Association of Geriatric Nutritional Risk Index with short-term mortality in patients with severe acute kidney injury: a retrospective cohort study.

OBJECTIVES: Geriatric Nutritional Risk Index (GNRI) is a new and simple index recently introduced to assess nutritional status, and its predictive value for clinical outcomes has been demonstrated in patients with chronic kidney disease. However, the association between the GNRI and prognosis has not been evaluated so far in patients with acute kidney injury (AKI), especially in those receiving continuous renal replacement therapy (CRRT). METHODS: A total of 1096 patients with severe AKI initiating CRRT were identified for inclusion in this retrospective observational study. Patients were divided into three groups according to GNRI tertiles, with tertile 1 as the reference. The outcomes of interest were the 28- and 90-days of all-cause mortality. The associations between GNRI and clinical outcomes were estimated using multivariate Cox proportional hazards model analysis. RESULTS: The overall mortality rates at 28- and 90-days were 61.6% (675/1096) and 71.5% (784/1096), respectively. After adjusting for multiple confounding factors, GNRI was identified as an independent prognostic factor for 28-days all-cause mortality (HR, 0.582; 95% CI, 0.467-0.727; p < .001 for tertile 3 vs. tertile 1) as well as 90-days all-cause mortality (HR, 0.540; 95% CI, 0.440-0.661; p < .001 for tertile 3 vs. tertile 1). The observed inverse associations were robust across subgroup analysis, and were more pronounced in elderly patients over 65 years of age. Finally, incorporating GNRI in a model with established risk factors might significantly improve its predictive power for the short-term death. CONCLUSIONS: GNRI is considered to be a useful prognostic factor in patients with severe AKI initiating CRRT, especially in elderly patients.

Effect of probiotics on the intestinal microbiota of hemodialysis patients: a randomized trial.

BACKGROUND: Intestinal dysbiosis contributes to the progression of renal failure and cardiovascular diseases in patients with chronic kidney disease. Probiotics is a promising intervention to improving intestinal dysbiosis. A double-blind clinical trial to investigate the ability of probiotics to modulate gut microbiota compositions in patients receiving hemodialysis (HD) was undertaken. METHODS: Fifty HD patients were enrolled and randomized, receiving either probiotics or placebo for 6 months. The responses to the interventions on gut microbiome, serum and fecal metabolome, serum albumin and endotoxin, endothelial activation markers and inflammatory markers were assessed. RESULTS: Totally, 22 in the probiotics group (11 males; 14 non-diabetic) and 23 in the placebo group (13 males; 17 non-diabetic) completed the study. Compared to that in the placebo group, probiotics did not significantly alter species diversity of the fecal microbiome. Probiotics did, however, restore the community composition, with particular significance in non-diabetic HD patients (P = 0.007 by Adonis analysis). Specifically, according to the results of linear discriminate analysis effect size, probiotics raised the proportions of family Bacteroidaceae and Enterococcaceae, and reduced Ruminococcaceae, Halomonadaceae, Peptostreptococcaceae, Clostridiales Family XIII. Incertae Sedis and Erysipelotrichaceae in non-diabetic HD patients. Additionally, probiotics reduced the abundances of several uremic retention solutes in serum or feces, including indole-3-acetic acid-O-glucuronide, 3-guanidinopropionic acid, and 1-methylinosine (P < 0.05). In the probiotic arm, no significant changes were observed in other secondary outcomes. CONCLUSIONS: Taken together, outcomes from this study suggest that probiotics do have benefits on improving intestinal imbalances and lowering exposure to several uremic toxins in HD patients.

Effect of Early Cannulation with Plastic Cannula on Arteriovenous Fistula Patency in Hemodialysis Patients.

