited lower levels of Bip and CHOP proteins than Ad-LacZ-infected cells. Moreover, a stronger and rapid activation of PERK, and a prolonged activation of JNK and p38 were observed in Ad-LacZ-infected cells. The generation of intracellular reactive oxygen species from ER stress was also diminished by the overexpression of DHCR24. Additionally, intracellular cholesterol level was also elevated in the Ad-DHCR24-infected cells, accompanied by a well-organized formation of caveolae (cholesterol-rich microdomain) on the plasma membrane, and improved colocalization of caveolin-1 and insulin-like growth factor 1 receptor. These results demonstrated for the first time that DHCR24 could protect neuronal cells from apoptosis induced by ER stress.
PLoS ONE 01/2014; 9(1):e86753. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract Background: In view of their high level of variability, autosomal short tandem repeats (STRs) are very useful as markers in the disciplines of forensic and population genetics studies. Aim: To investigate the diversity distributions of allelic frequencies of 15 loci from a sample from the Chinese Xibe ethnic group in Liaoning. Subjects and methods: Fifteen STR loci for 150 unrelated Xibe individuals from Liaoning, China were amplified simultaneously in a fluorescence-based reaction using a 2720 Thermal cycler (ABI). Separation and detection of the amplified product were conducted with the Li-COR 4300 DNA Analyzer. Results: In total, 117 alleles were observed, with the corresponding allele frequencies ranging from 0.001 to 0.507. D18S51 had the highest polymorphism (PIC = 0.840) among all 15 STR loci, whereas TPOX had the lowest (PIC = 0.590). The power of discrimination ranged from 0.801 for TH01 locus to 0.957 for D18S51 locus, whereas the power of exclusion ranged from a minimum 0.316 for TPOX locus to a maximum 0.720 for D21S11 locus. The phylogenetic tree established among worldwide populations showed that the Xibe population is far from other populations. Conclusion: Databases for the 15 STR loci will be useful for personal identification and paternity tests in the Xibe population and for the establishment of phylogenetic relationships between populations.
Annals of Human Biology 11/2013; · 1.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this study was to estimate the allelic frequencies of the 19 STR loci with the Goldeneye™ DNA ID system 20A kit in a sample of 150 Manchu individuals from China to be used for forensic purposes and population studies. The observed heterozygosity(HO)values of these 19 STR loci ranged from 0.600 (D3S1358) to 0.914 (D18S51), the expected (HE) ranged from 0.615 (TPOX) to 0.876 (D16S1043). The power of discrimination (PD) values were found to range from 0.793 (TPOX) to 0.950 (D16S1043) and the probability of exclusion (PE) varies between 0.291 (D3S1358) and 0.825 (D18S51 and Penta E ). Among all the 19 loci, D16S1043 had the highest polymorphism (PIC=0.860), whereas TPOX had the lowest (PIC=0.550). For the 19 loci, the combined power of discrimination and the combined probability of exclusion are 0.9999999999999999999942 and 0.999999996777, respectively. The phylogenetic tree established among worldwide populations shows different populations who say the same language usually have a close genetic relationship with each other across the three language families studied (Sino-Tibetan, Altaic and Arabic).
[show abstract][hide abstract] ABSTRACT: To clarify the association between regional variations in urolithiasis incidence and nutrition intake, we evaluated associated data from Japanese national surveys. The incidence of urolithiasis in 12 regions of Japan was calculated from 2005 patient data obtained from 430 hospitals (n = 92,797). Nutrition intake data were obtained from the National Health and Nutrition Survey. We examined the association between urolithiasis incidence and average intake of various types of food or nutrients by region. Continuing surveys in Japan reveal fixed variations in urolithiasis incidence among geographic regions. The national average of patients with urolithiasis was estimated as 203.1 per 100,000 citizens. Regarding food, intake of fruit correlated negatively with the incidence of urolithiasis (r = -0.721, p = 0.008), while intake of eggs (r = 0.537, p = 0.072) and sugar (r = 0.475, p = 0.119) tended to positively correlate with incidence. Regarding nutrients, intake of potassium (r = -0.500, p = 0.098), vitamin K (r = -0.562, p = 0.057), and pantothenic acid (r = -0.560, p = 0.058) tended to negatively correlate with incidence. The incidence of urolithiasis is higher in geographic areas with populations having low fruit and high sugar intake.
[show abstract][hide abstract] ABSTRACT: Background : Matrix Gla protein (MGP) is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods : Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG) for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC) method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR) and Western blot. Results : Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL) and the distal convoluted tubule (DCT) in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD) in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion : Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.
