Heterogeneous effects of hospital competition on inpatient quality: an analysis of five common diseases in China.

BACKGROUND: Many countries has introduced pro-competition policies in the delivery of healthcare to improve medical quality, including China. With the increasing intensity of competition in China's healthcare market, there are rising concerns among policymakers about the impact of hospital competition on quality. This study investigated heterogeneous effects of hospital competition on inpatient quality. METHODS: We analyzed the inpatient discharge dataset and selected chronic obstructive pulmonary disease (COPD), ischemic stroke, pneumonia, hemorrhagic stroke, and acute myocardial infarction (AMI) as representative diseases. A total of 561,429 patients in Sichuan Province in 2017 and 2019 were included. The outcomes of interest were in-hospital mortality and 30-day unplanned readmissions. The Herfindahl-Hirschman Index was calculated using predicted patient flows to measure hospital competition. To address the spatial correlations of hospitals and the structure of the dataset, the multiple membership multiple classification model was employed for analysis. RESULTS: Amid intensifying competition in the hospital market, our study discerned no marked statistical variance in the risk of inpatient quality across most diseases examined. Amplified competition exhibited a positive correlation with heightened in-hospital mortality for both COPD and pneumonia patients. Elevated competition escalated the risk of 30-day unplanned readmissions for COPD patients, while inversely affecting the risk for AMI patients. CONCLUSIONS: There is the heterogeneous impact of hospital competition on quality across various diseases in China. Policymakers who intend to leverage hospital competition as a tool to enhance healthcare quality must be cognizant of the possible influences of it.

Ganshuang granule plays a pharmacological role in anti-alcoholic and anti-hangover via regulating alcohol metabolism and affecting neurotransmitters.

BACKGROUND: To explore the effect of Ganshuang granule on anti-alcoholic and anti-hangover and its potential mechanism. METHODS: SPF SD rats' drunken model and SPF Kunming mice's hangover model were used as models. RESULTS: Ganshuang granule could significantly reduce sleep time, the time to climb in mice, and significantly prolong the tolerance time and shorten sleep time in rats (p < 0.05). The blood ethanol concentration of rats in each administration group was lower than that in the model group at each time point (p < 0.05). Compared with the control group, the activities of ADH and ALDH in the liver of the model group were significantly decreased (p < 0.05); the content of DA and 5-HT in the striatum of the model group was significantly increased (p < 0.05); and the activity of AchE in the hippocampus was significantly decreased (p < 0.05). The above processes could be improved and regulated in the drug administration group. Compared with the control group, there was no significant difference between ADH and ALDH in the serum of the model group (p > 0.05). However, the activities of ADH and ALDH in the liver of drunk rats could be upregulated by Ganshuang granule (p < 0.05). CONCLUSION: Ganshuang granule has the pharmacological effects of anti-alcoholic and anti-hangover, which is related to regulating the activities of ADH and ALDH in the liver, the contents of DA and 5-HT in striatum, and the activity of AchE in the hippocampus.

The Glomerulus Multiomics Analysis Provides Deeper Insights into Diabetic Nephropathy.

Although diabetic nephropathy (DN) is the leading cause of the end-stage renal disease, the exact regulation mechanisms remain unknown. In this study, we integrated the transcriptomics and proteomics profiles of glomeruli isolated from 50 biopsy-proven DN patients and 25 controls to investigate the latest findings about DN pathogenesis. First, 1152 genes exhibited differential expression at the mRNA or protein level, and 364 showed significant association. These strong correlated genes were divided into four different functional modules. Moreover, a regulatory network of the transcription factors (TFs)-target genes (TGs) was constructed, with 30 TFs upregulated at the protein levels and 265 downstream TGs differentially expressed at the mRNA levels. These TFs are the integration centers of several signal transduction pathways and have tremendous therapeutic potential for regulating the aberrant production of TGs and the pathological process of DN. Furthermore, 29 new DN-specific splice-junction peptides were discovered with high confidence; these peptides may play novel functions in the pathological course of DN. So, our in-depth integrative transcriptomics-proteomics analysis provided deeper insights into the pathogenesis of DN and opened the potential avenue for finding new therapeutic interventions. MS raw files were deposited into the proteomeXchange with the dataset identifier PXD040617.

