Dapagliflozin Improves Angiogenesis after Hindlimb Ischemia through the PI3K-Akt-eNOS Pathway.

Recently, the vascular protective effect of anti-diabetic agents has been receiving much attention. Sodium glucose cotransporter 2 (SGLT2) inhibitors had demonstrated reductions in cardiovascular (CV) events. However, the therapeutic effect of dapagliflozin on angiogenesis in peripheral arterial disease was unclear. This study aimed to explore the effect and mechanism of dapagliflozin on angiogenesis after hindlimb ischemia. We first evaluated the effect of dapagliflozin on post-ischemic angiogenesis in the hindlimbs of rats. Laser doppler imaging was used to detect the hindlimb blood perfusion. In addition, we used immunohistochemistry to detect the density of new capillaries after ischemia. The relevant signaling pathways of dapagliflozin affecting post-ischemic angiogenesis were screened through phosphoproteomic detection, and then the mechanism of dapagliflozin affecting post-ischemic angiogenesis was verified at the level of human umbilical vein endothelial cells (HUVECs). After subjection to excision of the left femoral artery, all rats were randomly distributed into two groups: the dapagliflozin group (left femoral artery resection, receiving intragastric feeding with dapagliflozin (1 mg/kg/d), for 21 consecutive days) and the model group, that is, the positive control group (left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days). In addition, the control group, that is the negative control group (without left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days) was added. At day 21 post-surgery, the dapagliflozin-treatment group had the greatest blood perfusion, accompanied by elevated capillary density. The results showed that dapagliflozin could promote angiogenesis after hindlimb ischemia. Then, the ischemic hindlimb adductor-muscle tissue samples from three rats of model group and dapagliflozin group were taken for phosphoproteomic testing. The results showed that the PI3K-Akt-eNOS signaling pathway was closely related to the effect of dapagliflozin on post-ischemic angiogenesis. Our study intended to verify this mechanism from the perspective of endothelial cells. In vitro, dapagliflozin enhanced the tube formation, migration, and proliferation of HUVECs under ischemic and hypoxic conditions. Additionally, the dapagliflozin administration upregulated the expression of angiogenic factors phosphorylated Akt (p-Akt) and phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor A (VEGFA), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. dapagliflozin could promote angiogenesis after ischemia. This effect might be achieved by promoting the activation of the PI3K-Akt-eNOS signaling pathway. This study provided a new perspective, new ideas, and a theoretical basis for the treatment of peripheral arterial disease.

COVID-19 vaccination coverage and its cognitive determinants among older adults in Shanghai, China, during the COVID-19 epidemic.

Objectives: This study aimed to examine the coverage of coronavirus disease 2019 (COVID-19) vaccination and its cognitive determinants among older adults. Methods: A cross-sectional study was conducted using a questionnaire to conduct a survey among 725 Chinese older adults aged 60 years and above in June 2022, 2 months after the mass COVID-19 outbreak in Shanghai, China. The questionnaire covered demographic characteristics, COVID-19 vaccination status, internal risk perception, knowledge, and attitude toward the efficacy and safety of COVID-19 vaccines. Results: The vaccination rate was 78.3% among the surveyed individuals. Self-reported reasons for unwillingness to get vaccinated (multiple selections) were "concerns about acute exacerbation of chronic diseases after vaccination (57.3%)" and "concerns regarding vaccine side effects (41.4%)." Compared to the unvaccinated group, the vaccinated group tended to have a higher score in internal risk perception (t = 2.64, P < 0.05), better knowledge of COVID-19 vaccines (t = 5.84, P < 0.05), and a more positive attitude toward the efficacy and safety of COVID-19 vaccines (t = 7.92, P < 0.05). The path analysis showed that the cognitive effect on vaccination behavior is relatively large, followed by the internal risk perception, and then the attitude toward COVID-19 vaccines. The more knowledgeable the participants were about COVID-19 vaccines, the more likely they were to receive the COVID-19 vaccines. In the multivariate logistic regression, the increased coverage of COVID-19 vaccination was associated with reduced age (OR = 0.53 95% CI 0.43-0.66, P < 0.001), being a resident in other places than Shanghai (OR = 0.40, 95% CI 0.17-0.92, P < 0.05), a shorter time of lockdown (OR = 0.33, 95% CI 0.13-0.83, P < 0.05), a history of other vaccines (OR = 2.58, 95% CI 1.45-4.60, P < 0.01), a fewer number of chronic diseases (OR = 0.49, 95% CI 0.38-0.62, P < 0.001), better knowledge about COVID-19 vaccines (OR = 1.60, 95% CI 1.17-2.19, P < 0.01), and a positive attitude toward COVID-19 vaccines (OR = 9.22, 95% CI 4.69-18.09, P < 0.001). Conclusion: Acquiring accurate knowledge and developing a positive attitude toward COVID-19 vaccines are important factors associated with COVID-19 vaccination. Disseminating informed information on COVID-19 vaccines and ensuring efficacious communication regarding their efficacy and safety would enhance awareness about COVID-19 vaccination among older adults and consequently boost their vaccination coverage.

