Changes of gut microbiota structure in rats infected with Toxoplasma gondii.

Toxoplasma gondii (T. gondii) infection can cause intestinal inflammation in rodents and significantly alters the structure of gut microbiota. However, the effects of different T. gondii genotypes on the gut microbiota of rats remain unclear. In this study, acute and chronic T. gondii infection in Fischer 344 rats was induced artificially by intraperitoneal injection of tachyzoites PYS (Chinese 1 ToxoDB#9) and PRU (Type II). Fecal 16S rRNA gene amplicon sequencing was employed to analyze the gut microbiota structure at different stages of infection, and to compare the effects of infection by two T. gondii genotypes. Our results suggested that the infection led to structural changes of gut microbiota in rats. At the acute infection stage, the microbiota diversity increased, while both diversity and abundance of beneficial bacteria decreased at the chronic infection stage. The differences of microbiota structure were caused by strains of different genotypes. However, the diversity changes were consistent. This study demonstrates that the gut microbiota plays an important role in T. gondii infection in rats. The data will improve our understanding of the association between T. gondii infection and gut microbiota in rodents.

Characterization of the complete mitochondrial genomes of Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae): Comparison with the members of the superfamily Paramphistomoidea and phylogenetic implication.

Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae) are two important parasites in wood frogs, which have large infection rates and essential importance of ecology, economy and society. In this study, the complete mitochondrial (mt) genomes of D. japonicus and D. mehari were sequenced, then compared with other related trematodes in the superfamily Paramphistomoidea. The complete circular mt sequence of D. japonicus and D. mehari were 14,210 bp and 14,179 bp in length, respectively. Both mt genomes comprised 36 functional subunits, consisting of 12 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one non-coding region. The mt genes of D. japonicus and D. mehari were transcribed in the same direction, and the gene arrangements were identical to those of Paramphistomoidea trematodes. In the 12 PCGs, GTG was the most common initiation codon, whereas TAG was the most common termination codon. All tRNAs had a typical cloverleaf structure except tRNA Ser1. A comparison with related Paramphistomoidea trematode mt genomes suggested that the cox1 gene of D. mehari was the longest in these trematodes. Phylogenetic analyses revealed that Paramphistomoidea trematodes formed a monophyletic branch, Paramphistomidae and Gastrothylacidae were more closely related than Diplodiscidae. And the further analysis with Pronocephalata branch found that the flukes parasitic in amphibians (frogs) formed one group, and the flukes from ruminants (cattle, sheep, ect) formed another group. Our study demonstrated the importance of sequencing mt genomes of D. japonicus and D. mehari, which will provide significant molecular resources for further studies of Paramphistomoidea taxonomy, population genetics and systematics.

Prevalence of Theileria in cattle in China: A systematic review and meta-analysis.

Theileria, one of the causative agents of blood protozoan, has brought a huge economic loss to the cattle industry worldwide. However, the epidemiology of Theileria in Chinese cattle has not been systematically investigated. This comprehensive review aimed at investigating the prevalence of Theileria infection in cattle in China. A total of 48 published papers on Theileria infection in cattle in China (including data from 21,366 animals) from inception to October 8, 2021 met the inclusion standard after searching in five databases (Technology Periodical Database, Wan Fang Database, China National Knowledge Infrastructure, PubMed, and ScienceDirect). The pooled prevalence of Theileria in cattle in China was 32.4% identified by using a random effects model. The prevalence in Northeastern China (45.3%) was higher than that in other regions. In the sex subgroup, the prevalence of Theileria was higher in females (48.9%) than that in males (45.8%). The prevalence of Theileria was higher in cattle of free range (34.4%) compared with that of intensive farming (22.3%). The prevalence prior to 2013 (36.1%) was higher than that after 2013 (33.6%). Among three cattle species, dairy cows had the lowest prevalence (21.5%). The prevalence of Theileria (T.) annulata (22.2%) and T. sergenti (26.2%) was higher than other species of Theileria (T. buffeli: 17.5%, T. luwenshuni: 0.9%, T. orientalis: 15.5%, T. ovis: 0.21%, T. sinensis: 20.2%, T. uilenbergi: 6.2%, Others: 0.9%). We also analyzed the impact of different geographic factor subgroups (longitude, latitude, precipitation, temperature, humidity, and altitude) on the prevalence of Theileria in cattle. Among them, climatic factors of longitude, latitude, precipitation, humidity, temperature were associated with the prevalence of Theileria. These analyses suggested that Theileria was common in cattle in China. Targeted prevention programs based on geographic and climatic conditions in different areas may play an important role in reducing Theileria infection among cattle.