[Chrysin alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis in rats].

The purpose of this study is to investigate whether chrysin reduces cerebral ischemia-reperfusion injury(CIRI) by inhi-biting ferroptosis in rats. Male SD rats were randomly divided into a sham group, a model group, high-, medium-, and low-dose chrysin groups(200, 100, and 50 mg·kg~(-1)), and a positive drug group(Ginaton, 21.6 mg·kg~(-1)). The CIRI model was induced in rats by transient middle cerebral artery occlusion(tMCAO). The indexes were evaluated and the samples were taken 24 h after the operation. The neurological deficit score was used to detect neurological function. The 2,3,5-triphenyl tetrazolium chloride(TTC) staining was used to detect the cerebral infarction area. Hematoxylin-eosin(HE) staining and Nissl staining were used to observe the morphological structure of brain tissues. Prussian blue staining was used to observe the iron accumulation in the brain. Total iron, lipid pero-xide, and malondialdehyde in serum and brain tissues were detected by biochemical reagents. Real-time quantitative polymerase chain reaction(RT-qPCR), immunohistochemistry, and Western blot were used to detect mRNA and protein expression of solute carrier fa-mily 7 member 11(SLC7A11), transferrin receptor 1(TFR1), glutathione peroxidase 4(GPX4), acyl-CoA synthetase long chain family member 4(ACSL4), and prostaglandin-endoperoxide synthase 2(PTGS2) in brain tissues. Compared with the model group, the groups with drug intervention showed restored neurological function, decreased cerebral infarction rate, and alleviated pathological changes. The low-dose chrysin group was selected as the optimal dosing group. Compared with the model group, the chrysin groups showed reduced content of total iron, lipid peroxide, and malondialdehyde in brain tissues and serum, increased mRNA and protein expression levels of SLC7A11 and GPX4, and decreased mRNA and protein expression levels of TFR1, PTGS2, and ACSL4. Chrysin may regulate iron metabolism via regulating the related targets of ferroptosis and inhibit neuronal ferroptosis induced by CIRI.

A potential link between fibroblast growth factor-23 and the progression of AKI to CKD.

BACKGROUND: Patients who recover from acute kidney injury (AKI) have a 25% increase in the risk of chronic kidney disease (CKD) and a 50% increase in mortality after a follow-up of approximately 10 years. Circulating FGF-23 increases significantly early in the development of AKI, is significantly elevated in patients with CKD and has become a major biomarker of poor clinical prognosis in CKD. However, the potential link between fibroblast growth factor-23 levels and the progression of AKI to CKD remains unclear. METHOD: Serum FGF-23 levels in AKI patients and ischaemia‒reperfusion injury (IRI) mice were detected with ELISA. Cultured HK2 cells were incubated with FGF-23 and PD173074, a blocker of FGFR, and then TGFß/Smad and Wnt/ß-catenin were examined with immunofluorescence and immunoblotting. Quantitative real-time polymerase chain reaction was used to detect the expression of COL1A1 and COL4A1. Histologic staining confirmed renal fibrosis. RESULTS: The level of serum FGF-23 was significantly different between AKI patients and healthy controls (P < 0.01). Moreover, serum FGF-23 levels in the CKD progression group were significantly higher than those in the non-CKD progression group of AKI patients (P < 0.01). In the AKI-CKD mouse model, serum FGF-23 levels were increased, and renal fibrosis occurred; moreover, the protein expression of ß-catenin and p-Smad3 was upregulated. PD173074 downregulated the expression of ß-catenin and p-Smad3 and reduced fibrosis in both mice and HK2 cells. CONCLUSION: The increase in FGF-23 may be associated with the progression of AKI to CKD and may mediate renal fibrosis via TGF-ß and Wnt/ß-catenin activation.

Chrysin protects against cerebral ischemia-reperfusion injury in hippocampus via restraining oxidative stress and transition elements.

