Asymmetries of Left and Right Adrenal Glands in Neural Innervation and Glucocorticoids Production.

The adrenal gland is paired peripheral end organs of the neuroendocrine system and is responsible for producing crucial stress hormones from its two functional compartments, the adrenal cortex, and the adrenal medulla under stimuli. Left-right asymmetry in vertebrates exists from the central nervous system to peripheral paired endocrine glands. The sided difference in the cerebral cortex is extensively investigated, while the knowledge of asymmetry of paired endocrine glands is still poor. The present study aims to investigate the asymmetries of bilateral adrenal glands, which play important roles in stress adaptation and energy homeostasis via steroid hormones produced from the distinct functional zones. Left and right adrenal glands from male C57BL/6J mice were initially histologically analyzed, and high-throughput RNA sequencing was then used to detect the gene transcriptional difference between left and right adrenal glands. Subsequently, the enrichment of functional pathways and ceRNA regulatory work was validated. The results demonstrated that the left adrenal gland had higher tissue mass and levels of energy expenditure, whereas the right adrenal gland appeared to be more potent in glucocorticoid secretion. Further analysis of adrenal stem/progenitor cell markers predicted that Shh signaling might play an important role in the left-right asymmetry of adrenal glands. Of the hub miRNAs, miRNA-466i-5p was identified in the left-right differential innervation of the adrenal glands. Therefore, the present study provides evidence that there are asymmetries between the left and right adrenal glands in glucocorticoid production and neural innervation, in which Shh signaling and miRNA-466i-5p play an important role.

Single-Cell Transcriptional Landscape Reveals the Regulatory Network and Its Heterogeneity of Renal Mitochondrial Damages in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is one of the common chronic microvascular complications of diabetes in which mitochondrial disorder plays an important role in its pathogenesis. The current study delved into the single-cell level transcriptome heterogeneity of mitochondrial homeostasis in db/db mice, an animal model for study of type 2 diabetes and DKD, with single-cell RNA sequencing (scRNA-Seq) and bulk RNA-seq analyses. From the comprehensive dataset comprising 13 meticulously captured and authenticated renal cell types, an unsupervised cluster analysis of mitochondria-related genes within the descending loop of Henle, collecting duct principal cell, endothelial, B cells and macrophage, showed that they had two types of cell subsets, i.e., health-dominant and DKD-dominant clusters. Pseudotime analysis, cell communication and transcription factors forecast resulted in identification of the hub differentially expressed genes between these two clusters and unveiled that the hierarchical regulatory network of receptor-TF-target genes was triggered by mitochondrial degeneration. Furthermore, the collecting duct principal cells were found to be regulated by the decline of Fzd7, which contributed to the impaired cellular proliferation and development, apoptosis and inactive cell cycle, as well as diminished capacity for material transport. Thereby, both scRNA-Seq and bulk RNA-Seq data from the current study elucidate the heterogeneity of mitochondrial disorders among distinct cell types, particularly in the collecting duct principal cells and B cells during the DKD progression and drug administration, which provide novel insights for better understanding the pathogenesis of DKD.

Evaluation of the association between maternal folic acid supplementation and the risk of congenital heart disease: a systematic review and meta-analysis.

BACKGROUND: Folic acid (FA), as a synthetic form of folate, has been widely used for dietary supplementation in pregnant women. The preventive effect of FA supplementation on the occurrence and recurrence of fetal neural tube defects (NTD) has been confirmed. Incidence of congenital heart diseases (CHD), however, has been parallelly increasing worldwide. The present study aimed to evaluate whether FA supplementation is associated with a decreased risk of CHD. METHODS: We searched the literature using PubMed, Web of Science and Google Scholar, for the peer-reviewed studies which reported CHD and FA and followed with a meta-analysis. The study-specific relative risks were used as summary statistics for the association between maternal FA supplementation and CHD risk. Cochran's Q and I2 statistics were used to test for the heterogeneity. RESULTS: Maternal FA supplementation was found to be associated with a decreased risk of CHD (OR = 0.82, 95% CI: 0.72-0.94). However, the heterogeneity of the association was high (P < 0.001, I2 = 92.7%). FA supplementation within 1 month before and after pregnancy correlated positively with CHD (OR 1.10, 95%CI 0.99-1.23), and high-dose FA intake is positively associated with atrial septal defect (OR 1.23, 95%CI 0.64-2.34). Pregnant women with irrational FA use may be at increased risk for CHD. CONCLUSIONS: Data from the present study indicate that the heterogeneity of the association between maternal FA supplementation and CHD is high and suggest that the real relationship between maternal FA supplementation and CHD may need to be further investigated with well-designed clinical studies and biological experiments.