Molecular Detection of Cryptosporidium spp. and Enterocytozoon bieneusi Infection in Wild Rodents From Six Provinces in China.

Enterocytozoon (E.) bieneusi and Cryptosporidium spp. are the most important zoonotic enteric pathogens associated with diarrheal diseases in animals and humans. However, it is still not known whether E. bieneusi and Cryptosporidium spp. are carried by wild rodents in Shanxi, Guangxi, Zhejiang, Shandong, and Inner Mongolia, China. In the present study, a total of 536 feces samples were collected from Rattus (R.) norvegicus, Mus musculus, Spermophilus (S.) dauricus, and Lasiopodomys brandti in six provinces of China, and were detected by PCR amplification of the SSU rRNA gene of Cryptosporidium spp. and ITS gene of E. bieneusi from June 2017 to November 2020. Among 536 wild rodents, 62 (11.6%) and 18 (3.4%) samples were detected as E. bieneusi- and Cryptosporidium spp.-positive, respectively. Differential prevalence rates of E. bieneusi and Cryptosporidium spp. were found in different regions. E. bieneusi was more prevalent in R. norvegicus, whereas Cryptosporidium spp. was more frequently identified in S. dauricus. Sequence analysis indicated that three known Cryptosporidium species/genotypes (Cryptosporidium viatorum, Cryptosporidium felis, and Cryptosporidium sp. rat genotype II/III) and two uncertain Cryptosporidium species (Cryptosporidium sp. novel1 and Cryptosporidium sp. novel2) were present in the investigated wild rodents. Meanwhile, 5 known E. bieneusi genotypes (XJP-II, EbpC, EbpA, D, and NCF7) and 11 novel E. bieneusi genotypes (ZJR1 to ZJR7, GXM1, HLJC1, HLJC2, and SDR1) were also observed. This is the first report for existence of E. bieneusi and Cryptosporidium spp. in wild rodents in Shanxi, Guangxi, Zhejiang, and Shandong, China. The present study also demonstrated the existence of E. bieneusi and Cryptosporidium spp. in S. dauricus worldwide for the first time. This study not only provided the basic data for the distribution of E. bieneusi and Cryptosporidium genotypes/species, but also expanded the host range of the two parasites. Moreover, the zoonotic E. bieneusi and Cryptosporidium species/genotypes were identified in the present study, suggesting wild rodents are a potential source of human infections.

Molecular detection of Blastocystis in black bears and sika deer in northern China.

Protists of the Blastocystis genus are distributed worldwide and can infect a range of hosts. However, data concerning Blastocystis infection are limited for sika deer and are not available for black bears. Therefore, in the present study, a total of 312 black bears (Ursus thibetanus) from Heilongjiang Province and 760 sika deer (Cervus nippon) from four different northern Chinese provinces were investigated. Blastocystis infection in these animals was detected via PCR amplification of the small subunit rRNA gene in fecal samples. The prevalence of Blastocystis infection in black bears and sika deer was 14.4% (45/312 positive samples) and 0.8% (6/760 positive samples), respectively. Young black bears (18.3%) had a significantly higher Blastocystis prevalence than adult bears (9.1%). The prevalence of Blastocystis was significantly higher in black bears raised outdoors (24.6%) than in bears raised indoors (12.2%). Blastocystis-positive sika deer were only found in Jilin Province (1.3%, 6/480). Female sika deer (0%, 0/61) had a significantly lower Blastocystis prevalence than males (0.9%, 6/699). Sanger sequencing was used to determine the small subunit rRNA gene sequences of the Blastocystis-positive PCR products. A neighbor-joining phylogenetic tree based on the small subunit rRNA gene sequences showed that only Blastocystis subtype (ST)1 was identified in black bears, whereas ST10 and ST14 were found in sika deer. This is the first report of Blastocystis ST1 infection in black bears. These findings also extend the distribution information of Blastocystis subtypes, which will provide a foundation for further study of Blastocystis in different hosts in China.