Chrysin is a natural flavonoid compound that has antioxidant and neuroprotective effects. Cerebral ischemia reperfusion (CIR) is closely connected with increased oxidative stress in the hippocampal CA1 region and homeostasis disorder of transition elements such as iron (Fe), copper (Cu) and zinc (Zn). This exploration was conducted to elucidate the antioxidant and neuroprotective effects of chrysin based on transient middle cerebral artery occlusion (tMCAO) in rats. Experimentally, sham group, model group, chrysin (50.0 mg/kg) group, Ginaton (21.6 mg/kg) group, Dimethyloxallyl Glycine (DMOG, 20.0 mg/kg) + chrysin group and DMOG group were devised. The rats in each group were performed to behavioral evaluation, histological staining, biochemical kit detection, and molecular biological detection. The results indicated that chrysin restrained oxidative stress and the rise of transition element levels, and regulated transition element transporter levels in tMCAO rats. DMOG activated hypoxia-inducible factor-1 subunit alpha (HIF-1α), reversed the antioxidant and neuroprotective effects of chrysin, and increased transition element levels. In a word, our findings emphasize that chrysin plays a critical role in protecting CIR injury via inhibiting HIF-1α against enhancive oxidative stress and raised transition metal levels.

Transcriptomics-proteomics Integration reveals alternative polyadenylation driving inflammation-related protein translation in patients with diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a complex disease involving the upregulation of many inflammation-related proteins. Alternative polyadenylation (APA), a crucial post-transcriptional regulatory mechanism, has been proven to play vital roles in many inflammatory diseases. However, it is largely unknown whether and how APA exerts function in DN. METHODS: We performed transcriptomics and proteomics analysis of glomeruli samples isolated from 50 biopsy-proven DN patients and 25 control subjects. DaPars and QAPA algorithms were adopted to identify APA events from RNA-seq data. The qRT-PCR analysis was conducted to verify 3'UTR length alteration. Short and long 3'UTRs isoforms were also overexpressed in podocytes under hyperglycemia condition for examining protein expression. RESULTS: We detected transcriptome-wide 3'UTR APA events in DN, and found that APA-mediated 3'UTR lengthening of genes (APA genes) increased their expression at protein but not mRNA level. Increased protein level of 3'UTR lengthening gene was validated in podocytes under hyperglycemia condition. Pathway enrichment analysis showed that APA genes were enriched in inflammation-related biological processes including endoplasmic reticulum stress pathways, NF-κB signaling and autophagy. Further bioinformatics analysis demonstrated that 3'UTR APA of genes probably altered the binding sites for RNA-binding proteins, thus enhancing protein translation. CONCLUSION: This study revealed for the first time that 3'UTR lengthening of APA genes contributed to the progression of DN by elevating the translation of corresponding proteins, providing new insight and a rich resource for investigating DN mechanisms.

[Exploration of mechanism of "simultaneous treatment of brain and heart" of Naoxintong Capsules based on Toll-like receptor signaling pathway].

This study aims to explore the mechanism of "simultaneous treatment of the brain and the heart" of Naoxintong Capsules(NXT) under cerebral ischemia based on Toll-like receptor(TLR) signaling pathway.Male SD rats were randomized into sham operation group, model group, NXT group, and positive drug group.Middle cerebral artery occlusion(MCAO) model rats were used in model group, NXT group, and positive drug group, respectively.Neurological function was scored with the Bederson scale, and brain infarct rate was measured by 2,3,5-triphenyltetrazolium chloride(TTC) staining.Brain edema was detected with wet-dry weight method.Hematoxylin-eosin(HE) staining and TdT-mediated dUTP nick-end labeling(TUNEL) staining were used to observe and count apoptotic cardiocytes.In addition, serum myocardial enzymes were measured.The expression of 8 TLR signaling pathway-related proteins interferon-α(IFN-α), interferon regulatory factor-3(IRF3), interferon regulatory factor-7(IRF7), TLR2, TLR4, TLR7, TLR9, and tumor necrosis factor-α(TNF-α) in the cerebral cortex and heart of rats was detected by Western blot. Brain infarct rate, neurological function score, and brain water content in NXT group decreased significantly compared with those in the model group. At the same time, the reduction in apoptosis rate of cardiocytes and the content of serum aspartate aminotransferase(AST), alanine aminotransferase(ALT), creatine kinase(CK), and lactate dehydrogenase(LDH) were decreased in the NXT group.Systems pharmacological results and previous research showed that TLR signaling pathway played an important role in immune inflammatory response.The study of TLR signaling pathway and related proteins is helpful to elucidate the mechanism of "simultaneous treatment of the brain and the heart". Western blot results showed that NXT significantly inhibited the expression of IRF3, IRF7, TLR2, TLR7, and TNF-α in cerebral cortex and heart under cerebral ischemia.Cerebral ischemia influences cardiac functions, and TLR signaling pathway is one of the pathways for "simultaneous treatment of the brain and the heart" of NXT.