Chemical constituents, clinical efficacy and molecular mechanisms of the ethanol extract of Abelmoschus manihot flowers in treatment of kidney diseases.

Abelmoschus manihot, also called as "Huangkui" in Chinese, is an annual flowering herb plant in the family of Malvaceae. As a traditional Chinese medicine, the ethanol extract of the flower in Abelmoschus manihot is made as Huangkui capsule and has been used for medication of the patients with kidney diseases. Its efficacy in clinical symptoms is mainly improving renal function and reducing proteinuria among the patients with chronic kidney disease, diabetic kidney disease or IgA nephropathy. The possible mechanism of Huangkui capsule treatment in kidney diseases may include reducing inflammation and anti-oxidative stress, improving immune response, protecting renal tubular epithelial cells, ameliorating podocyte apoptosis, glomerulosclerosis and mesangial proliferation, as well as inhibiting renal fibrosis. In this review, we first described chemical constituents and pharmacokinetic characteristics in ethanol extract of the flower of Abelmoschus manihot. We then summarized the clinical and epidemiological relevancies of kidney diseases particularly in the mainland of China and discussed the possible molecular mechanisms of Huangkui capsule in the treatment of kidney diseases. Finally, we prospected further research on cellular and molecular mechanisms and application of this Chinese natural medicine in kidney diseases.

Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism.

Bone morphogenetic protein (BMP)-9 has been reported to regulate energy balance in vivo. However, the mechanisms underlying BMP9-mediated regulation of energy balance remain incompletely understood. Here, we investigated the role of BMP9 in energy metabolism. In the current study, we found that hepatic BMP9 expression was down-regulated in insulin resistance (IR) mice and in patients who are diabetic. In mice fed a high-fat diet (HFD), the overexpression of hepatic BMP9 improved glucose tolerance and IR. The expression of gluconeogenic genes was down-regulated, whereas the level of insulin signaling molecule phosphorylation was increased in the livers of Adenovirus-BMP9-treated mice and glucosamine-treated hepatocytes. Furthermore, BMP9 overexpression ameliorated triglyceride accumulation and inhibited the expression of lipogenic genes in both human hepatocellular carcinoma HepG2 cells treated with a fatty acid mixture as well as the livers of HFD-fed mice. In hepatocytes isolated from sterol regulatory element-binding protein (SREBP)-1c knockout mice, the effects of BMP9 were ablated. Mechanistically, BMP9 inhibited SREBP-1c expression through the inhibition of liver X receptor response element 1 activity in the SREBP-1c promoter. Taken together, our results show that BMP9 is an important regulator of hepatic glucose and lipid metabolism.-Yang, M., Liang, Z., Yang, M., Jia, Y., Yang, G., He, Y., Li, X., Gu, H. F., Zheng, H., Zhu, Z., Li, L. Role of bone morphogenetic protein-9 in the regulation of glucose and lipid metabolism.

Gut ghrelin regulates hepatic glucose production and insulin signaling via a gut-brain-liver pathway.