Prevalence and Risk Factors of Toxoplasma gondii Infection Among Five Wild Rodent Species from Five Provinces of China.

Toxoplasma gondii, an intracellular zoonotic parasite, can infect humans and various animals worldwide. Wild rodents plan an important role as intermediate hosts of T. gondii. Some studies on T. gondii from wild rodents have been published, but the investigation data of T. gondii in wild rodents in China are limited. Therefore, brain tissue samples from 382 wild rodents in four provinces and one autonomous region of China were screened by PCR amplification of T. gondii B1 gene. Furthermore, the wild rodents were identified as five species based on their morphological characteristics, including Citellus dauricus (n = 35 from Heilongjiang), Lasiopodomys brandti (n = 81 from Inner Mongolia), Apodemus agrarius (n = 12 from Heilongjiang), Rattus norvegicus (n = 2 from Heilongjiang; n = 99 from Zhejiang; n = 54 from Shanxi), and Mus musculus (n = 99 from Guangxi). The overall prevalence of T. gondii in these wild rodents was 5.24% (20/382) in this study. At different regions, the highest prevalence of T. gondii was in Guangxi (12.12%) compared with other regions (0% in Heilongjiang; 2.47% in Inner Mongolia; 2.02% in Zhejiang; 7.41% in Shanxi). At different climates, the higher prevalence was found in temperate/mesothermal climates (7.07%) compared with continental/microthermal climates (3.26%). Also, the results showed that M. musculus had the highest prevalence of T. gondii infection (12.12%) among the rodent species sampled. Moreover, sampling year was significantly related to the prevalence of T. gondii in investigated wild rodents (p = 0.0117). This is the first report of T. gondii in wild rodents from Zhejiang, Guangxi, Shanxi, Heilongjiang provinces and Inner Mongolia autonomous region in China, providing the fundamental information for further prevention and control of toxoplasmosis in China.

Molecular detection of Cryptosporidium spp. and Enterocytozoon bieneusi in Longjiang Wagyu cattle in Northeastern China.

Cryptosporidium spp. and Enterocytozoon bieneusi are two important zoonotic pathogens that can cause diarrhea and other gastrointestinal illnesses in humans and animals. However, the prevalence and genotype of the parasites in Longjiang Wagyu cattle in Heilongjiang Province, Northeast China have not been reported. In the present study, a total of 423 fecal samples of Longjiang Wagyu cattle collected from different farms in Heilongjiang Province, Northeast China, were examined for Cryptosporidium spp. and E. bieneusi using nested PCR. The overall infection rates for Cryptosporidium spp. and E. bieneusi were 6.38% (n = 27) and 7.09% (n = 30), respectively. The prevalence in different age groups ranged from 3.80% (95% confidence interval (CI) 1.01-6.59) to 8.36% (95% CI 4.83-11.90) for Cryptosporidium spp. and 5.97% (95% CI 2.52-9.43) to 7.94% (95% CI 4.49-11.40) for E. bieneusi. By analyzing the DNA sequences of the small subunit (SSU) rRNA gene, two Cryptosporidium species were detected in this study, namely C. parvum (n = 25) and C. ryanae (n = 2). The IIdA20G1 subtype was further identified by using the 60-kDa glycoprotein (gp60) gene of C. parvum. E. bieneusi was identified using three known sequences through the analysis of internal transcribed spacer (ITS) sequences: J (n = 23), I (n = 5), and BEB4 (n = 2), and all belonged to group 2. The results indicated that some of the Cryptosporidium species and E. bieneusi genotypes identified in Longjiang Wagyu cattle in the study areas might have zoonotic potential.

Prevalence of Clonorchis sinensis infection in fish in South-East Asia: A systematic review and meta-analysis.

