Grain yield improvement by genome editing of TaARF12 that decoupled peduncle and rachis development trajectories via differential regulation of gibberellin signalling in wheat.

Plant breeding is constrained by trade-offs among different agronomic traits by the pleiotropic nature of many genes. Genes that contribute to two or more favourable traits with no penalty on yield are rarely reported, especially in wheat. Here, we describe the editing of a wheat auxin response factor TaARF12 by using CRISPR/Cas9 that rendered shorter plant height with larger spikes. Changes in plant architecture enhanced grain number per spike up to 14.7% with significantly higher thousand-grain weight and up to 11.1% of yield increase under field trials. Weighted Gene Co-Expression Network Analysis (WGCNA) of spatial-temporal transcriptome profiles revealed two hub genes: RhtL1, a DELLA domain-free Rht-1 paralog, which was up-regulated in peduncle, and TaNGR5, an organ size regulator that was up-regulated in rachis, in taarf12 plants. The up-regulation of RhtL1 in peduncle suggested the repression of GA signalling, whereas up-regulation of TaNGR5 in spike may promote GA response, a working model supported by differential expression patterns of GA biogenesis genes in the two tissues. Thus, TaARF12 complemented plant height reduction with larger spikes that gave higher grain yield. Manipulation of TaARF12 may represent a new strategy in trait pyramiding for yield improvement in wheat.

Matrilineal empowers wheat pollen with haploid induction potency by triggering postmitosis reactive oxygen species activity.

Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). Chemical assays detected a second wave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.

InDels Identification and Association Analysis with Spike and Awn Length in Chinese Wheat Mini-Core Collection.

Diversity surveys of germplasm are important for gaining insight into the genomic basis for crop improvement; especially InDels, which are poorly understood in hexaploid common wheat. Here, we describe a map of 89,923 InDels from exome sequencing of 262 accessions of a Chinese wheat mini-core collection. Population structure analysis, principal component analysis and selective sweep analysis between landraces and cultivars were performed. Further genome-wide association study (GWAS) identified five QTL (Quantitative Trait Loci) that were associated with spike length, two of them, on chromosomes 2B and 6A, were detected in 10 phenotypic data sets. Assisted with RNA-seq data, we identified 14 and 21 genes, respectively that expressed in spike and rachis within the two QTL regions that can be further investigated for candidate genes discovery. Moreover, InDels were found to be associated with awn length on chromosomes 5A, 6B and 4A, which overlapped with previously reported genetic loci B1 (Tipped 1), B2 (Tipped 2) and Hd (Hooded). One of the genes TaAGL6 that was previously shown to affect floral organ development was found at the B2 locus to affect awn length development. Our study shows that trait-associated InDels may contribute to wheat improvement and may be valuable molecular markers for future wheat breeding.

Receptor activator of NF-κB mediates podocyte injury in diabetic nephropathy.

Receptor activator of NF-κB (RANK) expression is increased in podocytes of patients with diabetic nephropathy. However, the relevance of RANK to diabetic nephropathy pathobiology remains unclear. Here, to evaluate the role of podocyte RANK in the development of diabetic nephropathy, we generated a mouse model of podocyte-specific RANK depletion (RANK-/-Cre T), and a model of podocyte-specific RANK overexpression (RANK TG), and induced diabetes in these mice with streptozotocin. We found that podocyte RANK depletion alleviated albuminuria, mesangial matrix expansion, and basement membrane thickening, while RANK overexpression aggravated these indices in streptozotocin-treated mice. Moreover, streptozotocin-triggered oxidative stress was increased in RANK overexpression but decreased in the RANK depleted mice. Particularly, the expression of NADPH oxidase 4, and its obligate partner, P22phox, were enhanced in RANK overexpression, but reduced in RANK depleted mice. In parallel, the transcription factor p65 was increased in the podocyte nuclei of RANK overexpressing mice but decreased in the RANK depleted mice. The relevant findings were largely replicated with high glucose-treated podocytes in vitro. Mechanistically, p65 could bind to the promoter regions of NADPH oxidase 4 and P22phox, and increased their respective gene promoter activity in podocytes, dependent on the levels of RANK. Taken together, these findings suggested that high glucose induced RANK in podocytes and caused the increase of NADPH oxidase 4 and P22phox via p65, possibly together with the cytokines TNF- α, MAC-2 and IL-1 ß, resulting in podocyte injury. Thus, we found that podocyte RANK was induced in the diabetic milieu and RANK mediated the development of diabetic nephropathy, likely by promoting glomerular oxidative stress and proinflammatory cytokine production.