INTRODUCTION: Plastic cannulas have been used to cannulate arteriovenous fistulas (AVFs) for hemodialysis (HD) in Japan for many years. However, the effect of early cannulation with plastic cannulas on AVF patency is not known. OBJECTIVE: We analyzed the relationship between first cannulation time (FCT) and patency rates for AVFs cannulated with plastic cannulas and investigated whether early cannulation with plastic cannulas affects AVF patency. METHODS: In total, 122 patients who underwent primary AVF construction were divided into an early cannulation group (FCT <10 days) and a late cannulation group (FCT ≥10 days). The Kaplan-Meier method and multivariable Cox regression models were used to investigate AVF patency. RESULTS: Median FCT was 6 days. There was no statistically significant between-group difference in primary (p = 0.643) or secondary (p = 0.453) patency rates. Early or late cannulation was not significantly associated with primary patency (hazard ratio [HR] 1.21; 95% CI 0.71-2.05) or secondary patency (HR 0.46; 95% CI 0.08-2.77) after adjustment for age, sex, presence of diabetes mellitus or hypertension, and HD at baseline. CONCLUSIONS: Early AVF cannulation (<10 days from creation) with plastic cannulas does not affect access patency, and it may be possible to cannulate AVFs earlier than 10 days to decrease the need for use of a central venous catheter.

Macrophages Are Involved in Gut Bacterial Translocation and Reversed by Lactobacillus in Experimental Uremia.

BACKGROUND: Uremia causes gut microbiome dysbiosis, which is characterized by a reduction in beneficial bacteria. Intestinal bacterial translocation (BT) contributes to microinflammation in uremia, which is associated with adverse outcomes. Whether macrophages are involved in BT remains unclear. AIMS: We investigated the involvement of macrophages in BT and microinflammation in uremic rats and whether Lactobacillus LB can influence macrophage activity. METHODS: Male Sprague-Dawley rats were randomly divided into three groups: sham, uremia, and uremia + probiotic. Macrophages and GFP-labeled tracer bacteria in intestinal and extraintestinal tissues were observed by fluorescence microscopy. The macrophage ultrastructure was examined by transmission electron microscopy. Immunochemistry was used to analyze the expression of cluster of differentiation 11a (CD11a), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1). RT-PCR and Western blot were employed to assess the mRNA and protein expression of early growth response gene 1 (EGR1) and toll-like receptor 4 (TLR4). RESULTS: In uremic rats, the colocalization of GFP-labeled tracer bacteria and macrophages was visible in intestinal and extraintestinal tissues. Compared with the sham group, the uremic macrophages showed fewer cytoplasmic protrusions and pseudopodia. Administration of Lactobacillus LB restored the protrusions and pseudopodia. Compared with the sham group, the uremia group exhibited macrophages with higher staining intensities for CD11a, iNOS, and ICAM-1, and higher mRNA and protein expression of TLR4 and EGR1. CONCLUSIONS: Intestinal macrophages in the uremic rats are polarized toward a proinflammatory phenotype, resulting in microinflammation. Macrophages with impaired phagocytic function are associated with BT. Lactobacillus LB reduces BT by enhancing macrophage phagocytosis.

miR149 rs71428439 polymorphism and risk of clear cell renal cell carcinoma: a case-control study.

Clear cell renal cell carcinoma (CCRCC) is the most common subtype of renal cell cancer and accounts for 70 % of renal cell cancer. CCRCC remains an enigmatic tumor type, as the molecular genetic mechanisms are still unclear. MicroRNA (miR) 149 functions as a tumor suppressor and plays an important role in the carcinogenesis of renal cells. In this study, we enrolled 1,000 CCRCC patients and 1,000 cancer-free controls to evaluate the association of miR149 rs71428439 with the risk of CCRCC by a case-control study to determine the effects on CCRCC risk. miR149 rs71428439 was significantly associated with increased CCRCC risk (odds ratio (OR) for trend = 1.53, P for trend = 4.04 × 10(-11)), with ORs (95 % confidence intervals (CIs)) of 1.42 (1.17-1.72) associated with AG genotype and 2.27 (1.76-2.94) associated with GG genotype, compared with subjects with AA genotype. The expression levels of miR149 in cancer tissues were significantly lower than those in adjacent normal tissues (P = 0.005), and per G allele has significantly lower miR149 levels in both tumor tissues and adjacent normal tissues. Our data suggest that the GG genotypes of miR149 rs71428439 predispose their carriers to CCRCC, and miR149 rs71428439 may be a new biomarker for predicting the risk of CCRCC.