Clonorchis sinensis, an important fish-borne zoonotic trematode, is widely distributed in South-East Asia, especially in China. Infections from human and animal reservoir hosts occur due to the consumption of raw or undercooked fish with C. sinensis metacercariae. This study aimed to evaluate the prevalence of C. sinensis metacercariae in fish in South-East Asia via systematic review and meta-analysis. We searched PubMed, ScienceDirect, China National Knowledge Infrastructure, Wanfang and Chongqing VIP databases for studies published between 1976 and 2020 that are related to the prevalence of C. sinensis metacercariae in fish. Studies were screened with keywords based on inclusion and exclusion criteria. Seventy-one eligible articles were identified, covering three countries: China, Korea and Vietnam. The pooled prevalence of C. sinensis metacercariae in fish from South-East Asia was 30.5%, with 35.1% in China, 29.7% in Korea and 8.4% in Vietnam. In subgroup analyses of climate, season, water source and publication date, the highest prevalence was identified in the Dwb climate type (43.3%), summer (70.2%), river (34.5%) and pre-2001 publications (38.9%), respectively. In comparison, the lowest prevalence was found in the Dfa climate type (14.5%), winter (19.5%), lake (8.0%) and post-2001 publications (23.8%). Meta-regression results indicated that country (p = .009), the published time (p = .035) and water source subgroups (p = .003) may be the source of heterogeneity. Overall, our study indicates that a high prevalence of C. sinensis infections occurs in fish in China, Korea and Vietnam, illuminating a significant public health concern in these countries.

Toxoplasma gondii Seropositivity and Associated Risk Factors in Cats (Felis catus) in Three Provinces in Northeastern China from 2013 to 2019.

Toxoplasma gondii is an important protozoan parasite that can infect a range of animals. Cats are the most important definitive hosts for T. gondii. Therefore, an assessment of the level of T. gondii infection in cats is of public health importance. As limited information about seroprevalence of T. gondii infection in cats in northeastern China was available, 854 serum samples, including 235 cats in Jilin province, 267 cats in Liaoning province, and 352 cats in Heilongjiang province, were examined by an indirect hemagglutination assay (IHA) test, between September 2013 and July 2019. The overall seroprevalence of T. gondii was 19.09% (163/854) at the cutoff 1:64, with 19.60% (69/352), 18.29% (43/235), and 19.10% (51/267) in Heilongjiang, Jilin and Liaoning, respectively. Logistic regression analysis showed that mode of life was highly related to T. gondii infection in cats at the investigation areas. These results could provide foundation data for prevention and control of T. gondii prevalence in these regions. Moreover, the results also suggested that the effective control strategies should continue to be performed.

Comparative analyses of complete ribosomal DNA sequences of Clonorchis sinensis and Metorchis orientalis: IGS sequences may provide a novel genetic marker for intraspecific variation.

Both Clonorchis sinensis and Metorchis orientalis are the fish-borne zoonotic trematodes, and have a wide distribution of southeastern Asia, especially in China. Due to the similar morphology, life cycle, and parasitic positions are difficult to differentiate between both metacercariae. In the present study, the complete rDNA sequences of five C. sinensis and five M. orientalis were obtained and compared for the first time. And the IGS rDNA sequences were tested as a genetic marker. The results showed complete rDNA lengths of C. sinensis were range from 8049 bp to 8391 bp, including 1991 bp, 1116 bp, 3854 bp, and 1088-1430 bp belonging to 18S, ITS, 28S and IGS, respectively. And the complete rDNA lengths of M. orientalis were range from 7881 bp to 9355 bp, including 1991 bp, 1077 bp, 3856 bp, and 957-2431 bp belonging to 18S, ITS, 28S and IGS, respectively. Comparative analyses reveal length difference main in IGS, which has higher intraspecific and interspecific variations than other ribosomal regions. Forty four repeat (forward and inverted) sequences were found in the complete rDNAs of C. sinensis and M. orientalis. The phylogenetic analyses showed that the sequences of ITS1, ITS2, 18S and 28S could be used as different level genetic markers. In IGS phylogenetic tree, Opisthorchiidae, Paramphistomidae, Dicrocoeliidae, and Schistosomatidae formed monophyletic groups, and the same length sequences were clustered together in the same species. These findings of the present study provide the new molecular data for studying the complete rDNA of C. sinensis and M. orientalis, and indicate IGS sequences may used as a novel genetic marker for studying intraspecific variation in trematodes.

Molecular detection of Cryptosporidium and Enterocytozoon bieneusi in dairy calves and sika deer in four provinces in Northern China.