BACKGROUND: Ghrelin modulates many physiological processes. However, the effects of intestinal ghrelin on hepatic glucose production (HGP) are still unclear. The current study was to explore the roles of intestinal ghrelin on glucose homeostasis and insulin signaling in the liver. METHODS: The system of intraduodenal infusion and intracerebral microinfusion into the nucleus of the solitary tract (NTS) in the normal chow-diet rats and pancreatic-euglycemic clamp procedure (PEC) combined with [3-3H] glucose as a tracer were used to analyze the effect of intestinal ghrelin. Intraduodenal co-infusion of ghrelin, tetracaine and Activated Protein Kinase (AMPK) activator (AICAR), or pharmacologic and molecular inhibitor of N-methyl-D-aspartate receptors within the dorsal vagal complex, or hepatic vagotomy in rats were used to explore the possible mechanism of the effect of intestinal ghrelin on HGP. RESULTS: Our results demonstrated that gut infusion of ghrelin inhibited duodenal AMP-dependent protein kinase (AMPK) signal pathways, increased HGP and expression of gluconeogenic enzymes, and decreased insulin signaling in the liver of the rat. Intraduodenal co-infusion of ghrelin receptor antagonist [D-Lys3]-GHRP-6 and AMPK agonist with ghrelin diminished gut ghrelin-induced increase in HGP and decrease in glucose infusion rate (GIR) and hepatic insulin signaling. The effects of gut ghrelin were also negated by co-infusion with tetracaine, or MK801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, and adenovirus expressing the shRNA of NR1 subunit of NMDA receptors (Ad-shNR1) within the dorsal vagal complex, and hepatic vagotomy in rats. When ghrelin and lipids were co-infused into the duodenum, the roles of gut lipids in increasing the rate of glucose infusion (GIR) and lowering HGP were reversed. CONCLUSIONS: The current study provided evidence that intestinal ghrelin has an effect on HGP and identified a neural glucoregulatory function of gut ghrelin signaling.

High Circulating Alarin Levels Are Associated with Presence of Metabolic Syndrome.

BACKGROUND/AIMS: Alarin has been reported to be related with increased food intake and body weight. The relationship of circulating Alarin with insulin resistance or metabolic syndrome (MetS), however, is unknown. This study aimed to investigate the physiological role of Alarin and its association with MetS in humans. METHODS: Newly diagnosed MetS patients (n=237) and age-matched healthy subjects (n=192) were recruited for this study. Oral glucose tolerance test, treadmill exercise, lipid infusions and euglycemic-hyperinsulinemic clamp (EHCs) were performed. Circulating Alarin and TNFα levels were measured by ELISA. RESULTS: Circulating Alarin levels were significantly higher in MetS patients compared with healthy subjects (0.46 ± 0.22 vs. 0.41 ± 0.14 µg/L, P < 0.01). In all studied subjects, circulating Alarin levels were positively correlated with WC, blood pressure, FBG, triglyceride, HbA1c, HOMA-IR, AUCglucose, and TNFα (P < 0.05 or P < 0.01). Multivariate logistic regression analyses revealed that circulating Alarin levels were correlated with MetS and insulin resistance. There was no significant change of circulating Alarin levels in the subjects with treadmill exercise for 45 min. In healthy individuals, however, glucose challenge, acute hyperglycemia and lipid infusions resulted in increased circulating Alarin levels, while acute hyperinsulinaemia transiently decreased circulating Alarin levels. CONCLUSION: The present study provides the evidence that circulating Alarin levels are associated with MetS and insulin resistance.

Protective Effect of Znt7 on High Glucose-Induced Epithelial-to-Mesenchymal Transition in Renal Tubular Epithelial Cells.

BACKGROUND/AIMS: Evidence from our and other groups has demonstrated that zinc transporter 7 in SLC30 family (ZnT7) inhibited epithelial-to-mesenchymal transition (EMT) and apoptosis in rat peritoneal mesothelial cells (RPMCs) under high glucose (HG) concentration. In the present study, we investigated the effect of ZnT7 on EMT of renal tubular epithelial cells (RTECs) in an in vitro model of diabetic nephropathy (DN). METHODS: A dual-fluorescent staining protocol was used for detection of ZnT7 in a normal rat kidney tubular epithelial cell line (NRK-52E cells). EMT was induced with HG (30 mM). NRK-52E cells were transfected with plasmids codifying for hZnT7-EGFP and interfering RNA for determination of the effect of ZnT7 over-expression and silencing, respectively. Expression of ZnT7, activation of the MAPK/ERK and TGF-ß/Smad pathways were analyzed with by means of Western blot. RESULTS: ZnT7 was localized in the perinuclear region and Golgi apparatus. In HG-induced EMT of NRK-52E cells, ZnT7 was up-regulated. Over-expression of ZnT7 led to inhibition of HG-induced EMT, while knock-down of ZnT7 increased EMT. Furthermore, knock-down of ZnT7 and increased HG-induced EMT was accompanied by activation of the MAPK/ERK and TGF-ß/Smad pathways. CONCLUSION: The present study provides evidence that ZnT7 has a protective effect over EMT of RTECs in DN and suggests that the inhibition of HG-induced EMT may be achieved through the MAPK/ERK and TGF-ß/Smad pathways. Thereby, ZnT7 could be a potential target for translation medicine and prevention program in DN.