Advanced glycation end-products suppress autophagic flux in podocytes by activating mammalian target of rapamycin and inhibiting nuclear translocation of transcription factor EB.

Insufficient autophagy in podocytes is related to podocyte injury in diabetic nephropathy (DN). Advanced glycation end-products (AGEs) are major factors of podocyte injury in DN. However, the role and mechanism of AGEs in autophagic dysfunction remain unknown. We investigated autophagic flux in AGE-stimulated cultured podocytes using multiple assays: western blotting, reverse transcription-quantitative PCR, immunofluorescence staining, and electron microscopy. We also utilized chloroquine and a fluorescent probe to monitor the formation and turnover of autophagosomes. Mice of the db/db strain were used to model diabetes mellitus (DM) with high levels of AGEs. To mimic DM with normal levels of AGEs as a control, we treated db/db mice with pyridoxamine to block AGE formation. AGEs impaired autophagic flux in the cultured podocytes. Compared with db/db mice with normal AGEs but high glucose levels, db/db mice with high AGEs and high glucose levels exhibited lower autophagic activity. Aberrant autophagic flux was related to hyperactive mammalian target of rapamycin (mTOR), a major suppressor of autophagy. Pharmacologic inhibition of mTOR activity restored impaired autophagy. AGEs inhibited the nuclear translocation and activity of the pro-autophagic transcription factor EB (TFEB) and thus suppressed transcription of its several autophagic target genes. Conversely, TFEB overexpression prevented AGE-induced autophagy insufficiency. Attenuating mTOR activity recovered TFEB nuclear translocation under AGE stimulation. Co-immunoprecipitation assays further demonstrated the interaction between mTOR and TFEB in AGE-stimulated podocytes and in glomeruli from db/db mice. In conclusion, AGEs play a crucial part in suppressing podocyte autophagy under DM conditions. AGEs inhibited the formation and turnover of autophagosomes in podocytes by activating mTOR and inhibiting the nuclear translocation of TFEB. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Soluble Urokinase Plasminogen Activator Receptor is Associated with Coronary Artery Calcification and Cardiovascular Disease in Patients Undergoing Hemodialysis.

BACKGROUND/AIMS: Cardiovascular disease (CVD) is an important cause of morbidity and mortality in hemodialysis patients. Vascular calcification is thought to play an important role in causing CVD. Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker strongly predictive of cardiovascular outcomes in the pathogenesis of diabetic patients with renal disease treated with hemodialysis. We investigated the relationship between suPAR and coronary artery calcification (CAC) in patients undergoing maintenance hemodialysis. METHODS: A total of 99 adult hemodialysis patients were enrolled in this study. Plasma samples were analyzed for suPAR with an enzyme-linked immunosorbent assay and the CAC score was determined with multidetector computed tomography. The occurrence of cardiovascular events and all-cause mortality during follow-up were recorded from January 1, 2010 to June 1, 2016. RESULTS: In 99 patients treated with maintenance hemodialysis, 91 (91.9%) had varying degrees of CAC, and suPAR correlated positively with the CAC score in a Spearman analysis. In a mean follow-up period of 33 months, 36 patients (36.4%) experienced at least one cardiovascular event. When the quartiles of suPAR concentrations were used as the cutoff points for a subgroup analysis, the incidence of CVD and all-cause mortality was much higher in the higher quartiles of suPAR. In a univariate Cox regression analysis, high suPAR was a risk factor for CVD and all-cause mortality. CONCLUSION: suPAR is associated with the CAC score and is a risk factor for new-onset CVD in patients undergoing hemodialysis.

RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal.

BACKGROUND: Podocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown. METHODS: Culture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes. RESULTS: We found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis. CONCLUSION: RhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases.