The protistan pathogens Cryptosporidium and Enterocytozoon bieneusi can cause significant intestinal diseases in animals and humans. However, limited information is available regarding prevalence and molecular characterization of Cryptosporidium and E. bieneusi in ruminants in Northern China. In this study, the overall prevalence of Cryptosporidium and E. bieneusi was 19.3% (62/321) and 28.97% (93/321) in dairy calves and 1.10% (9/818) and 13.57% (111/818) in sika deer (Cervus nippon) in four provinces in Northern China, respectively. The prevalence of Cryptosporidium and E. bieneusi in different factor groups was various. Five Cryptosporidium species/genotypes were identified, of which C. parvum, C. ryanae, C. bovis, and C. andersoni were only found in dairy calves, and only Cryptosporidium deer genotype was found in sika deer. Moreover, J, I, and BEB4 ITS genotypes of E. bieneusi were found in dairy calves, and six known genotypes (JLD-III, JLD-IX, JLD-VII, EbpC, BEB6, and I) and ten novel genotypes (namely LND-I and JLD-XV to JLD-XXIII) were found in sika deer in this study. Cryptosporidium parvum and E. bieneusi genotype J were identified as the predominant species/genotypes in dairy calves, whereas the predominance of Cryptosporidium spp. and E. bieneusi in sika deer was Cryptosporidium deer genotype and BEB6, respectively. The present study reported the prevalence and genotypes of Cryptosporidium and E. bieneusi in dairy calves and sika deer in four provinces in northern China. The present findings also suggest that investigated dairy calves and sika deer may play an important role in the transmission of E. bieneusi and Cryptosporidium to humans and other animals, and also in an effort to better understand the epidemiology of these enteric pathogens in China.

Immunoglobulin G4-related Kidney Disease Associated With Autoimmune Hemolytic Anemia.

Awareness of the uncommon associated clinical manifestations of immunoglobulin G4 (IgG4)-related kidney disease is essential for the early diagnosis and effective treatment of patients. To the best of our knowledge, there have been few reports of patients with IgG4-related kidney disease associated with autoimmune hemolytic anemia. We here report a rare case of IgG4-related kidney disease associated with autoimmune hemolytic anemia. A 70-year-old man with kidney dysfunction and severe anemia had been diagnosed with chronic kidney disease and treated without any improvement. On admission, he had a high serum creatinine level, low hemoglobin level, positive direct Coombs test, and mild proteinuria. Serum IgG and IgG4 were elevated. Kidney biopsy showed marked infiltration of IgG4-positive plasma cells and storiform fibrosis in the interstitial compartment, which confirmed the diagnosis of IgG4-related kidney disease. Corticosteroid therapy was initiated, and subsequently, the kidney dysfunction and anemia dramatically improved.

Influence of HbA1c on short-term blood pressure variability in type 2 diabetic patients with diabetic nephropathy.

The aim of this study was to understand the characteristics of blood pressure (BP) variability in subjects with diabetic nephropathy (DN), and identify the probable predictors affecting BP variability. Fifty-one chronic kidney disease (CKD)-hypertensive patients without diabetes (NDN group) and sixty type 2 diabetic patients with overt DN (DN group) were enrolled in this study. The values of short-term BP variability were obtained from 24 h ambulatory BP monitoring (ABPM). Variance analysis or nonparametric analysis revealed that 24-h systolic BP variability and nighttime systolic BP variability of the DN group were significantly higher than those of the NDN group [(12.23±3.66) vs. (10.74±3.83) mmHg, P<0.05; (11.23±4.82) vs. (9.48±3.69) mmHg, P<0.05]. Then the patients of the DN group were divided into two groups according to glycated hemoglobin (HbA1c) level: Group A (HbA1c<7%) and Group B (HbA1c≥7%), and the t-test showed that patients in Group B had larger 24-h diastolic, daytime diastolic, and nighttime systolic/diastolic BP variability compared with Group A. In the DN group, partial correlation analysis revealed that HbA1c exhibited a strong association with 24-h diastolic, daytime diastolic, nighttime systolic and diastolic BP variability (P<0.001, P<0.001, P<0.05, and P<0.001, respectively). Taken together, larger short-term BP variability was detected in hypertensive type 2 diabetic patients with overt nephropathy and renal insufficiency. It may imply that the optimal BP variability level could benefit from a better glycaemic control.

Amelioration of rhabdomyolysis-induced renal mitochondrial injury and apoptosis through suppression of Drp-1 translocation.