Assessment of global long interspersed nucleotide element-1 (LINE-1) DNA methylation in a longitudinal cohort of type 2 diabetes mellitus (T2DM) individuals.

INTRODUCTION: Recent studies have indicated that methylation of the LINE-1 elements is associated with an increased risk of worsening carbohydrate metabolism. It has been shown that overall DNA methylation of LINE-1 elements could be considered as a risk factor for T2DM and its complications, independent of other established risk factors. METHODS: A total of 794 T2DM individuals from Salford, UK were included in this study (60% men n = 470). All patients had clinical and metabolic variables measured in 2002 (baseline outcomes) and annually through to 2016. Global LINE-1 DNA methylation was measured at four CpG sites. The QIAGEN PyroMark Q96 MD pyrosequencer was used to quantify methylation. RESULTS: The overall mean ± SD global LINE-1 methylation was 75.81 ± 3.25%. Cross-sectional linear regression analysis at baseline year 2002 showed that LINE-1 methylation was a significant predictor of diastolic BP (adjusted beta coefficient ß = -0.25), estimated glomerular filtration rate (eGFR) (ß = -0.48) and cholesterol HDL ratio (ß = -0.04). A 10% increase in LINE-1 methylation was associated with a lower diastolic BP by 2.5 mm Hg, a lower eGFR by 4.8 ml/min/1.73 m2 and decreased cholesterol/HDL ratio by 0.4 mmol/L. Longitudinal analysis over the 14-year-follow-up periods showed that global LINE-1 methylation at baseline was associated with lower BMI in women [ß = -0.25] and lower cholesterol: HDL ratio [ß = -0.07]. A 10% increase in LINE-1 methylation was associated with reduction in BMI by 2.5 kg/m2 in women and reduction in cholesterol:HDL ratio by 0.7 mmol/L. CONCLUSION: In a 14-year longitudinal cohort of T2DM individuals, relations between global LINE-1 DNA methylation status and specific metabolic markers were seen. Also, a higher degree of DNA methylation was predictive of less weight gain over time in women.

Genetic and biological effects of sodium-chloride cotransporter (SLC12A3) in diabetic nephropathy.

BACKGROUND/AIMS: Solute carrier family 12 member 3 (SLC12A3) encodes a sodium/chloride transporter in kidneys. Previous reports suggest that Arg913Gln polymorphism in this gene is associated with diabetic nephropathy (DN), but the data appear to be inconsistent. Up to now, there is no biological evidence concerning the effects of SLC12A3 in DN. In this study, we aim to evaluate the genetic effects of the SLC12A3 gene and its Arg913Gln polymorphism with genetic and functional analyses. METHODS: We genotyped SLC12A3 genetic polymorphisms including Arg913Gln in 784 non-diabetes controls and 633 type 2 diabetes (T2D) subjects with or without DN in a Malaysian population and performed a meta-analysis of the present and previous studies. We further analyzed the role of slc12a3 in kidney development and progress of DN in zebrafish and db/db mice. RESULTS: We found that SLC12A3 Arg913Gln polymorphism was associated with T2D (p = 0.028, OR = 0.772, 95% CI = 0.612-0.973) and DN (p = 0.038, OR = 0.547, 95% CI = 0.308-0.973) in the Malaysian cohort. The meta-analysis confirmed the protective effects of SLC12A3 913Gln allele in DN (Z-value = -1.992, p = 0.046, OR = 0.792). Furthermore, with knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage. Slc12a3 mRNA and protein expression levels were upregulated in kidneys of db/db mice from 6, 12, and 26 weeks at the age. CONCLUSION: The present study provided the first biological and further genetic evidence that SLC12A3 has genetic susceptibility in the development of DN, while the minor 913Gln allele in this gene confers a protective effect in the disease.