Expression of Cofilin-1 and Transgelin in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) has attracted much research attention around the world, and the number of ESCC cases has increased gradually in recent years. Identifying the specific biomarkers of ESCC is an effective approach for the early diagnosis of tumors. MATERIAL AND METHODS: Immunohistochemical streptavidin-peroxidase method was used to determine the expressions of Cofilin-1 and transgelin in 68 patients with esophageal squamous cell carcinoma (ESCC) and 48 individuals with normal esophageal tissues. In addition to the relationships between the expression of Cofilin-1 and transgelin, the clinicopathologic features of ESCC were also discussed. The correlation between Cofilin-1 and transgelin protein expression in ESCC was analyzed. RESULTS: (1) The positive expression rates of Cofilin-1 and transgelin were 60.3% (41/68) and 54.4% (37/68) in esophageal carcinoma tissue, respectively. The positive expression rates of Cofilin-1 and transgelin in normal esophageal tissue were 27.1% (13/48) and 29.1% (14/48), respectively. The differences were statistically significant (P<0.05). (2) The positive expression rate of Cofilin-1 did not differ significantly (P>0.05) with sex, age, ethnicity, tumor size, or infiltration depth; but did have a statistically significant (P<0.05) difference with various degrees of tumor differentiation, lymph node metastasis, and clinical stages. (3) The positive expression rate of transgelin did not differ significantly (P>0.05) with sex, age, ethnicity, tumor size, infiltration depth, and clinical stage, but did significantly (P<0.05) differ with degree of tumor differentiation and lymph node metastasis. CONCLUSIONS: Cofilin-1 and transgelin may play roles in the carcinogenesis and development of esophageal squamous cell carcinoma. Cofilin-1 may be useful as an important biomarker for indicating the degree of malignancy of esophageal squamous cell carcinoma, and the detection of transgelin is valuable in early diagnosis of esophageal squamous cell carcinoma.

[Changes of Intestinal Mucosal Barrier and Intestinal Flora in Rats with Severe Acute Pancreatitis].

This paper is to explore changes of intestinal mucosal barrier, intestinal flora, and bacterial translocation in rats with severe acute pancreatitis (SAP). Twenty four male SD rats were randomly divided into the control group (n = 10) and the experimental group (n = 14). The model of severe acute pancreatitis of rats was induced by the method of injecting adversely 5% sodium taurocholate into the common biliary-pancreatic duct. All of the rats were killed after 24 hours and the level of the serum amylase and the plasma endotoxin was determined after that. The pathological changes of pancreas and small intestine were observed through hematoxylin-eosin staining (HE staining) and the abdominal viscera bacterial translocation rates were tested. With the method of real-time polymerase chain reaction (RT-PCR) the quantity of the intestinal flora was analyzed. In the control group, the level of Escherichia coli, Lactobacillus and Bifidobacterium were 2.08 ± 1.29, 11.04 ± 7.55 and 12.21 ± 4.95, respectively. On the contrast, the level of Escherichia coli in the cecum contents was much higher (9.72 ± 3.58, P < 0.01), while the Lactobacillus number was decreased significantly (0.67 ± 0.34, P < 0.01), and the Bifidobacterium number was also decreased (4.59 ± 3.42, P < 0.05) in the experimental group, so the ratio of Bifidobacterium/Escherichia coli was reversed. Besides, in the experimental group, the plasma endotoxin positive rates and the bacterial translocation rates were much higher (P < 0.01 or P < 0.05) and the pathology scores of pancreas and small intestines were also significantly higher (P < 0.01) than those in the control group. These results indicated that in severe acute pancreatitis rats, the intestinal mucosal barrier was severely damaged and the dysbacteriosis occurs in the intestinal canal. And these might relate to the occurrence and development of multiple organ infection.

A Multi-Omics Approach for Rapid Identification of Large Genomic Lesions at the Wheat Dense Spike (wds) Locus.