INTRODUCTION: Mitochondrial dysfunction plays an important role in acute kidney injury (AKI). Mitochondrial fission regulated by dynamin-related protein 1 (Drp-1) impairs the function of the mitochondria and the survival of cells. This study was conducted to explore the effects of suppression of Drp-1 accumulation in the mitochondria, on mitochondrial function and renal tubular cell apoptosis in rhabdomyolysis (RM)-induced AKI. METHODS: An RM model was induced by intramuscular injection of glycerol in Sprague Dawley rats. Twenty-four and 48 hours after intraperitoneal injections of mitochondrial division inhibitor 1 (Mdivi-1), we observed the functions of the kidney, changes in pathology, expressions of Drp-1 in tubular tissues (by immunohistochemistry and Western blot) and accumulation of Drp-1 and mitofusin 2 in tubular mitochondria (by Western blot). Mitochondrial function (ATP and ROS) and tubular epithelial cell apoptosis (by TUNEL) were also measured. RESULTS: RM induced Drp-1 accumulation, decreased ATP production and increased ROS in mitochondria. With increasing cytochrome c expression, cell apoptosis increased, whereas kidney function decreased. These changes were time-dependent. At different time points, despite not significantly influencing the overall expression of Drp-1, Mdivi-1 suppressed the accumulation of Drp-1, inhibited the insertion of proapoptotic Bax in mitochondria and inhibited the release of cytochrome c, thus ameliorating cell apoptosis. CONCLUSIONS: To conclude, in RM-induced AKI, suppression of Drp-1 accumulation in mitochondria favors the maintenance of mitochondrial function and reduces the apoptosis of tubular cells. Regulation of the mitochondrial fusion-fission balance may offer a novel strategy for the prevention and treatment of RM-induced AKI.

[Comparison of the effects of alpha-keto/ amino acid supplemented low protein diet and diabetes diet in patients with diabetic nephropathy].

OBJECTIVE: To investigate if a-keto/amino acid supplemented low protein diet can slow down the progression of diabetic nephrophathy in comparison with non-supplemented diabetes diet. METHODS: A prospective, randomized, controlled clinical study was conducted. Twenty three cases of type 2 diabetic nephropathy in IV stage were randomly divided into alpha-keto/amino acid supplemented diet group (trial group) and conventional diabetes diet group (control group), The treatment duration was 52 weeks. 24 h urine protein was measured at 0, 12, 20, 36 and 52 weeks. Before and after the 52 weeks treatment, all the patients received the measurement of glomerular filtration rate (GFR), blood glucose, blood lipids, inflammatory markers, as well as nutritional status. RESULTS: After the treatment for 20, 36, 52 weeks, mean 24 h urine protein decreased significantly in trial groups (P < 0.05), and 24 h urine protein in trial group were significantly decreased (P < 0.05) compared with control group in 20 weeks after treatment. Either in trial group or in control group, GFR remained relatively stable during the observation period. Nutrition status, inflammatory markers, and serum calcium, phosphorus levels between the two groups were no significantly difference. The adverse events experienced by the patients in trial group were similar and consistent with the patients underlying renal diseases. CONCLUSION: Alpha-keto/amino acid can reduce proteinuria more effectively, while improve renal function and nutritional status in diabetic nephropathy patients with well-toleration.

[Influence of mannose binding lectin to expression of TGF-beta1 and NF-kappaB in high concentration of glucose cultured human renal glomerular endothelial cells].

OBJECTIVE: To investigate the effect of mannose binding lectin (MBL) complement pathway on expression of transforming growth factor-beta1 (TGF-beta1) and NF-kappaB in cultured human renal glomerular endothelial cells (HRGECs) stimulated by high concentration of glucose. METHODS: Human glomerular endothelial cells in culture were randomly divided into 5 groups according to different managements: normal concentration of glucose as controlled group, MBL + normal concentration of glucose group, high concentration of glucose, MBL + high glucose and MBL + high glucose + MBL blocker respectively. Flow cytometry was used to detect the depositions of MBL and C3 on the surfaces of HRGECs. Real-time PCR method was used to detect the mRNA levels of TGF-beta1. Human TGF-beta1 ELISA kit was used to detect the concentration of TGF-beta1 in supernatant fluid. ESMA was used to detect the activity of NF-kappaB in HRGECs. RESULTS: Compared with the normal glucose group and high glucose group, the depositions of MBL, C3 were apparently increased in MBL + high glucose group (P < 0.05). Expression of TGF-beta1 were significantly higher (P < 0.05) in MBL + high concentration of glucose groups than the normal glucose group and the high concentration of glucose group. Compared with the high glucose group, the activity of NF-kappaB in HRGECs was apparently increased in MBL + high glucose group, which could be significantly downregulated by MBL blocking antibody. CONCLUSION: High concentration of glucose can increase the expression of TGF-beta1 of cultured human glomerular endothelial cells. At the same time, high glucose together with MBL can up regulate the expression of TGF-beta1 and the activity of NF-kappaB in HRGECs.