Kidney and Blood Pressure Research 02/2013; 37(1):15-23. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: PURPOSE: Matrix Gla protein (MGP) is a molecular determinant regulating the extracellular matrix calcification. To further confirm whether the MGP genetic polymorphism was universally associated with the risk of kidney stone, we investigated the association of genetic polymorphisms of MGP with kidney stone in the Chinese Han population. MATERIALS AND METHODS: 728 subjects were recruited for the study. We firstly re-sequenced the human genomic MGP gene including the 1500bp promoter, 5'-UTR, 4 exons and 3'-untranslated regions, identified single nucleotide polymorphisms (SNPs) in MGP, and performed an association analysis with kidney stones in 54 subjects of the Chinese Han population. A candidate tag SNP was genotyped in total subjects using an allele specific PCR, and further analyzed the association with kidney stone. RESULTS: We identified 18 polymorphisms including four tag SNPs. A tag SNPrs4236 was associated with kidney stones. The G allele carrier had a 1.373-fold reduced kidney stone risk compared with A allele carriers in SNPrs4236 (odds ratios (OR)=1.373; 95%CI, 1.051-1.793; p=0.019). However, we did not find an association between the polymorphism and clinical characteristics of kidney stones. CONCLUSIONS: Our findings showed that SNPrs4236 of the MGP gene is associated with kidney stones in the Chinese Han population, and influences the genetic susceptibility to kidney stones. In the future, functional assays of the polymorphism should permit a better understanding of the role of MGP genetic variants and kidney stones.
[show abstract][hide abstract] ABSTRACT: The objective of this study is to understand pathogenesis of melamine-related kidney stone formation. We investigated the characterization of renal tubular cell under exposure to a mixture of melamine and cyanuric acid in vivo. Male Sprague-Dawley rats were separated into two experimental groups. Treatment group was administered daily with a standard commercial diet mixing with melamine and cyanuric acid, and control group was given a normal diet. Rat kidney specimens were stained with hematoxylin/eosin and the crystals were examined using a polarizing microscope. Renal tubular epithelial cells were observed by transmission electron microscopy. Semiquantitative RT-PCR assay was performed to determine monocyte chemoattractant protein-1 (MCP-1) mRNA expression, a protein in response to various proinflammatory stimuli. Apoptotic cells were examined by TUNEL assay. Melamine-associated crystals formed in glomerulus and wide renal tubule segment including proximal convoluted renal tubules, distal convoluted renal tubules, the limb loops of Henle and medullary collecting ducts in the cortex and medulla. Light microscopy results showed that the crystals lead to tubular lumen dilatation and tubular epithelial cell necrosis. It was observed that nucleus of renal tubular epithelial cells became irregular outlines and condensed, lysosomal-related structures increased, and integrity of renal tubule was deficient under electron microscopy. Apoptotic cells were noted widely in cortex and medulla. MCP-1 mRNA expression was significantly increased in the melamine and cyanuric acid-administrated group. Renal tubular epithelial cell injury, apoptosis and inflammation are involved in melamine-related kidney stone formation. Our findings are important for understanding pathogenesis of melamine-related kidney stone formation and estimating its clinical prognosis.
[show abstract][hide abstract] ABSTRACT: DHCR24 encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.
Journal of Molecular Endocrinology 02/2012; 48(1):1-9. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the present study, we investigated the genetic polymorphisms of 6 autosomal STR loci Hum-CSF1PO, D13S317, D5S818, D16S539, TH01, and TPOX in the Xibo population of Liaoning, northeastern China as well as its genetic relationships with other populations in China. No significant deviations from Hardy-Weinberg equilibrium could be found for all loci. Allele frequencies in the Xibo population ranged from 0.001 to 0.507. Among all the 6 loci, D16S539 had the highest polymorphism (PIC=0.8632), whereas TPOX had the lowest (PIC=0.5179). A phylogenic tree was constructed using Poptree 2 software. In the phylogenic tree, Xibo population has a distant relationship with the other populations.
[show abstract][hide abstract] ABSTRACT: Melamine was known as a new risk for kidney stone due to recent incidences of milk powder contamination in China. Here, we performed a retrospective study to investigate whether age, gender, and urinary pH affect melamine-associated kidney stone risk.
A retrospective review was performed of 217 children aged less than 3 years old. All children had a history of being fed with Sanlu milk powder contaminated by melamine, and underwent a clinical screening on kidney stone in Shenyang from November 2008 to February 2009. A comparison with the Chi-square was conducted between 83 cases and 125 normal subjects. The difference between children's gender, age, and urinary pH was evaluated.