Gut bacterial translocation may aggravate microinflammation in hemodialysis patients.

BACKGROUND/AIMS: Bacterial translocation (BT) promotes microinflammation in predialysis patients with end-stage renal disease (ESRD). However, the change in BT has not been reported in ESRD patients undergoing regular hemodialysis treatment. The present study investigated whether hemodialysis promotes gut BT and microinflammation. METHODS: The blood, gut, and dialysate of hemodialysis patients were analyzed using bacterial 16S rDNA amplification and DNA pyrosequencing to determine the presence of bacteria and alteration in gut microbiomes. High-sensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), and endotoxin were also determined. Plasma D-lactate was tested for gut permeability. RESULTS: Bacteria were present in the plasma of 12 out of 52 ESRD patients. The majority of the bacteria detected in the blood were also distributed in the gut of ESRD patients on the basis of the phylogenetics of the blood and gut microbial specimens in the patients. In patient, groups treated with and without hemodialysis, the plasma hs-CRP, IL-6, and endotoxin levels differed between the positive and negative plasma bacterial DNA. In patients who were positive in blood bacteria, the bacterial DNA concentration was positively correlated with plasma levels of CRP and IL-6. The ESRD patients who underwent hemodialysis had a different flora and showed slightly higher levels of hs-CRP, IL-6, and plasma endotoxin, compared with those in ESRD patients who did not undergo hemodialysis. CONCLUSION: ESRD, rather than hemodialysis, primarily contributes to BT and microinflammation in ESRD patients. Hemodialysis may exaggerate microinflammation in ESRD patients to some extent.

Probiotic Bifidobacterium animalis subsp. lactis Bi-07 alleviates bacterial translocation and ameliorates microinflammation in experimental uraemia.

AIM: Uraemia is characterized by intestinal bacterial translocation, which contributes to the development of microinflammation. Probiotics enhance the intestinal barrier and overall health of the host. The present study investigated whether the probiotic Bifidobacterium animalis subsp. lactis Bi-07 alleviates bacterial translocation and ameliorates microinflammation in experimental uraemia. METHODS: Sixty Sprague-Dawley rats were divided into three groups of 20 rats each: the sham group, which underwent only laparotomy; the uraemia group, which underwent 5/6 nephrectomy; and the uraemia + probiotic group, which underwent 5/6 nephrectomy and daily intragastric administration of B. animalis subsp. lactis Bi-07 for 4 weeks. Bacterial translocation was evaluated by polymerase chain reaction amplification of the green fluorescent protein (GFP) gene from oral GFP-labelled Escherichia coli in the peripheral blood, mesenteric lymph nodes, liver, and spleen. Intestinal permeability, plasma inflammatory biomarker levels, and endotoxin levels were measured. Jejunum, ileum, and colon specimens were removed for histological examination. RESULTS: Uraemic rats exhibited a significantly higher incidence of bacterial translocation (70%) than did sham rats (10%). Probiotic treatment resulted in a decrease in bacterial translocation (20%). Intestinal permeability, inflammatory biomarker levels, and endotoxin levels in uraemic rats were significantly higher than those in the sham group. After treatment with the probiotic, inflammatory biomarker levels significantly decreased. Uraemic rats demonstrated superficial mucosal erosion and inflammatory cell infiltration in the small intestine, and administration of the probiotic alleviated these lesions. CONCLUSION: The probiotic B. animalis subsp. lactis Bi-07 alleviate bacterial translocation and ameliorate microinflammation through the recovery of intestinal mucosal integrity.

Gut bacterial translocation is associated with microinflammation in end-stage renal disease patients.