A single-cell profile reveals the transcriptional regulation responded for Abelmoschus manihot (L.) treatment in diabetic kidney disease.

BACKGROUND: Huangkui capsule (HKC), as an ethanol extract of Abelmoschus manihot (L.), has a significant efficacy in treatment of the patients with diabetic kidney disease (DKD). The bioactive ingredients of HKC mainly include the flavonoids such as rutin, hyperoside, hibifolin, isoquercetin, myricetin, quercetin and quercetin-3-O-robinobioside. PURPOSE: To explore the molecular mechanisms of A. manihot in treatment of DKD. STUDY DESIGN: A single-cell RNA sequencing analysis of kidneys in db/db mice with and without HKC administration. METHODS: Urinary biochemical and histopathological examination in C57BL/6 and db/db mice of DKD and HKC groups was done. Single-cell RNA sequencing pipeline was then performed. The regulatory mechanisms of seven flavonoids in HKC were revealed by cell communication, prediction of transcription factor regulatory network, and molecular docking. RESULTS: By constructing ligand-receptor regulatory network and performing molecular docking between 75 receptors with different activities and seven flavonoids. 11 key receptors in 4 cell types (segment 3 proximal convoluted tubular cell, ascending limbs of the loop of Henle, distal convoluted tubule, and T cell) in kidneys were found to be directly interacted with HKC. The interactions regulated 8 downstream regulons. The docking receptors in T cell led to transcriptional event differences in the regulons such as Cebpb, Rel, Tbx21 and Klf2 and consequently affected the activation, differentiation, and infiltration of T cell, while the receptors Tgfbr1 and Ldlr in stromal cells of kidneys were closely associated with the downstream transcriptional events of renal injury and proteinuria in DKD. CONCLUSION: The current study provides novel information of the key receptors and regulons in renal cells for a better understanding of the cell type specific molecular mechanisms of A. manihot in treatment of DKD.

Prediction of IVF/ICSI outcome based on the follicular output rate.

This study assessed the true accuracy of follicular output rate (FORT) as a prognostic indicator of response to FSH and reproductive competence after IVF/intracytoplasmic sperm injection. A total of 1643 cycles, including 140 polycystic ovary syndrome (PCOS) patients who underwent ovarian stimulation, were studied. FORT was calculated as the ratio of preovulatory follicle count on the day of stimulation×100/small antral follicle count (3-10mm in diameter) at baseline. Low, medium and high FORT groups were defined according to tertile values. Among 1503 non-PCOS cycles, numbers of retrieved oocytes and of all embryos that could be transferred, as well as rates of good-quality embryos, embryo implantations and clinical pregnancies, progressively increased with FORT. In PCOS patients, FORT were significantly lower in patients who achieved clinical pregnancy compared with those who did not (0.56±0.21 versus 0.66±0.29, P=0.031). Fertilization and good-quality embryo rates were significantly higher with medium FORT than low and high FORT (P=0.001 and P=0.047, respectively). Medium FORT in PCOS patients and high FORT in non-PCOS patients may predict better outcomes for IVF/ICSI.

Prediction of cellular targets in diabetic kidney diseases with single-cell transcriptomic analysis of db/db mouse kidneys.

Diabetic kidney disease is the leading cause of impaired kidney function, albuminuria, and renal replacement therapy (dialysis or transplantation), thus placing a large burden on health-care systems. This urgent event requires us to reveal the molecular mechanism of this disease to develop more efficacious treatment. Herein, we reported single-cell RNA sequencing analyses in kidneys of db/db mouse, an animal model for type 2 diabetes and diabetic kidney disease. We first analyzed the hub genes expressed differentially in the single cell resolution transcriptome map of the kidneys. Then we figured out the communication among the renal and immune cells in the kidneys. Data from this report may provide novel information for better understanding the cell-specific targets involved in the aetiologia of type 2 diabetic kidney disease and for cell communication and signaling between renal cells and immune cells of this complex disease.