[Influence of high glucose and mannose binding lectin complement pathway activation to IL-6 and TNF-alpha's expression by human renal glomerular endothelial cells].

OBJECTIVE: To investigate the effect of high glucose and mannose binding lectin (MBL) complement pathway activation's effect on expression of Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-alpha) from human renal glomerular endothelial cells (HRGEC), to explore unknown pathogenesy of diabetic nephropathy. METHODS: Normal HRGEC was divided randomly into normal glucose group(5 mmol/L D-glucose), manicol group (5 mmol/L D-glucose+25 mmol/L manicol) and high glucose group (30 mmol/L D-glucose). Real-time PCR was used to detect IL-6 and TNF-alpha mRNA expression in each group, Euzymelinked Immunosorbent Assay (ELISA) was performed to examine the protein expression of IL-6 and TNF-alpha in supernatant after 24 hours' culture. HRGEC was then randomly divided into two groups: single high glucose group and high glucose + MBL group. After 24 hours' culture with 30 mmol/L D-glucose, 30% MBL deficiency human serum was added into two groups, 1 microg/mL MBL was only added into high glucose + MBL group, continued the culturation for another 4 hours. Flow cytometry and immunofluorescence technique were applied to evaluate MBL, C3 and membrane attacks complex (MAC) deposition on cell surface respectively. Real-time PCR and ELISA were performed to examine mRNA and protein expression of both IL-6 and TNF-alpha in each group. RESULTS: Compared with normal glucose group and manicol group, the mRNA and protein expression of IL-6 and TNF-alpha in high glucose group were increased (P < 0.05). Flow cytometry confirmed obvious MBL and C3 co-deposition and Immunofluorescence confirmed obvious MAC deposition on cell surface in high glucose+ MBL group. Compared with single high glucose group, the mRNA and protein expression of IL-6 and TNF-alpha in high glucose+ MBL group were significantly higher (P < 0.05). CONCLUSION: High glucose can bring inflammatory factors' overexpression from cultured HRGEC; high glucose together with MBL can bring MBL complement pathway activation and inflammatory factors' overexpression, this indicates that the activation of MBL complement pathway may be a potential unknown pathogenesy of diabetic nephropathy and its proinflammatory status.

Effect of anaphylatoxin C3a, C5a on the tubular epithelial-myofibroblast transdifferentiation in vitro.

BACKGROUND: Tubulointerstitial renal fibrosis is the common end point of progressive kidney diseases, and tubular epithelial-myofibroblast transdifferentiation (TEMT) plays a key role in the progress of tubulointerstitial renal fibrosis. Anaphylatoxin C3a and C5a are identified as novel profibrotic factors in renal disease and as potential new therapeutic targets. The aim of this study was to investigate whether C3a, C5a can regulate TEMT by transforming growth factor-ß1 (TGF-ß)/connective tissue growth factor (CTGF) signaling pathway and the effects of C3a and C5a receptor antagonists (C3aRA and C5aRA) on C3a- and C5a-induced TEMT. METHODS: HK-2 cells were divided into C3a and C5a groups which were subdivided into four subgroups: control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C3a group, 50 nmol/L C3a plus 1 µmol/L C3aRA group; control group, 10 ng/ml TGF-ß1 group, 50 nmol/L C5a group, 50 nmol/L C5a plus 2.5 µmol/L C5aRA group. TGF-ß1 receptor antagonist (TGF-ß1RA) 10 µg/ml was used to investigate the mechanism of C3a- and C5a-induced TEMT. Electron microscopy was used to observe the morphological changes. Immunocytochemistry staining, real-time PCR and Western blotting were used to detect the expressions of a smooth muscle actin (α-SMA), E-cadherin, Col-I, C3a receptor (C3aR), C5aR, CTGF and TGF-ß1. RESULTS: HK-2 cells cultured with C3a and C5a for 72 hours exhibited strong staining of α-SMA, lost the positive staining of E-cadherin, and showed a slightly spindle-like shape and loss of microvilli on the cell surface. The expressions of α-SMA, E-cadherin, Col-I, C3aR, C5aR, TGF-ß1 and CTGF in C3a- and C5a-treated groups were higher than normal control group (P < 0.05). C3aRA and C5aRA inhibited the expressions of α-SMA, Col-I, C3aR, C5aR, and up-regulated the expression of E-cadherin (P < 0.05). TGF-ß1 and CTGF mRNA expressions induced by C3a and C5a were partly blocked by TGF-ß1RA (P < 0.05). CONCLUSION: C3a and C5a can induce TEMT via the up-regulations of C3aR and C5aR mRNA and the activation of TGF-ß1/CTGF signaling pathway in vitro.