AIM: To investigate whether gut bacteria translocation occurs in end-stage renal disease patients and contributes to microinflammation in end-stage renal disease (ESRD). METHODS: The subjects were divided into two groups: nondialysed ESRD patients (n = 30) and healthy controls (n = 10). Blood samples from all participants were subjected to bacterial 16S ribosomal DNA amplification and DNA pyrosequencing to determine the presence of bacteria, and the alteration of gut microbiomes were examined with the same methods. High-sensitive C-reactive protein and interleukin-6 were detected. Plasma D-lactate was tested for gut permeability. RESULTS: Bacterial DNAs were detected in the blood of 20% (6/30) of the ESRD patients. All the observed genera in blood (Klebsiella spp, Proteus spp, Escherichia spp, Enterobacter spp, and Pseudomonas spp) were overgrown in the guts of the ESRD patients. Plasma D-lactate, High-sensitive C-reactive protein, and interleukin-6 levels were significantly higher in patients with bacterial DNA than those without. The control group showed the same results as that of patients without bacterial DNA. CONCLUSION: Bacterial translocation occurs in ESRD patients and is associated with microinflammation in end stage renal disease.

Emerging roles of circRNAs in mice kidney with aging.

Circular RNA (circRNA) is a novel type of noncoding RNA expressed in different tissues and species. Up to now, little is known of the function and expression of circRNAs in kidney aging. In this research, we used RNA sequencing to identify 11,929 circRNAs in kidney from 3-, 12-, and 24-month-old mice, of which 12 circRNAs were validated by qPCR. Based on the validated circRNAs and their predicted miRNA-mRNA target pairs, a circRNA-miRNA-mRNA interactions network was conducted. Bioinformatics analysis for all the mRNAs in the ceRNA network showed that the most enriched gene ontology (GO) term and one of the most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were associated with endoplasmic reticulum (ER). The network also identified circNpas2, which was decreased significantly in mice kidney during aging, as a hub gene. Subsequently, we found that the cell cycle was arrested in G1 phase and the expression of P53 and P16 increased significantly in the circNpas2-knockdown cells. Moreover, knockdown of circNpas2 inhibited expression of ER-related proteins, HSPA5 and ERO1L. Taken together, our findings contribute to a better understanding of the role played by circRNA during kidney aging and provide potential therapeutic targets for the prevention of kidney aging. RESEARCH HIGHLIGHTS: This study is the first to systematically analyze the dysregulated circRNAs and ceRNA network during renal aging. The dysregulated circRNAs during renal aging are most enriched in ER stress-related pathway. CircNpas2 regulates senescence in TCMK-1.

Roxadustat Attenuates the Disruption of Epithelial Tight Junction in Caco2 Cells and a Rat Model of CKD Through MicroRNA-223.

Introduction: Increasing evidence supports the idea that the disruption of epithelial tight junction proteins (TJPs) caused by accumulation of uremia toxins, such as homocysteine (Hcy), is one of the most important mechanisms underlying the damage of intestinal barrier function (IBF) in chronic kidney disease (CKD). Since the decrease of hypoxia inducible factor-1α (HIF-1α) is reported to be involved in Hcy-induced cell injury, and the upregulation of microRNA-223 (miR-223) plays a vital protective role in the impairment of IBF in the experimental colitis, we investigated the effect of HIF-1α stabilizer roxadustat on the disruption of TJPs induced by Hcy and CKD and the underlying mechanism. Methods: Chronic kidney disease was induced in rats via 5/6 nephrectomy. In a series of experiments, the rats were treated orally with roxadustat of different doses. The expression of tight junction proteins, HIF-1α, and miR-223 was analyzed in different groups by western blotting analysis, RT-qPCR techniques and immunofluorescence. A series of experiments with cultured Caco2 cells was performed. Results: The results showed that the expression of TJPs (occludin, claudin-1, and ZO-1) decreased significantly, accompanied by the reduction of HIF-1α and miR-223 in Hcy-treated Caco2 cells and colonic mucosa of uremic rats. The reduction of HIF-1α and miR-223 was reversed by roxadustat and the decrease of TJPs expression was attenuated in both Caco2 cells induced by Hcy and colon tissue of CKD rats. Furthermore, transfection with miR-223 mimics increased the expression of TJPs, while transfection with miR-223 inhibitor decreased their expression in Caco2 cells. MiR-223 inhibitor applied before roxadustat treatment partly diminished the effect of roxadustat on TJPs expression in Caco2 cells. Conclusion: These results indicated that roxadustat attenuated the disruption of epithelial TJPs induced by Hcy in Caco2 cells and the damage of colonic epithelium in CKD rats through the upregulation of miR-223 induced by HIF-1α. A novel insight into the IBF dysfunction in CKD was provided, and it suggests a potential therapeutic use of roxadustat for the IBF dysfunction besides anemia in CKD.