Abelmoschus Manihot ameliorates the levels of circulating metabolites in diabetic nephropathy by modulating gut microbiota in non-obese diabetes mice.

Huangkui capsule (HKC), a traditional Chinese medicine, has been used for medication of kidney diseases, including diabetic nephropathy (DN). The current study aimed to evaluate the effects of HKC in the modulation of gut microbiota and the amelioration of metabolite levels by using non-obese diabetes (NOD) mice with DN. The microbiota from three parts of intestines (duodenum, ileum and colon) in NOD mice with and without HKC treatment were analysed using 16S rDNA sequencing techniques. Untargeted metabolomics in plasma of NOD mice were analysed with liquid mass spectrometry. Results showed that HKC administration ameliorated DN in NOD mice and the flora in duodenum were more sensitive to HKC intervention, while the flora in colon had more effects on metabolism. The bacterial genera such as Faecalitalea and Muribaculum significantly increased and negatively correlated with most of the altered metabolites after HKC treatment, while Phyllobacterium, Weissella and Akkermansia showed an opposite trend. The plasma metabolites, mainly including amino acids and fatty acids such as methionine sulfoxide, BCAAs and cis-7-Hexadecenoic acid, exhibited a distinct return to normal after HKC treatment. The current study thereby provides experimental evidence suggesting that HKC may modulate gut microbiota and subsequently ameliorate the metabolite levels in DN.

Effects of total flavones of Abelmoschus manihot (L.) on the treatment of diabetic nephropathy via the activation of solute carriers in renal tubular epithelial cells.

As a traditional Chinese medicine, Huangkui capsule (HKC) has been used to treat patients with kidney diseases, including diabetic nephropathy (DN). We have recently demonstrated that HKC could re-regulate the activities of solute carriers (SLC)s in proximal and distal convoluted tubules of kidneys in regression of the development of DN. The main active chemical constituents of HKC are the flavones of Abelmoschus manihot (L.). The current study aims to further evaluate the efficacy of total flavones of A. manihot (TFA) in the regression of DN by analyzing SLC activities in proximal and distal convoluted tubules of kidneys. TFA (0.076 g/kg/d) or vehicle was administered in db/db mice, the animal model of type 2 diabetes and DN, daily via oral gavage for four weeks. Blood glucose levels and urinary albumin-to-creatinine ratio (UACR) were measured and used for the determination of T2D and DN. Ten SLCs, including slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 were highly expressed in proximal and distinct convoluted tubules of kidneys. Their expression at mRNA and protein levels before and after TFA treatment were analyzed with real-time RT-PCR and immunohistochemistry. Data showed that UACR in the db/db mice after TFA treatment was significantly decreased. Compared with the group of non-diabetic control, slc2a2, slc4A1, slc5a2, slc5A3, slc5a8, slc6a20, slc27a2, slc12a3, slc34a1 and slc38a2 in the group of DN were down-regulated but up-regulated after TFA treatment. Further analyses of whole kidney sections indicated that the numbers and structures of the nephron in db/db mice was increased and improved after TFA treatment. Thereby, the current study provides further evidence that the flavones in A. manihot have pharmacological effects on the treatment of DN by improving the biological function of SLCs in kidneys.

Identification of the main flavonoids of Abelmoschus manihot (L.) medik and their metabolites in the treatment of diabetic nephropathy.