Optimal spike architecture provides a favorable structure for grain development and yield improvement. However, the number of genes cloned to underlie wheat spike architecture is extremely limited. Here, we obtained a wheat dense spike mutant (wds) induced by 60Co treatment of a common wheat landrace Huangfangzhu that exhibited significantly reduced spike and grain lengths. The shortened spike length was caused by longitudinal reduction in number and length of rachis cells. We adopted a multi-omics approach to identify the genomic locus underlying the wds mutant. We performed Exome Capture Sequencing (ECS) and identified two large deletion segments, named 6BL.1 at 334.8∼424.3 Mb and 6BL.2, 579.4∼717.8 Mb in the wds mutant. RNA-seq analysis confirmed that genes located in these regions lost their RNA expression. We then found that the 6BL.2 locus was overlapping with a known spike length QTL, qSL6B.2. Totally, 499 genes were located within the deleted region and two of them were found to be positively correlated with long spike accessions but not the ones with short spike. One of them, TraesCS6B01G334600, a well-matched homolog of the rice OsBUL1 gene that works in the Brassinosteroids (BR) pathway, was identified to be involved in cell size and number regulation. Further transcriptome analysis of young spikes showed that hormone-related genes were enriched among differentially expressed genes, supporting TraesCS6B01G334600 as a candidate gene. Our work provides a strategy to rapid locate genetic loci with large genomic lesions in wheat and useful resources for future wheat study.

Wheat breeding history reveals synergistic selection of pleiotropic genomic sites for plant architecture and grain yield.

Diversity surveys of crop germplasm are important for gaining insights into the genomic basis for plant architecture and grain yield improvement, which is still poorly understood in wheat. In this study, we exome sequenced 287 wheat accessions that were collected in the past 100 years. Population genetics analysis identified that 6.7% of the wheat genome falls within the selective sweeps between landraces and cultivars, which harbors the genes known for yield improvement. These regions were asymmetrically distributed on the A and B subgenomes with regulatory genes being favorably selected. Genome-wide association study (GWAS) identified genomic loci associated with traits for yield potential, and two underlying genes, TaARF12 encoding an auxin response factor and TaDEP1 encoding the G-protein γ-subunit, were located and characterized to pleiotropically regulate both plant height and grain weight. Elite single-nucleotide haplotypes with increased allele frequency in cultivars relative to the landraces were identified and found to have accumulated over the course of breeding. Interestingly, we found that TaARF12 and TaDEP1 function in epistasis with the classical plant height Rht-1 locus, leading to propose a "Green Revolution"-based working model for historical wheat breeding. Collectively, our study identifies selection signatures that fine-tune the gibberellin pathway during modern wheat breeding and provides a wealth of genomic diversity resources for the wheat research community.

Association between plasma S-adenosylmethionine and risk of mortality in patients with coronary artery disease: A cohort study.

BACKGROUND: S-adenosylmethionine (SAM) as methyl donors participates in methylation and is converted into S-adenosylhomocysteine (SAH), which is a precursor of homocysteine. Increased plasma SAH and homocysteine are associated with increased risk of cardiovascular disease. However, the relation of plasma SAM with cardiovascular risk is still unclear. OBJECTIVES: To determine the relation between plasma SAM and risk of mortality among patients with coronary artery disease (CAD). METHODS: Baseline plasma SAM concentrations were measured in 1553 patients with CAD from the Guangdong Coronary Artery Disease Cohort between October 2008 and December 2011. Proportional hazards Cox analyses were performed to ascertain associations between SAM and risk of all-cause and cardiovascular mortality. RESULTS: After a median follow-up of 9.2 (IQR: 8.5-10.2) y, of 1553 participants, 321 had died, including 227 deaths from cardiovascular diseases. Patients in the lowest quartile of SAM concentrations had a higher risk of all-cause death (HR, 1.59; 95% CI: 1.14, 2.21) and cardiovascular death (HR, 2.14; 95% CI: 1.41, 3.27) than those in the highest quartile in multivariable adjusted analysis. Each 1-SD decrease in the SAM concentration remained associated with a 42% greater risk of total death (HR, 1.42; 95% CI: 1.23, 1.64) and a 66% higher risk of cardiovascular death (HR, 1.66; 95% CI: 1.37, 2.01) after fully adjusting for other cardiovascular risk factors. Furthermore, each 1-SD decrease in plasma SAM/SAH ratio, as the methylation index, was also inversely associated with the risk of all-cause (HR, 1.80; 95% CI: 1.42, 2.29) and cardiovascular mortality (HR, 1.68; 95% CI: 1.29, 2.19) in fully adjusted analyses. CONCLUSIONS: Our data show a significant inverse relation between plasma SAM and risk of mortality in patients with CAD after adjustment for homocysteine, SAH, and other cardiovascular disease risk factors.