Systems pharmacology, proteomics and in vivo studies identification of mechanisms of cerebral ischemia injury amelioration by Huanglian Jiedu Decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Huanglian Jiedu Decoction (HLJDD) has the effect of clearing heat and detoxifying, and has been considered as an effective prescription for cerebral ischemia (CI) for thousands of years in traditional Chinese medicine (TCM). It can improve the quality of life of patients with ischemic stroke, but its pharmacological mechanism remains unclear. AIM OF THE STUDY: The study aimed to explore the pharmacological action and potential mechanism of HLJDD against CI by systems pharmacology, proteomics and in vivo experiments. MATERIALS AND METHODS: In this study, databases such as TCMIP V2.0 and Genecards were used to predict compounds, targets and CI related targets, and network topology criteria of protein-protein interaction (PPI) network was used to screen core targets. The Database for Annotation, Visualization and Integrated Discovery database (DAVID) was used to discover biological processes and pathways. In addition, molecular docking was performed between the screened core biological active compounds and targets to verify the binding activity. Finally, proteomics and Western blot were performed on cerebral cortex tissues of middle cerebral artery occlusion (MCAO) model rats with HLJDD intervention to further verify the predicted results. RESULTS: 77 compounds and 308 targets of HLJDD were identified, 54 of which were predicted to be associated with cerebral ischemia. PPI network and enrichment results showed that 8 targets, including AKT1, PTGS2 and TLR4, were core targets of HLJDD in CI. And 19 signaling pathways, including Rap1 signaling pathway, cAMP signaling pathway and arachidonic acid metabolism, were identified as key pathways to the therapeutic activity of HLJDD in CI. Combined with proteomics studies, we identified that Rap1 signaling pathway and upstream and downstream targets were the key mechanisms. Molecular biology experiments showed that RAP1A and AKT expression levels were significantly up-regulated in middle cerebral artery occlusion (MCAO) rats treated with HLJDD (P < 0.0001), GRIN1 expression level was significantly down-regulated (P < 0.0001). However, ACTB expression level was slightly down-regulated (P > 0.05), which may be related to the biological function. CONCLUSION: This study confirms the pharmacological effect of HLJDD on cerebral ischemia. These results indicate that HLJDD mediates various pathways such as inhibition of apoptosis, regulation of oxygen balance, inhibition of excitatory toxicity and maintenance of basic cell functions to improve CI by regulating Rap1 signaling pathway.

Quality Control of DNA Extracted from All-Cell Pellets After Cryopreservation for More Than 10 Years.

Background: Cryopreserved whole blood, all-cell pellets (ACPs), and buffy coats in biobanks are widely used to obtain DNA for genetic testing. However, there are few studies concerning the quality control of DNA extracted from them. Our research aimed to perform quality control of DNA extracted from ACPs after cryopreservation for >10 years. Materials and Methods: A total of 1377 ACP samples (separated from 3 mL of whole blood) were retrieved from our biobank, where they had been cryopreserved for 10-15 years. Chemagic STAR was used to extract the DNA. Absorbance at A260, A280, and A230 were measured by spectrophotometry, and integrity was analyzed by agarose gel electrophoresis. The quality thresholds for an Illumina Asian Screening Array (ASA) were yields greater than 0.5 µg, concentration of 25-150 ng/µL, A260/280 ratio of 1.6-2.1, and no degradation fragments in the electrophoresis gel. Results: The median yield of genomic DNA was 54.30 µg (interquartile range [IQR] 35.55-74.64). The median A260/280 and A260/230 ratios were 1.90 (IQR 1.87-1.94) and 1.98 (IQR 1.64-2.41), respectively. In total, 1377 samples (100%) had qualified yields, and 1366 samples (99.20%) had qualified integrity results. Finally, 1328 (96.44%) samples were used for ASA. Of the remaining samples, 34 needed to be repurified, 4 were obtained at an insufficient concentration, and 11 were unqualified for integrity. In addition, we analyzed the influence of hemolysis (90 samples) and clots (102 samples) on the quality of DNA samples. Hemolysis and clotting did not influence yield or integrity, but a significant difference was found in A260/230 compared to normal samples (p < 0.05). Furthermore, the samples (14 samples) with both hemolysis and clots had higher A260/280 (p < 0.05). Conclusion: ACP samples stored for >10 years at -80°C produced DNA with high quality for use in genetic analysis. Hemolysis and clots in the ACPs led to lower purity, but did not significantly affect yield or integrity.