Introduction: Huangkui capsule (HKC) is made from the ethanol extract of Abelmoschus manihot (L.) Medik [Malvaceae; abelmoschi corolla] and received approval from the China Food and Drug Administration (Z19990040) in 1999. Currently, HKC is used for treatment of the patients with diabetic nephropathy (DN) in China. The bioactive chemical constituents in HKC are total flavonoids of A. manihot (L.) Medik (TFA). The present study aims to identify the primary flavonoid metabolites in HKC and TFA and their metabolism fates in db/db mice, the animal model for the study of type 2 diabetes and DN. Methods: HKC (0.84 g/kg/d) and TFA (0.076 g/kg/d) or vehicle were respectively administered daily via oral gavage in db/db mice for 4 weeks. The metabolism fate of the main metabolites of HKC in serum, liver, kidney, heart, jejunum, colon, jejunal contents, colonic contents, and urine of db/db mice were analyzed with a comprehensive metabolite identification strategy. Results and Discussion: In db/db mice administered with HKC and TFA, 7 flavonoid prototypes and 38 metabolites were identified. The related metabolic pathways at Phases I and II reactions included dehydroxylation, deglycosylation, hydrogenation, methylation, glucuronidation, sulphation, and corresponding recombined reactions. Quercetin, isorhamnetin, quercetin sulphate, quercetin monoglucuronide, and isorhamnetin monoglucuronide presented a high exposure in the serum and kidney of db/db mice. Thereby, the present study provides a pharmacodynamic substance basis for better understanding the mechanism of A. manihot (L.) Medik for medication of DN.

Evaluation of the efficacy of Abelmoschus manihot (L.) on diabetic nephropathy by analyzing biomarkers in the glomeruli and proximal and distal convoluted tubules of the kidneys.

Introduction: As a traditional Chinese medicine, Abelmoschus manihot (L.) in the form of Huangkui (HK) capsule has been used as a medication for kidney diseases, including diabetic nephropathy (DN), in China. The most significant effect of HK capsule treatment in kidney diseases is the reduction of albuminuria and proteinuria. To evaluate the efficacy of HK capsule in the regression of DN, in the current study, we analyzed the biomarkers in the glomerulus and proximal and distal convoluted tubules in the kidneys of db/db mice, the animal model for type 2 diabetes and DN. Methods: Huangkui capsules (0.84 g/kg/d) or vehicle were administered daily via oral gavage for 4 weeks in db/db mice. Urinary albumin-to-creatinine ratio and blood glucose levels were measured during the whole experimental period. Five biomarkers in the glomerulus and proximal and distal convoluted tubules in the kidneys were selected, namely, col4a3, slc5a2, slc34a1, slc12a3, and slc4a1, and their activities at mRNA and protein levels before and after HK capsule treatment were analyzed by real-time RT-PCR and immunohistochemistry. Result and discussion: After HK capsule treatment for 4 weeks, the urinary albumin-to-creatinine ratio in db/db mice was found to be significantly decreased. The activities of col4a3, slc5a2, slc34a1, slc12a3, and slc4a1 in the kidneys were upregulated in db/db mice prior to the treatment but downregulated after HK capsule treatment. Further analyses of the fields of whole kidney tissue sections demonstrated that the number of nephrons in the kidneys of db/db mice with HK capsule treatment was higher than that in the kidneys of db/db mice without HK capsule treatment. Thereby, the current study provides experimental evidence confirming the medical efficacy of A. manihot in the reduction of albuminuria and proteinuria, suggesting that A. manihot may have pharmacological efficacy in the regression of the development of type 2 diabetes-DN.

Genetic and Biological Effects of SLC12A3, a Sodium-Chloride Cotransporter, in Gitelman Syndrome and Diabetic Kidney Disease.

The SLC12A3 (Solute carrier family 12 member 3) gene encodes a sodium-chloride cotransporter and mediates Na+ and Cl- reabsorption in the distal convoluted tubule of kidneys. An experimental study has previously showed that with knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage, suggesting that SLC12A3 may have genetic effects in renal disorders. Many clinical reports have demonstrated that the function-loss mutations in the SLC12A3 gene, mainly including Thr60Met, Asp486Asn, Gly741Arg, Leu859Pro, Arg861Cys, Arg913Gln, Arg928Cys and Cys994Tyr, play the pathogenic effects in Gitelman syndrome. This kidney disease is inherited as an autosomal recessive trait. In addition, several population genetic association studies have indicated that the single nucleotide variant Arg913Gln in the SLC12A3 gene is associated with diabetic kidney disease in type 2 diabetes subjects. In this review, we first summarized bioinformatics of the SLC12A3 gene and its genetic variation. We then described the different genetic and biological effects of SLC12A3 in Gitelman syndrome and diabetic kidney disease. We also discussed about further genetic and biological analyses of SLC12A3 as pharmacokinetic targets of diuretics.