Epigenetic regulation of TXNIP-mediated oxidative stress and NLRP3 inflammasome activation contributes to SAHH inhibition-aggravated diabetic nephropathy.

S-adenosylhomocysteine (SAH) is hydrolyzed by SAH hydrolase (SAHH) to homocysteine and adenosine. Increased plasma SAH levels were associated with disturbed renal function in patients with diabetes. However, the role and mechanism of SAHH in diabetic nephropathy is still unknown. In the present study, we found that inhibition of SAHH by using its inhibitor adenosine dialdehyde (ADA) accumulates intracellular or plasma SAH levels and increases high glucose-induced podocyte injury and aggravates STZ-induced diabetic nephropathy, which is associated with Nod-like receptor protein 3 (NLRP3) inflammasome activation. Inhibition or knockout of NLRP3 attenuates SAHH inhibition-aggravated podocyte injury and diabetic nephropathy. Additionally, SAHH inhibition increases thioredoxin-interacting protein (TXNIP)-mediated oxidative stress and NLRP3 inflammasome activation, but these effects were not observed in TXNIP knockout mice. Mechanistically, SAHH inhibition increased TXNIP by inhibiting histone methyltransferase enhancer of zeste homolog 2 (EZH2) and reduced trimethylation of histone H3 lysine 27 and its enrichment at promoter of early growth response 1 (EGR1). Moreover, EGR1 is activated and enriched at promoters of TXNIP by SAHH inhibition and is essential for SAHH inhibition-induced TXNIP expression. Inhibition of EGR1 protected against SAHH inhibition-induced NLRP3 inflammasome activation and oxidative stress and diabetic nephropathy. Finally, the harmful effects of SAHH inhibition on inflammation and oxidative stress and diabetic nephropathy were also observed in heterozygote SAHH knockout mice. These findings suggest that EZH2/EGR1/TXNIP/NLRP3 signaling cascade contributes to SAHH inhibition-aggravated diabetic nephropathy. Our study firstly provides a novel insight into the role and mechanism of SAHH inhibition in diabetic nephropathy.

Thromboxane A2 is involved in the development of hypertension in chronic kidney disease rats.

Hypertension is one of the most common complications of chronic kidney disease (CKD). Some research has indicated that changes in large artery function especially caused by thromboxane A2 (TXA2) may be a novel factor acting to induce hypertension in CKD. We studied the 5/6 nephrectomy rat model and measured serum levels of creatinine (Cr), calcium (Ca), phosphorus (P), TXA2-stable metabolites (thromboxane B2, TXB2), and caudal artery pressure after nephrectomy. The tension variations in thoracic aortas were measured after stimulating by vasoconstrictor/vasodilator using the cumulative concentration administration method and then tested the expression of TXA2 receptors in the thoracic aortas through western blots. The CKD rats developed uremia, electrolyte imbalances，and hypertension. They also exhibited a significant increase in TXB2 concentration. The aortic rings of CKD rats showed an increased contraction response to U46619 (a TXA2 analogue) and the expression of TXA2 receptors also enhanced. In the meanwhile, the diastolic function decreased in the CKD group. Our results demonstrate that the impairment of artery contractile function caused by the increase of TXA2 receptors on the wall of aortic rings may be involved in hypertension in CKD rats.

[Protective effect of low-dose hydrocortisone on myocardium in early septic shock].