[Diagnosis of a case of autosomal recessive polycystic kidney disease with combined prenatal imaging and genetic testing].

OBJECTIVE: To explore the genetic basis for a fetus with renal abnormalities through whole exome sequencing and imaging examination. METHODS: Clinical data and result of medical imaging of the fetus was collected. Amniotic fluid sample was collected for the extraction of fetal DNA. Whole exome sequencing was carried out. Candidate variants were verified by Sanger sequencing. RESULTS: Prenatal ultrasonography showed that the fetus had bilateral enlargement of the kidneys with hyperechogenicity and diffuse renal cysts. Whole exome sequencing revealed that the fetus carried compound heterozygous variants of the PKHD1 gene, namely c.5137G>T and c.2335_2336delCA, which were derived from its mother and father, respectively. CONCLUSION: The fetus was diagnosed with autosomal recessive polycystic kidney disease through combined prenatal ultrasonography and whole exome sequencing. The compound heterozygous variants of the PKHD1 gene probably underlay the pathogenesis in the fetus. The results have enabled prenatal diagnosis and genetic counseling for its parents.

MicroRNA-30 regulates left ventricular hypertrophy in chronic kidney disease.

Left ventricular hypertrophy (LVH) is a primary feature of cardiovascular complications in patients with chronic kidney disease (CKD). miRNA-30 is an important posttranscriptional regulator of LVH, but it is unknown whether miRNA-30 participates in the process of CKD-induced LVH. In the present study, we found that CKD not only resulted in LVH but also suppressed miRNA-30 expression in the myocardium. Rescue of cardiomyocyte-specific miRNA-30 attenuated LVH in CKD rats without altering CKD progression. Importantly, in vivo and in vitro knockdown of miRNA-30 in cardiomyocytes led to cardiomyocyte hypertrophy by upregulating the calcineurin signaling directly. Furthermore, CKD-related detrimental factors, such as fibroblast growth factor-23, uremic toxin, angiotensin II, and transforming growth factor-ß, suppressed cardiac miRNA-30 expression, while miRNA-30 supplementation blunted cardiomyocyte hypertrophy induced by such factors. These results uncover a potentially novel mechanism of CKD-induced LVH and provide a potential therapeutic target for CKD patients with LVH.

Efficacy and safety of laparoscopic pyelolithotomy versus percutaneous nephrolithotomy for treatment of large renal stones: a meta-analysis.

BACKGROUND: We aimed to compare the efficacy and safety of laparoscopic pyelolithotomy (LPL) versus percutaneous nephrolithotomy (PCNL) for treating renal stones larger than 2 cm. METHODS: We searched the PubMed, Embase, Web of Science, SinoMed, and Chinese National Knowledge Infrastructure databases for studies that compared the surgical outcomes of LPL and PCNL. We conducted a meta-analysis of the retrieved studies, expressed as weighted mean difference or risk ratios with 95% confidence intervals. RESULTS: We included 25 studies (1831 patients). LPL was associated with a significantly higher stone-free rate, lower rates of blood loss, complementary treatment, blood transfusion, and complications, and less reduction in hemoglobin level compared with PCNL. LPL and PCNL were similar in terms of duration of hospital stay, conversion rate, changes in glomerular filtration rate and creatinine level, and mean time of postoperative analgesia. However, LPL was associated with a longer operation time than PCNL. CONCLUSION: LPL appears to be more effective and safer than PCNL in patients with large renal stones, by increasing the stone-free rate and reducing blood loss, complementary treatment, blood transfusion, and complications compared with PCNL. LPL may thus be a useful modality for treating patients with large renal stones.