[Effect of high concentration of glucose on thickness of glycocalyx and expression of syndecan-1 and glypican-1 in cultured human renal glomerular endothelial cells].

OBJECTIVE: To investigate the influence of high concentration of glucose on the thickness of Glycocalyx and expression of core protein Sydecan-1 and Glypican-1 in cultured human renal glomerular endothelial cells (HRGECs). METHODS: HRGECs in culture were randomly divided into 3 groups, high concentration of glucose (30 mmol/L D-glucose, high glucose group), normal concentration of glucose as controlled group (5 mmol/L D-glucose+25 mmol/L mannitol, normal control group), and mannitol group (30 mmol/L mannitol) respectively. After 72 hours, confocal laser scanning microscopy (CLSM) was used to observe and characterize the fully hydrated glycoalyx of HRGECs. Real time quantitative PCR and Western blot were applied to detect the mRNA levels and protein expression of Syndecan-1 and Glypican-1, and the fluorescence microscope were used to observe the immunofluorescence change of Syndecan-1 and Glypican-1. RESULTS: Compared with normal control group, the thickness of Glycocalyx on the surface of HRGECs in high glucose group decreased to 36.8% (P < 0.05). Immunofluorescence shows the depositions of Syndecan-1 and Glypican-1 were weakened in high glucose group. The mRNA and protein expression of Syndecan-1 and Glypican-1 were significantly decreased (P < 0.05) compared with normal control group and mannitol group. CONCLUSION: High concentration of glucose can reduce thickness of Glycocalyx on the surface of human glomerular endothelial cell. At the same time, high glucose can decrease the expression of core protein Sydecan-1 and Glypican-1 of HRGECs.

[Effects of high glucose on expression of core binding factor alpha1 and osteocalcin in vascular smooth muscle cells].

OBJECTIVE: To investigate the effects of high glucose on expression of core binding factor alpha1 (cbfalpha-1) and osteocalcin (OC) in vascular smooth muscle cells (VSMCs), and discuss the mechanism of small vessels calcification induced by high glucose (GS) in vitro. METHODS: The primary cultured VSMCs from rats' aortic segments were divided into three groups, including normal control group (5 mmol/L D-glucose), high glucose group (25 mmol/L D-glucose) and mannitol group (5 mmol/L D-glucose plus 25 mmol/L mannitol). We measured quantitatively the calcium deposition in VSMCs and investigated the calcium extent of VSMCs by alizarin red stain in each group. The mRNA levels of cbfalpha-1 and OC were measured by real-time PCR, and the protein expression levels of cbfalpha-1 and OC were examined by Western blot. The activity of alkaline phosphatase was measured by alkaline phosphatase activity testing kit, and the protein level of alpha-smooth muscle actin (a-SMA) was detected by immunohistochemistry. RESULTS: When compared with the normal group and mannitol group, the high glucose group showed that the calcium deposition and calcium extent of VSMCs increased obviously, the mRNA and protein levels of cbfalpha-1 and OC also increased significantly (P < 0.05), while the protein level of alpha-SMA decreased (P < 0.05), which were in a dose-dependent manner. The level of alkaline phosphatase activity of VSMCs was approximately doubled in high glucose group. CONCLUSION: The mechanism of high glucose induced calcification in VSMCs may be due to the increased expression of cbfalpha-1 and OC. High glucose decrease the expression of alpha-SMA in VSMCs, which could induce the transdifferentiation from RVSMCs to osteoblast-like cells.