OBJECTIVE: To investigate the effects and mechanisms of low-dose hydrocortisone on myocardial injury in early septic shock rats. METHODS: Seventy-two healthy male Sprague-Dawley (SD) rats were divided into Sham group, lipopolysaccharide (LPS) model group (LPS group) and low dose hydrocortisone intervention group (LD group) according to the random number table method, with 24 rats in each group. The rat model of septic shock was produced by intravenous injection of LPS at 20 mg/kg. Sham group was injected with an equal amount of physiological saline. The LD group was injected 5 mg/kg of hydrocortisone via right femoral vein after model establishment. Sham group and LPS group were injected with an equal amount of physiological saline. Blood pressure and heart rate (HR) of rats in each group were continuously monitored. In each group, 8 rats were sacrificed for arterial blood gas analysis at 0, 3 and 6 hours after model establishment, and the level of plasma N-terminal B-type brain natriuretic peptide precursor (NT-proBNP) was detected by enzyme linked immunosorbent assay (ELISA). The expressions of cleaved-caspase-3 and nuclear factor-κB p65 (NF-κB p65) were detected by Western Blot. Myocardial tissue was harvested 6 hours after model establishment, the histopathological changes were observed by hematoxylin eosin (HE) staining, and the apoptosis rate of myocardial cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL). RESULTS: After LPS injection, mean arterial pressure (MAP) decreased significantly at 1 hour, then gradually increased, and was significantly higher than Sham group at 6 hours. There was no significant change in HR, and the difference was not statistically significant compared with Sham group. Blood lactic acid (Lac), base excess (BE), plasma NT-proBNP level, myocardial tissue caspase-3 and NF-κB p65 expression increased with the extension of time, all reach the peak in 6 hours, and significantly higher than Sham group. After early treatment with low-dose hydrocortisone in septic shock, MAP showed an increasing trend and Lac, BE decreased slowly. At 6 hours, MAP, Lac and BE were significantly lower than those in the LPS group [MAP (mmHg, 1 mmHg = 0.133 kPa): 98.6±7.5 vs. 106.1±8.5, Lac (mmol/L): 1.29±0.08 vs. 2.42±0.37, BE (mmol/L): 4.45±0.57 vs. 8.18±1.03, all P < 0.05]. The level of plasma NT-proBNP, and the expressions of caspase-3 and NF-κB p65 in myocardial tissue were significantly lower than those in LPS group at 3 hours and 6 hours after low-dose hydrocortisol treatment [NT-proBNP (ng/L): 2 740.56±97.31 vs. 4 425.60±743.32 at 3 hours, 2 638.81±205.12 vs. 4 993.01±373.78 at 6 hours; caspase-3/GAPDH: 0.567±0.045 vs. 0.841±0.162 at 3 hours, 0.496±0.071 vs. 1.116±0.172 at 6 hours; NF-κB p65/GAPDH: 0.852±0.734 vs. 1.232±0.115 at 3 hours, 0.783±0.047 vs. 1.383±0.215 at 6 hours, all P < 0.05]. HE staining results showed that myocardial cells in the LPS group were broken and inflammatory cells infiltrated. The myocardial histopathological changes in LD group were significantly less than those in LPS group. TUNEL staining showed that the apoptosis of myocardial cells in LPS group increased, and the apoptosis rate was significantly higher than that in Sham group [(82.41±1.57)% vs. (5.77±0.69)%, P < 0.05]. The apoptosis rate in LD group was significantly lower than that in LPS group [(27.82±1.77)% vs. (82.41±1.57)%, P < 0.05]. CONCLUSIONS: Low-dose hydrocortisone plays a protective role in the myocardial injury of early septic shock, and its mechanism may be related to the inhibition of caspase-3 and NF-κB p65 expression, the reduction of apoptosis rate and myocardial inhibition.

Protein A immunoadsorption combination with immunosuppressive therapy improves neuropsychiatric systemic lupus erythematosus: A case report.

We described protein A immunoadsorption combination with immunosuppressive treatment improved rapidly a patient with Neuropsychiatric systemic lupus erythematosus.

Amino acid starvation promotes podocyte autophagy through mammalian target of rapamycin inhibition and transcription factor EB activation.

Autophagy is important for maintaining normal physiological functions and podocyte cell homeostasis. Amino acid signaling is an important upstream signaling pathway for autophagy regulation. However, the function and the associated mechanism of amino acid signaling in podocyte autophagy is unclear. The present study used normal culture medium and amino acid deprivation medium to culture podocytes in vitro. Multiple methods were utilized to detect autophagic activity including western blot analysis to measure the levels of microtubule­associated protein 1 light chain 3 (LC3) II and beclin1, reverse transcription­quantitative polymerase chain reaction was performed to evaluate the levels of LC3 mRNA and transmission electron microscopy was conducted to observe autophagosomes. In addition, tandem green fluorescent protein (GFP)­monomeric red fluorescent protein (mRFP)­LC3 adenoviruses were employed to transduce podocytes to observe autophagic flux. Furthermore, the present study examined the effects of amino acid signaling on mammalian target of rapamycin (mTOR) activity and the nuclear translocation of transcription factor EB (TFEB), a core regulator of autophagy, using western blotting and immunofluorescence. The results revealed that amino acid starvation promoted the expression of LC3II and beclin1, and increased the number of autophagosomes and autolysosomes. Amino acid starvation inhibited mTOR activity, and promoted nuclear translocation and TFEB activity. Inhibition of TFEB blocked amino acid starvation­induced autophagy. These results indicated that amino acid starvation stimulated podocyte autophagy, and thus suggested that mTOR suppression and TFEB activation may mediate amino acid starvation­induced autophagy in podocytes.