Interaction between Plasma Metabolomics and Intestinal Microbiome in db/db Mouse, an Animal Model for Study of Type 2 Diabetes and Diabetic Kidney Disease.

Evidence has demonstrated that either metabolites or intestinal microbiota are involved in the pathogenesis of type 2 diabetes (T2D) and diabetic kidney disease (DKD). To explore the interaction between plasma metabolomics and intestinal microbiome in the progress of T2D-DKD, in the current study, we analyzed metabolomics in the plasma of db/db mice with liquid chromatography-mass spectrometry and also examined intestinal prokaryotes and entire gut microbiome dysbiosis at the genus level with both 16S rDNA and metagenomic sequencing techniques. We found that Negativibacillus and Rikenella were upregulated, while Akkermansia, Candidatus, Erysipelatoclostridium and Ileibacterium were downregulated in the colon of db/db mice compared with non-diabetic controls. In parallel, a total of 91 metabolites were upregulated, while 23 were downregulated in the plasma of db/db mice. The top five upregulated metabolites included D-arabinose 5-phosphate, estrone 3-sulfate, L-theanine, 3'-aenylic acid and adenosine 5'-monophosphate, and the five most significantly downregulated metabolites were aurohyocholic acid sodium salt, calcium phosphorylcholine chloride, tauro-alpha-muricholic acid sodium salt, galactinol and phosphocholine. These plasma metabolites were interacted with intestinal microbiomes, which are mainly involved in the pathways related to the biosynthesis of unsaturated fatty acids, fatty acid elongation, steroid biosynthesis, and D-arginine and D-ornithine metabolism. In the differential metabolites, N-acetyl-L-ornithine, ornithine and L-kyn could be metabolized by the correspondingly differential ontology genes in the intestinal metagenome. The current study thereby provides evidence for a gut-metabolism-kidney axis in the metabolism of db/db mice, in which the gut microbiome and circulating metabolomics interact, and suggests that information from this axis may contribute to our understanding of T2D and DKD pathogenesis.

Evaluation of Cardiovascular Toxicity of Folic Acid and 6S-5-Methyltetrahydrofolate-Calcium in Early Embryonic Development.

Folic acid (FA) is a synthetic and highly stable version of folate, while 6S-5-methyltetrahydrofolate is the predominant form of dietary folate in circulation and is used as a crystalline form of calcium salt (MTHF-Ca). The current study aims to evaluate the toxicity and safety of FA and MTHF-Ca on embryonic development, with a focus on cardiovascular defects. We began to analyze the toxicity of FA and MTHF-Ca in zebrafish from four to seventy-two hours postfertilization and assessed the efficacy of FA and MTHF-Ca in a zebrafish angiogenesis model. We then analyzed the differently expressed genes in in vitro fertilized murine blastocysts cultured with FA and MTHF-Ca. By using gene-expression profiling, we identified a novel gene in mice that encodes an essential eukaryotic translation initiation factor (Eif1ad7). We further applied the morpholino-mediated gene-knockdown approach to explore whether the FA inhibition of this gene (eif1axb in zebrafish) caused cardiac development disorders, which we confirmed with qRT-PCR. We found that FA, but not MTHF-Ca, could inhibit angiogenesis in zebrafish and result in abnormal cardiovascular development, leading to embryonic death owing to the downregulation of eif1axb. MTHF-Ca, however, had no such cardiotoxicity, unlike FA. The current study thereby provides experimental evidence that FA, rather than MTHF-Ca, has cardiovascular toxicity in early embryonic development and suggests that excessive supplementation of FA in perinatal women may be related to the potential risk of cardiovascular disorders, such as congenital heart disease.