Tob1 controls dorsal development of zebrafish embryos by antagonizing maternal beta-catenin transcriptional activity.

Maternal beta-catenin and Nodal signals are essential for the formation of the dorsal organizer, which, in turn, induces neural and other dorsal tissue development in vertebrate embryos. Tob (Transducer of ErbB2) proteins possess antiproliferative properties and are known to influence BMP signaling, but their relationship to other signaling pathways and to embryonic patterning in general was unclear. In this study, we demonstrate that zebrafish tob1a is required for correct dorsoventral patterning. Mechanistically, Tob1a inhibits beta-catenin transcriptional activity by physically associating with beta-catenin and preventing the formation of beta-catenin/LEF1 complexes. Although Tob1a can also inhibit the transcriptional activity of the Nodal effector Smad3, its role in limiting dorsal development is executed primarily by antagonizing the beta-catenin signal. We further demonstrate that Tob family members across species share similar biochemical properties and biological activities.

Synthesis and fluorescence properties of 5,7-diphenylquinoline and 2,5,7-triphenylquinoline derived from m-terphenylamine.

Synthesis of 5,7-phenylquinoline from the Skraup reaction of m-terphenyl-amine and glycerol in the presence of acid is reported. Further reaction of 5,7-diphenyl-quinoline with phenyl lithium prepared in situ led to the formation of 2,5,7-triphenyl-quinoline. All of the products and their intermediates were characterized and the UV-Vis and photo-luminescence (PL) spectra of m-terphenylamine, 5,7-diphenylquinoline and 2,5,7-triphenylquinoline are also reported.

RACK1 silencing attenuates renal fibrosis by inhibiting TGF-ß signaling.

The receptor for activated C-kinase 1 (RACK1) is a member of the WD40-repeat family of proteins and has been reported to be implicated in the development of liver fibrosis. However, the role of RACK1 in renal fibrosis remains unclear. Therefore, in this study, we investigated the effects of RACK1 on transforming growth factor-ß1 (TGF-ß1)-treated human proximal tubular epithelial cells and aimed to elucidate the possible mechanisms responsible for its anti-fibrotic effects. Our results revealed that RACK1 was highly expressed in the renal fibrotic tissues and TGF-ß1-treated HK-2 cells. RACK1 silencing inhibited TGF-ß1­induced α-smooth muscle actin and connective tissue growth factor expression in the HK-2 cells. Furthermore, RACK1 silencing inhibited the expression of phosphorylated Smad3 in the TGF-ß1-treated HK-2 cells. To the best of our knowledge, these data demonstrate for the first time the role of RACK1 in renal fibrosis. The present findings indicate that RACK1 silencing attenuates renal fibrosis by suppressing the activation of TGF-ß1/Smad3 signaling pathway in HK-2 cells. Thus, RACK1 may serve as a novel regulator of renal fibrosis.

Liquid Chromatography/Mass Spectrometry Reveals the Effect of Lactobacillus Treatment on the Faecal Metabolite Profile of Rats with Chronic Renal Failure.