Ensemble-Based Assimilation of Satellite All-Sky Microwave Radiances Improves Intensity and Rainfall Predictions for Hurricane Harvey (2017).

Ensemble-based data assimilation of radar observations across inner-core regions of tropical cyclones (TCs) in tandem with satellite all-sky infrared (IR) radiances across the TC domain improves TC track and intensity forecasts. This study further investigates potential enhancements in TC track, intensity, and rainfall forecasts via assimilation of all-sky microwave (MW) radiances using Hurricane Harvey (2017) as an example. Assimilating Global Precipitation Measurement constellation all-sky MW radiances in addition to GOES-16 all-sky IR radiances reduces the forecast errors in the TC track, rapid intensification (RI), and peak intensity compared to assimilating all-sky IR radiances alone, including a 24-hr increase in forecast lead-time for RI. Assimilating all-sky MW radiances also improves Harvey's hydrometeor fields, which leads to improved forecasts of rainfall after Harvey's landfall. This study indicates that avenues exist for producing more accurate forecasts for TCs using available yet underutilized data, leading to better warnings of and preparedness for TC-associated hazards in the future.

The Effects of Storage Time and Repeated Freeze-Thaw Cycles on Intact Fibroblast Growth Factor 23 Levels.

Background: Fibroblast growth factor 23 (FGF23) has become increasingly important in chronic kidney diseases (CKDs), cardiovascular calcification, and metabolic bone diseases. Fresh or stored blood samples are widely used for the FGF23 assay. Clarifying the factors influencing the FGF23 assay can help to quantify FGF23 more accurately. This study explored the effects of low-temperature storage time and repeated freeze-thaw cycles on the measurement of serum intact FGF23 (iFGF23). Materials and Methods: We selected 60 serum samples from patients with CKD stages 3-5 and hemodialysis patients. An enzyme-linked immunosorbent assay was used to measure the changes in serum iFGF23 levels after 6 years of storage at -80°C. In total, 18 fresh serum samples were frozen and thawed for 0, 1, 3, and 5 cycles to explore the effects of repeated freeze-thaw cycles on serum iFGF23 levels. Results: Median serum iFGF23 concentrations were 252.17 (interquartile range [IQR] 113.82-592.38) pg/mL and 203.85 (IQR 64.76-545.39) pg/mL before and after 6 years. There were no significant differences between them. However, we found a downward trend of 48% in the samples close to the normal level of iFGF23 (<150.34 pg/mL) after 6 years of storage (p = 0.160). In addition, the iFGF23 levels of samples frozen and thawed for 0, 1, 3, and 5 cycles were 278.41 ± 39.51 (mean ± standard deviation) pg/mL, 262.84 ± 38.42 pg/mL, 252.97 ± 34.65 pg/mL and 250.49 ± 37.12 pg/mL, respectively. A slight downward trend in iFGF23 levels was observed with increasing freeze-thaw times; however, no significant differences were found among different freeze-thaw cycles. Conclusion: Serum iFGF23 levels remained stable after storage at -80°C for 6 years. In addition, five freeze-thaw cycles had no significant effects on serum iFGF23 levels.

Quality Control of RNA Extracted from PAXgene Blood RNA Tubes After Different Storage Periods.

Background: The PAXgene Blood RNA tube is used to protect RNA of whole blood, and the stability of RNA in this tube has already been reported. However, there are few reports on the quality of RNA from long-term preservation of these tubes. Materials and Methods: Our biobank conducted quality control of RNA extracted from the tubes after varying storage periods. A total of 300 blood samples of renal disease patients were randomly selected at each time point (from 1 month to 7 years). Results: Median RNA yields were 3.46 (2.65-4.87) µg, 4.34 (3.20-5.87) µg, 4.77 (2.88-6.29) µg, 4.19 (2.65-6.26) µg, 3.85 (2.43-6.13) µg, and 3.21 (1.85-6.61) µg at 1 month, 1, 2, 3, 6, and 7 years, respectively. There were no significant differences in RNA yields among all the storage periods. A260/280 ratios were 2.02 ± 0.04, 2.05 ± 0.04, 2.05 ± 0.04, 2.08 ± 0.03, 2.12 ± 0.10, and 2.11 ± 0.05, all of which were ≥1.8. However, A260/280 of the samples stored for 6 and 7 years had a rising trend, compared with the other time points (p < 0.05). Median RNA integrity number (RIN) values were 8.4 (7.6-9.1), 8.3 (7.7-8.9), 8.3 (7.8-8.8), 8.5 (8.3-8.9), 7.6 (7.1-8.1), and 7.9 (7.2-8.3) at each time point. Lower RIN values were found at 6 and 7 years compared with the other storage periods (p < 0.05). The rates of RIN values ≥7.0 were 92%, 84%, 96%, 92%, 86%, and 84%, which exhibited no differences across all the storage periods. In addition, the yields and RIN values of RNA from samples with blood clots were significantly lower than those without (p < 0.001). Conclusions: The quantity and quality of RNA extracted from PAXgene Blood RNA tubes are stable throughout cryopreservation for 7 years.