A total of 208 subjects, 136 boys and 72 girls, were included in the study. Significant association was observed between melamine-associated kidney stone risk and gender [odds ratio (OR), 2.03; 95% confidence interval (CI), 1.11-3.74; P=0.02] and urinary pH (OR, 1.78; 95% CI, 1.01-3.11; P=0.04), respectively. Male children were at about twofold increased melamine-associated kidney stone risk compared with female children. Acidic urine showed about 1.78-fold increased melamine-associated kidney stone risk compared with normal urine.
Our investigation results showed an association of gender and urinary pH with melamine-associated kidney stone formation risk.
[show abstract][hide abstract] ABSTRACT: Type 2 diabetes is often associated with high blood cholesterol. Here, we investigated the effect of cholesterol loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to cholesterol-induced apoptosis in time- and dose-dependent manner. Treatment with methyl-β-cyclodextrin that removes cholesterol from plasma membrane prevented the cells from cholesterol-induced apoptosis. Western blot analysis revealed that the levels of phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and c-Jun N-terminal kinases (P-JNK) were significantly increased after the cholesterol loading, suggesting that the stress-activated protein kinase signaling was stimulated. A specific p38 inhibitor rescued MIN6 cells from cholesterol-induced apoptosis, while JNK inhibitor failed, suggesting the importance of activation of p38 MAPK signaling in response to cholesterol. The expression of Bip and CHOP, the endoplasmic reticulum (ER) stress markers, remained unaffected, indicating that the ER stress may not be involved in the cytotoxicity of cholesterol on the ΜΙΝ6 cells. The intracellular concentration of reactive oxygen species measured by use of 2',7'-dichlorofluorescin diacetate was significantly increased after cholesterol loading, demonstrating the induced apoptosis was mediated through oxidative stress. Addition of reduced form of glutathione in the medium rescued MIN6 cells from apoptosis induced by cholesterol loading. Taken together, these results demonstrate that the free cholesterol loading can induce apoptosis of MIN6 cells mediated by oxidative stress and the activation of p38 MAPK signaling.
Cell Stress and Chaperones 04/2011; 16(5):539-48. · 2.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: The growing evidences demonstrated hyperlipidemia in obesity and type 2 diabetes is characterized by high levels of free fatty acids, low-density lipoprotein (LDL), triglyceride, and cholesterol.
We investigated the effect of LDL particles (LDLs) loading on MIN6 cells derived from pancreatic β cells. Exposure of MIN6 cells to LDLs induced apoptosis in dose and time-dependent manner, demonstrated by the TUNEL in situ apoptotic assay. The immunocytochemical analysis and Western blotting revealed that the LDLs-induced apoptosis is associated with the activation of caspase 3 and upregulation of p53. The intracellular concentration of Reactive Oxygen Species (ROS) measured by use of DCFHDA was significantly increased after loading LDLs, demonstrating the induced apoptosis by LDLs loading was mediated through oxidative stress. Addition of reduced form of Glutathione (GSH) in the medium rescued MIN6 cells from apoptosis. The Cellular cholesterol level was increased significantly after LDLs loading, suggesting that the excess cholesterol induced by LDLs loading might contribute to the apoptosis in MIN6s. Agarose electrophoresis demonstrated that the LDLs added to the medium were not oxidized.
Taken together, these results demonstrate that the LDLs loading can induce apoptosis of MIN6 cells mediated by the excess cellular cholesterol and generation of oxidative stress.
Lipids in Health and Disease 01/2011; 10:123. · 2.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Matrix Gla protein (MGP), a potent calcification inhibitor in extracellular matrix, is a molecular determinant regulating vascular calcification. Here, we characterized MGP expression in cultured renal tubular epithelial cells exposed to oxalate and calcium oxalate monohydrate (COM) crystals.
NRK-52E cells were cultured and exposed to medium containing oxalate or COM for 1, 3, 6, 12 and 24 h. The levels of MGP mRNA expression were quantified using real-time PCR (RT-PCR), and the levels of protein expression were characterized by Western blot.
MGP was expressed rapidly and intensively following exposure to COM and was time-dependent with exposure to oxalate.
Our results have given a profile of MGP expression in renal tubular epithelial cells exposed to oxalate and crystals and provided support for an association between MGP and kidney stones. Further investigations on the mechanism of the MGP in the process of crystal-cell interaction may be important in the future.
Urologia Internationalis 01/2010; 85(2):237-41. · 1.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Matrix Gla protein, a potent calcification inhibitor in arterial vessels, is also expressed in the kidney and is up-regulated following the administration of ethylene glycol, a precursor of oxalate. Considering the analogous characteristics between arterial calcification and kidney stones, we identified variants of the human matrix Gla protein gene and investigated whether there is an association between MGP genetic polymorphisms and kidney stones.