BACKGROUND/AIM: Chronic kidney disease is accompanied by changes in the gut microbiome and by an increase in the number of gut pathogenic bacteria. The aim of this study was to investigate the difference of the faecal metabolic profiles in rats with uremia, and to determine whether the altered metabolites in the rats with uremia can be restored by Lactobacillus. METHODS: Thirty rats were randomly divided into 3 groups: sham, uremia and uremia + probiotic (UP) groups. The rats in uremia and UP groups were prepared through surgical renal mass 5/6 ablation. The rats in the UP group received Lactobacillus LB (1 ml, 109 CFU/ml) through gavage every day for 4 weeks. The rats were fed with a standard diet. Faecal samples were analysed through ultra performance liquid chromatography/mass spectrometry. Statistical analyses were performed using MetaboAnalyst and MATLAB. RESULTS: A total of 99, 324 and 177 significantly different ion peaks were selected between sham and uremia groups; sham and UP groups; and uremia and UP groups, respectively. In the 3 groups, 35 significantly altered metabolites were identified; of the 35 metabolites, 27 initially increased and then decreased; by contrast, 8 metabolites initially decreased and then increased. The 35 metabolites were subjected to pathway analysis in MetaboAnalyst. CONCLUSIONS: Faecal metabolites were significantly altered in rats with uremia; these changes were partially reversed by Lactobacillus.

Significantly reduced adsorption and activation of blood components in a membrane oxygenator system coated with crosslinkable zwitterionic copolymer.

UNLABELLED: A crosslinkable zwitterionic copolymer PMBT was coated onto the surfaces of polypropylene hollow fiber membrane (PP-HFM) oxygenator and its connecting tubes. The PMBT copolymer coating on the oxygenator circuit formed a cell outer membrane mimetic surface with excellent stability. The hemocompatibility of the PMBT copolymer coated PP-HFM oxygenator circuit was evaluated by animal extracorporeal circulation. The concentrations of clotting components fibrinogen and platelet in the blood were almost unchanged during the circulation through the PMBT copolymer coated oxygenator circuits. By contrast, the concentrations of fibrinogen and platelet were significantly reduced to 52% and 56% respectively in the uncoated oxygenator group due to adsorption and thrombogenesis of the blood during 2h circulation. Moreover, concentration of activation marker beta-thromboglobulin for platelet in the blood was remarkably lower in the PMBT group than the uncoated control group (p<0.01). All the results strongly supported that the hemocompatibility of the PP-HFM oxygenator circuit could be improved significantly by coating a stable and densely assembled zwitterionic polymer film. This simple, stable and highly effective cell membrane mimetic coating strategy may be applicable in developing advanced oxygenator systems and other artificial organs. STATEMENT OF SIGNIFICANCE: Although a number of studies have reported the fabrication of zwitterionic phosphorylcholine coated oxygenators to resist the adsorption and activation of blood components and eliminate heparin-induced thrombocytopenia, none of them have fabricated stable and densely assembled film, especially with crosslinkable amphiphilic random copolymer described in our manuscript. The novel features of our work include.

A novel zinc finger transcription factor resembles krox-20 in structure and in expression pattern in zebrafish.

Krx-20, a zinc finger protein, plays an essential role in hindbrain segmentation of vertebrates. Zebrafish gene frb35 encodes a novel zinc finger protein that shares a high homology with Krx-20. The expression domains of frb35 overlap those of krx-20. The expression of frb35 first occurs in the 3rd rhombomere primordium at the onset of segmentation, which is soon followed by expression in the 5th rhombomere. From mid-segmentation onward, the 5th rhombomere has a higher frb35 expression level than the 3rd rhombomere. Unlike krx20, frb35 expression persists in the 5th rhombomere until the end of pharyngula period.

[Effects of nuclear factor of kappa B decoy oligodeoxynucleotides on murine with multiple myeloma].