MiR-30 family prevents uPAR-ITGB3 signaling activation through calcineurin-NFATC pathway to protect podocytes.

Urokinase plasminogen activator receptor (uPAR) is upregulated in podocytes of glomerular diseases and crucially mediates podocyte injury through integrin ß3 (ITGB3). We previously showed that the miR-30 family maintains podocyte structure and function by inhibiting injurious calcineurin signaling through nuclear factor of activated T cells C (NFATC). Here, we tested whether the miR-30-calcineurin-NFATC and uPAR-ITGB3 pathways, two of the major pathways leading to podocyte injury, could interact. We found that podocyte-specific miR-30 knockdown in mice induced uPAR upregulation and ITGB3 activation, accompanied by proteinuria and podocyte injury. These effects of miR-30 knockdown were reduced using inhibitors of ITGB3, calcineurin, and NFATC, respectively, which are known to be antiproteinuric. These results indicate that miR-30 deficiency leads to calcineurin-NFATC signaling activation, which in turn activates the uPAR-ITGB3 pathway. In cultured podocytes, miR-30 knockdown also activated uPAR-ITGB3 signaling, leading to Rho GTPase activation, synaptopodin downregulation and podocyte injury. To explore uPAR-ITGB3 signaling regulation by miR-30 in podocytopathy development, we treated mice with lipopolysaccharide (LPS) and found that miR-30 was downregulated in podocytes, accompanied by uPAR upregulation and ITGB3 activation. We obtained the same results in cultured podocytes treated with LPS. Podocyte-specific transgenic miR-30 abolished uPAR-ITGB3 signaling and ameliorated podocyte injury and proteinuria in mice. Taken together, these experiments show that uPAR-ITGB3 signaling is negatively regulated by miR-30 through calcineurin-NFATC pathway, a novel mechanism underlying podocyte injury in glomerular diseases. Our study has elucidated the relationship among the crucial players governing podocyte pathophysiology and the antiproteinuric actions of drugs commonly used for podocytopathies.

Fibrinogen links podocyte injury with Toll-like receptor 4 and is associated with disease activity in FSGS patients.

AIM: Fibrinogen (Fg) is reported to participate in inflammation through Toll-like receptor 4 (TLR4). However, it remains unknown whether Fg might induce podocyte damage through TLR4 and be related to disease activity in patients with focal segmental glomerulosclerosis (FSGS). METHODS: We observed Fg-induced alterations in actin and apoptosis in cultured human podocytes transfected with or without TLR4 siRNA. Expression of TLR4, phospho-p38 MAPK and phospho-NF-κB p65 was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or western blotting, and we analysed urinary Fg levels in adriamycin-treated mice and double immunofluorescence staining for TLR4, Fg and podocin. Urinary Fg changes were also analyzed in FSGS patients under prednisone treatment. RESULTS: First, Fg dose-dependently induced actin damage and apoptosis in cultured human podocytes, with an Fg-induced increase in TLR4 expression, and TLR4 siRNA transfection prevented these effects. TLR4 knockdown inhibited activation of p38 MAPK and NF-κB p65 in podocytes. Elevated urinary Fg levels were positively correlated with albuminuria in adriamycin-treated mice, in which Fg and TLR4 colocalized and exhibited increased expression in podocytes. Additionally, elevated urinary Fg levels were positively correlated with 24-h proteinuria and foot process width in FSGS patients. Urinary Fg levels were significantly decreased in patients with complete remission but not in those without remission. CONCLUSIONS: Fg induced podocytes injury via the TLR4-p38 MAPK-NF-κB p65 pathway. In FSGS patients, urinary Fg levels reflect therapeutic response to prednisone and disease activity.