We studied single nucleotide polymorphisms in matrix Gla protein in 122 kidney stone cases and 125 controls. We resequenced the human genomic MGP gene, including the 1,000 bp promoter 5'-untranslated region, 4 exons and 3'-untranslated regions, and we performed systematic genetic analysis. A single nucleotide polymorphism was genotyped using a fluorescent 5'endonuclease assay and its association with kidney stones was analyzed.
We observed 19 polymorphisms. A single nucleotide polymorphism was associated with kidney stones (OR 0.51, 95% CI 0.30-0.87; p = 0.012). The G allele carrier had a 2-fold decreased kidney stone risk compared with A allele carriers in single nucleotide polymorphism 11 (OR 0.55, 95% CI 0.31-1.00, p = 0.047). We found no association between the polymorphism and kidney stone clinical characteristics.
Our findings show that an MGP gene polymorphism is associated with kidney stones and influences genetic susceptibility to kidney stones. In the future functional assays of the polymorphism should permit better understanding of the role of matrix Gla protein genetic variants and kidney stones.
The Journal of Urology 07/2007; 177(6):2361-5. · 3.70 Impact Factor
[show abstract][hide abstract] ABSTRACT: The establishment of an experimental animal model would be useful to study the mechanism of kidney stone formation. A calcium kidney stone model in rats induced by ethylene glycol has been used for research; however, to investigate the genetic basis affecting kidney stone formation, which will contribute to preventive medicine, the establishment of a kidney stone model in mice is essential. This study indicates the optimum conditions for inducing calcium oxalate stones in normal mouse kidney. Various doses of oxalate precursors, ethylene glycol, glycolate and glyoxylate, were administered either by free drinking or intraabdominal injection for 2 months as a preliminary study. Stone formation was detected with light microscopy, polarized light optical microscopy and electron microscopy. Stone components were detected with X-ray diffraction analysis. The expression of osteopontin (OPN), a major stone-related protein, was detected with immunohistochemical staining, in situ hybridization and quantitative reverse transcriptase polymerase chain reaction. Kidney stones were not detected in ethylene glycol- or glycolate-treated groups even at the highest dose of LD(50). Whereas, numerous kidney stones were detected in glyoxylate-treated mice (more than 60 mg/kg) at 3, 6 and 9 days after glyoxylate were administered intraabdominally. However, the number of kidney stones decreased gradually at day 12, and was hardly detected at day 15. The stone component was further analyzed as calcium oxalate monohydrate. A dramatic increase in the expression of OPN was observed by the administration of glyoxylate. We established a mouse kidney stone experimental system in this study. The difficulty of inducing kidney stones suggested that mice have greater intrinsic ability to prevent stone formation with hyperoxaluric stress than rats. The differing response to hyperoxaluric stress between mice and rats possibly contributes to the molecular mechanism of kidney stone formation and will aid preventive medicine in the future.
Urological Research 05/2007; 35(2):89-99. · 1.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: We investigated polymorphisms of the osteopontin (OPN) gene to explore whether they could be used as a gene marker for determining the risk of urinary calcium stones.
A total of 76 patients with urinary calcium stones and 124 controls were studied. Two single nucleotide polymorphisms with nonsynonymous amino acid located at positions 9,402 (Arg/His) and 9,171 (Asn/Ser) in the OPN gene were genotyped using the TaqMan 5' nuclease assay in a PRISM 7700 sequence detection system. Results were analyzed with Fisher's exact test.
Results revealed a significant different polymorphism at OPN gene position 9,402 between patients with urinary calcium stones and control subjects. The frequency of the A/G genotype at position 9,402 in patients with urinary calcium stones was significantly higher than that in control subjects (p <0.01). The frequency of the A allele at position 9,402 was significantly higher in the patient than in the control group (4.6% vs 0.4%, p <0.01). There was no statistical difference in this polymorphism at OPN gene position 9,402 between genotype distribution and clinical characteristics associated with urinary calcium stones. There was no difference in the polymorphism at OPN gene position 9,171 between the patient and control groups.
Polymorphism at position 9,402 of the OPN gene is highly associated with urinary calcium stones and it is a candidate genetic marker for evaluating the genetic risk of urinary calcium stone disease, whereas polymorphism at position 9,171 of the OPN gene is not associated with urinary calcium stone disease.
The Journal of Urology 11/2005; 174(4 Pt 1):1472-6. · 3.70 Impact Factor