OBJECTIVE: The present study was to evaluate the effects of nuclear factor of kappa B decoy oligodeoxynucleotides on murine multiple myeloma models. METHODS: The severe combined immunodeficient mice were injected subcutaneously with RPMI-8226 myeloma cells. When tumors became measurable, the mice were divided into 2 treatment groups who respectively received 5 µg/g or 10 µg/g liposome-NF-κB decoy ODN compounds, and one control group was selected; the control group received 10 µg/g liposome-NF-κB mutant decoy ODN compounds, twice per week for 4 weeks. The mice were killed when they died or the tumor diameter became >2 cm. RESULTS: The liposome-NF-κB decoy ODN could efficiently suppress NF-κB DNA binding activity and inhibited the expression of IL-6. As compared with the control group, the two liposome-NF-κB decoy ODN-treated groups showed more remarkably survival time and smaller tumor volume. CONCLUSION: In vivo transfection of NF-κB decoy ODN may provide a new therapeutic strategy for multiple myeloma.

Inhibition of macrophage migration inhibitory factor reduces diabetic nephropathy in type II diabetes mice.

Macrophage migration inhibitory factor (MIF) plays a critical role in inflammation and is elevated in diabetic kidney. However, whether MIF plays a causative role in diabetic nephropathy (DN) remains unclear. In the present study, we have demonstrated that after treatment of 8-week-old diabetic db/db and nondiabetic db/m mice with the MIF inhibitor ISO-1 (20 mg/kg) for 8 weeks, there was a significant decrease in blood glucose, albuminuria, extracellular matrix accumulation, epithelial-mesenchymal transition (EMT), and macrophage activation in the kidney of db/db mice. Incubation of macrophages with MIF induced the production of proinflammatory cytokines, including interleukin (IL) 6, IL-1ß, tumor necrosis factor α (TNF-α). The conditioned media (CM) of MIF-activated macrophages and TNF-α induced by MIF caused podocyte damage. Moreover, CM from MIF-activated macrophages induced EMT of renal tubular cells, and this effect was blocked by ISO-1. Thus, MIF inhibition may be a potential therapeutic strategy for DN. This effect may be attributable to its inhibitory effect on macrophage activation in the diabetic kidney.

Identification of evolutionarily conserved, functional noncoding elements in the promoter region of the sodium channel gene SCN8A.

SCN8A is a major neuronal sodium channel gene expressed throughout the central and peripheral nervous systems. Mutations of SCN8A result in movement disorders and impaired cognition. To investigate the basis for the tissue-specific expression of SCN8A, we located conserved, potentially regulatory sequences in the human, mouse, chicken, and fish genes by 5' RACE of brain RNA and genomic sequence comparison. A highly conserved 5' noncoding exon, exon 1c, is present in vertebrates from fish to mammals and appears to define the ancestral promoter region. The distance from exon 1c to the first coding exon increased tenfold during vertebrate evolution, largely by insertion of repetitive elements. The mammalian gene acquired three novel, mutually exclusive noncoding exons that are not represented in the lower vertebrates. Within the shared exon 1c, we identified four short sequence elements of 10-20 bp with an unusually high level of evolutionary conservation. The conserved elements are most similar to consensus sites for the transcription factors Pou6f1/Brn5, YY1, and REST/NRSF. Introduction of mutations into the predicted Pou6f1 and REST sites reduced promoter activity in transfected neuronal cells. A 470-bp promoter fragment containing all of the conserved elements directed brain-specific expression of the LacZ reporter in transgenic mice. Transgene expression was highest in hippocampal neurons and cerebellar Purkinje cells, consistent with the expression of the endogenous gene. The compact cluster of conserved regulatory elements in SCN8A provides a useful target for molecular analysis of neuronal gene expression.

Cloning and expression analysis of zebrafish tob1 gene.

Many members of the Btg/Tob family, which possess anti-proliferation activity, have been identified in mammals but their physiological functions are poorly understood. We have identified a zebrafish tob1 gene, which encodes a peptide of 322 amino acids. The expression pattern of zebrafish tob1 during early embryogenesis is examined by RT-PCR and whole-mount in situ hybridization. The maternal tob1 transcript is abundant in fertilized eggs and blastulas. The expression of tob1 ceases during midgastrulation and early segmentation, when it is expressed and restricted to the notochord and somites. On day 2, expression occurs in the pectoral fin bud but not in other tissues. It will be interesting to investigate whether Tob1 is required for controlling cell proliferation during vertebrate embryogenesis.