Urinary Fibrinogen as a Predictor of Progression of CKD.

BACKGROUND AND OBJECTIVES: Fibrinogen has been reported to be involved in kidney tubulointerstitial fibrosis and podocyte injury in mouse models. However, the relationship between urinary fibrinogen and kidney outcomes has not been clarified in patients with CKD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We evaluated 402 patients with CKD and kidney biopsies, including 101 with diabetic nephropathy, 94 with idiopathic membranous nephropathy, 55 with idiopathic FSGS, and 152 with IgA nephropathy. We quantified urinary fibrinogen by ELISA and tested associations with kidney histology and progression to ESRD. RESULTS: Median (interquartile range) urinary fibrinogen-to-creatinine ratio was 536 (191-1461) ng/mg for patients with CKD, significantly higher than 2 (2-3) ng/mg for healthy controls (P<0.001). Urinary fibrinogen was positively correlated with urine protein (r=0.64; P<0.001) and interstitial fibrosis and tubular atrophy (r=0.10; P=0.04), and it was negatively correlated with eGFR (r=-0.20; P<0.001). Over a median follow-up period of 35 months (interquartile range, 24-78 months), 68 of 402 patients (17%) developed ESRD. Higher urinary fibrinogen level was associated with increased risk of ESRD (hazard ratio, 2.12; 95% confidence interval, 1.31 to 3.26) per log10 higher urinary fibrinogen-to-creatinine ratio (P=0.003) adjusting for age, sex, BP, urine protein, disease type, eGFR, and interstitial fibrosis and tubular atrophy. For prediction of ESRD, the addition of urinary fibrinogen to eGFR, urine protein, and BP increased the area under the receiver operating curve from 0.73 to 0.76, and the Akaike information criterion improved from 333.6 to 327.0. CONCLUSIONS: Urinary fibrinogen correlated with interstitial fibrosis and tubular atrophy and was an independent risk factor for progression of CKD to ESRD.

Apolipoprotein A-1-related amyloidosis 2 case reports and review of the literature.

RATIONALE: Apolipoprotein A-1 (ApoA-1)-related amyloidosis is characterized by the deposition of ApoA-1 in various organs and can be either hereditary or nonhereditary. It is rare and easily misdiagnosed. Renal involvement is common in hereditary ApoA-1 amyloidosis, but rare in the nonhereditary form. PATIENT CONCERNS: We reported two cases with ApoA-1 amyloidosis, a 64-year-old man suffering from nephrotic syndrome and a 40-year-old man with nephrotic syndrome and splenomegaly. Renal biopsies revealed glomerular, interstitial and vascular amyloid deposits and positive phospholipase A2 receptor staining in the glomerular capillary loop in case 1, and mesangial amyloid deposits in case 2. DIAGNOSES: After immunostaining failed to determine the specific amyloid protein, proteomic analysis of amyloid deposits by mass spectrometry was performed and demonstrated the ApoA-1 origin of the amyloid. Genetic testing revealed no mutation of the APOA1 gene in case 1 but a heterozygous mutation, Trp74Arg, in case 2. Case 1 was thus diagnosed as nonhereditary ApoA-1 associated renal amyloidosis with membranous nephropathy, and case 2 as hereditary ApoA-1 amyloidosis with multiorgan injuries (kidney and spleen) and a positive family history. INTERVENTIONS: Case 1 was treated with glucocorticoid combined with cyclosporine. Case 2 was treated with calcitriol and angiotensin converting enzyme inhibitors. OUTCOMES: Two cases were followed up for 5 months and 2 years, respectively; and case 1 was found to have attenuated proteinuria while case 2 had an elevation of cholestasis indices along with renal insufficiency. LESSONS: Proteomic analysis by mass spectrometry of the amyloid deposits combined with genetic analysis can provide accurate diagnosis of ApoA-1 amyloidosis. Besides, these 2 cases expand our knowledge of ApoA-1-related renal amyloidosis.