WWC1 promotes podocyte survival via stabilizing slit diaphragm protein dendrin.

Previous studies have indicated that glomerular podocyte injury serves a crucial role in proteinuria during the process of chronic kidney disease. The slit diaphragm of podocytes forms the final barrier to proteinuria. Dendrin, a constituent of the slit diaphragm protein complex, has been observed to relocate from the slit diaphragm to the nuclei in injured podocytes and promote podocyte apoptosis. However, the exact mechanism for nuclear relocation of dendrin remains unclear. The expression of WWC1 in podocyte injury induced by lipopolysaccharides (LPS) or adriamycin (ADR) was detected by reverse transcription­quantitative polymerase chain reaction (RT­qPCR), western blotting and the immunofluorescence assay. The role of WWC1 in podocyte apoptosis was detected by knockdown of WWC1 and flow cytometry. The mRNA and protein expression levels of apoptosis­associated genes Bcl­2­associated X (Bax) and Bcl­2 were measured by RT­qPCR and western blotting. The impact of WWC1 on dendrin nucleus relocation in vitro in podocytes was further evaluated by knockdown of WWC1. Expression of WWC1 significantly decreased in injured podocytes in vitro. The loss­of­function assay indicated that knockdown of WWC1 gene in vitro promoted podocyte apoptosis, accompanied with increased levels of the pro­apoptotic protein Bax and decreased levels of the anti­apoptotic protein Bcl­2. Furthermore, the relocation of dendrin protein was significantly promoted by knockdown of the WWC1 gene. In conclusion, the study indicated that loss of WWC1 may contribute to podocyte apoptosis by inducing nuclear relocation of dendrin protein, which provided novel insight into the molecular events in podocyte apoptosis.

Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis.

OBJECTIVE: Several studies have reported that tacrolimus (TAC) significantly reduced proteinuria in lupus nephritis (LN) patients and mouse models. However, the mechanism for this effect remains undetermined. This study explored the mechanism of how TAC protects podocytes from injury to identify new targets for protecting renal function. METHODS: MRL/lpr mice were given TAC at a dosage of 0.1 mg/kg per day by intragastric administration for 8 weeks. Urine and blood samples were collected. Kidney sections (2 µm) were stained with hematoxylin-eosin (HE), periodic acid-Schiff base (PAS) and Masson's trichrome stain. Mouse podocyte cells (MPC5) were treated with TAC and/or TGF-ß1 for 48 h. The mRNA levels and protein expression of synaptopodin and Wilms' tumor 1 (WT1) were determined by real-time PCR, Western blotting and/or immunofluorescence, respectively. Flow cytometry was used to detect cell apoptosis with annexin V. Podocyte foot processes were observed under transmission electron microscopy. IgG and C3 deposition were assessed with immunofluorescence assays and confocal microscopy. RESULTS: Synaptopodin expression significantly decreased in MRL/lpr disease control mice, accompanied by increases in 24-h proteinuria, blood urea nitrogen, and serum creatinine. TAC, however, reduced proteinuria, improved renal function, attenuated renal pathology, restored synaptopodin expression and preserved podocyte numbers. In MPC5 cells, TGF-ß1 enhanced F-actin damage in podocytes and TAC stabilized it. TAC also decreased TGF-ß1-induced podocyte apoptosis in vitro and inhibited foot process fusion in MRL/lpr mice. In addition, our results also showed TAC inhibited glomerular deposition of IgG and C3. CONCLUSION: This study demonstrated that TAC reduced proteinuria and preserved renal function in LN through protecting podocytes from injury partly by stabilizing podocyte actin cytoskeleton and inhibiting podocyte apoptosis.