High sodium promotes the secretion and synthesis of PTH through PiT-1-IKKß pathway in parathyroid gland in vitro.

Parathyroid hormone (PTH) is secreted by the parathyroid glands (PTGs) and is an important hormone regulating mineral metabolism. Previous studies reported that high sodium diet will cause the increase in serum PTH, but the specific mechanism is unknown. Consequently, the present study aims to investigate the effects and mechanisms of high sodium on PTH synthesis and secretion from PTGs. We developed a tissue culture model using normal rat PTGs, discovered that sodium elicited and promoted concentration-dependent and time-dependent PTH secretion. Changes in sodium-associated transporters from PTGs incubated with high sodium were thoroughly examined. Increased expression of sodium-phosphate cotransporter Slc20a1 (also known as PiT-1) was observed. Further tests revealed that PiT-1 activated the NF-κB signaling pathway, resulting in increased IKKß phosphorylation, IKBα degradation, and increased p65 phosphorylation followed by nuclear entry, which led to increased PTH transcription. Meanwhile, IKKß phosphorylated SNAP23, promoting exocytosis and eventually led to increased PTH secretion. In conclusion, our findings indicate that PiT-1 plays an important role in the increased secretion and synthesis of PTH directly induced by high sodium under physiological conditions, and may provide a potential therapeutic target for secondary hyperparathyroidism (SHPT).

Effects of a high-phosphorus diet on the gut microbiota in CKD rats.

OBJECTIVE: To investigate whether high-phosphorus diets alter gut microbiota in healthy rats and chronic kidney disease (CKD) rats. METHODS: In this 4-week randomized controlled trial, healthy rats and CKD rats were fed a regular-phosphorus (Pi: 0.8%) and high-phosphorus (Pi: 1.2%) diet. The subjects were divided into four groups: sham-group rats with regular-phosphorus diet intervention (CTL group), sham-group rats with high-phosphorus diet intervention (CTLP group), CKD model rats with regular-phosphorus diet intervention (CKD group), and CKD model rats with high-phosphorus diet intervention (CKDP group). The V3-V4 region of the 16S rRNA gene was sequenced to study the effect of a high-phosphorus diet on gut microbiota. RESULTS: A high-phosphorus intervention increased systolic blood pressure (SBP) and parathyroid hormone (PTH) in CTL and CKD rats but did not change serum creatinine and 25(OH)D levels. After the high-phosphorus diet, serum phosphate and fibroblast growth factor 23 (FGF23) increased in the CKDP group compared with the CKD group. The gut microbiota was significantly altered after intervention with a high-phosphorus diet in CTL and CKD group rats. A high-phosphorus diet reduced the Shannon index values of gut microbiota in all rats. The Chao1 and Ace indexes were decreased in the CTL group after high-phosphorus diet intervention. Some microbial genera were elevated significantly after high-phosphorus dietary intervention, such as Blautia and Allobaculum. The main bacteria linked to SBP and FGF23 also correlated directly with creatinine. After high-phosphorus diet intervention, the bacteria Prevotella were positively related to SBP in CTLP and CKDP groups. CONCLUSIONS: High-phosphorus diets were associated with adverse changes in gut microbiota and elevated SBP, which may have adverse consequences for long-term health outcomes.

Higher one-year achievement rate of serum phosphate associated with lower cardiovascular mortality in hemodialysis patients.

BACKGROUND: Estimation of phosphate load in hemodialysis patients is always controversial in clinical practice. The aim of this study was to verify individual achievement rate of serum phosphate as the evaluation of phosphate load through investigating its impact on cardiovascular mortality in hemodialysis patients. METHODS: This was a single-center, retrospective cohort study. A total of 251 maintenance hemodialysis patients were enrolled. The individual achievement rate of serum phosphate was defined as the times of tests within the target range divided by total times of tests over a period of time. Cox regression model was used to examine the relationship between individual achievement rate of serum phosphate and cardiovascular mortality. RESULTS: The mean age of the study population was 61 ± 13 years old. A total of 44 (17.5%) patients died from cardiovascular disease (CVD) during a median follow-up of 65 months. Multivariable Cox analysis showed that one-year serum phosphate achievement rate of 0% (HR = 4.117, P = 0.016) and 25% (HR = 3.343, P = 0.023) increased the risk of cardiovascular mortality while the achievement rate of 50% (HR = 2.129, P = 0.162) and 75% (HR = 1.080, P = 0.902) did not, compared to the rate of 100%. Urea reduction ratio (URR) was positively, while serum intact parathyroid hormone (iPTH), alkaline phosphatase (ALP), normalized protein catabolic rate (nPCR), and total phosphate-binding capacity of drug were negatively associated with achievement in target of serum phosphate. CONCLUSIONS: Keeping one-year achievement rate of serum phosphate higher than 50% provides significant clinical benefits in reducing cardiovascular mortality.

Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human.

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Dominant factors of the phosphorus regulatory network differ under various dietary phosphate loads in healthy individuals.

BACKGROUND: The purpose of this study was to explore the contribution of each factor of the phosphorus metabolism network following phosphorus diet intervention via Granger causality analysis. METHODS: In this study, a total of six healthy male volunteers were enrolled. All participants sequentially received regular, low-, and high-phosphorus diets. Consumption of each diet lasted for five days, with a 5-day washout period between different diets. Blood and urinary samples were collected on the fifth day of consumption of each diet at 9 time points (00:00, 04:00, 08:00, 10:00, 12:00, 14:00, 16:00, 20:00, 24:00) for measurements of serum levels of phosphate, calcium, PTH, FGF23, BALP, α-Klotho, and 1,25 D and urinary phosphorus excretion. Granger causality and the centrality of the above variables in the phosphorus network were analyzed by pairwise panel Granger causality analysis using the time-series data. RESULTS: The mean age of the participants was 28.5 ± 2.1 years. By using Granger causality analysis, we found that the α-Klotho level had the strongest connection with and played a key role in influencing the other variables. In addition, urinary phosphorus excretion was frequently regulated by other variables in the network of phosphorus metabolism following a regular phosphorus diet. After low-phosphorus diet intervention, serum phosphate affected the other factors the most, and the 1,25 D level was the main outcome factor, while urinary phosphorus excretion was the most strongly associated variable in the network of phosphorus metabolism. After high-phosphorus diet intervention, FGF23 and 1,25 D played a more critical role in active regulation and passive regulation in the Granger causality analysis. CONCLUSIONS: Variations in dietary phosphorus intake led to changes in the central factors involved in phosphorus metabolism.

Comparative Performance of Creatinine-Based GFR Estimation Equations in Exceptional Longevity: The Rugao Longevity and Ageing Study.

Purpose: Reduced kidney function has been associated with an increased risk for adverse outcomes. Accurate assessment of glomerular filtration rate (GFR) is key to diagnosis and management of kidney disfunction. Debate exists on the best GFR estimation equation for elderly people. This study aimed to compare the predictive validity and discriminative ability of four GFR equations in relation to 2-year and 6-year mortality in exceptional longevity (EL) (those over 95 years old with intact health) individuals and is an ideal model to address factors relating to life span and age-related diseases. Patients and Methods: This study used 6 years' data of 278 EL from the Rugao longevity cohort. Baseline GFR was estimated using four equations: Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, Modification of Diet in Renal Disease Study (MDRD) equation, Berlin Initiative Study-1 (BIS) equation, and modified MDRD equation. Predictive validity was tested using Cox proportional hazards analysis. Overall improvement in reclassification based on estimated GFR (eGFR) was assessed applying net reclassification improvement (NRI). Results: Mean age of participants was 97±2 years with median follow-up of 2.6 years. Median (IQR) eGFR by CKD-EPI, MDRD, BIS, and modified MDRD equations were 73.9 (62.2-77.6), 82.3 (67.4-98.6), 56.4 (47.9-63.9), and 101.5 (83.1-121.6) mL/min per 1.73 m2, respectively. Higher eGFREPI was associated with lower mortality after multivariate adjustment (for continuous eGFREPI, HRtwo-year 1.018, 95% CI 1.002-1.033, P=0.023; HRsix-year 1.013, 95% CI 1.002-1.025, P=0.022), while eGFR from other equations did not show any associations with mortality. NRI for two-year mortality was 0.14 and approximately significant, which may favor the CKD-EPI when compared to BIS equation (P=0.052). Conclusion: The CKD-EPI equation showed more accurate estimation of kidney function in the elderly with respect to GFR distribution and predictability of mortality risk.

Calcium-sensing receptor activation attenuates collagen expression in renal proximal tubular epithelial cells.

316: F1006-F1015, 2019. First published March 6, 2019; doi: 10.1152/ajprenal.00413.2018 .-Experimental studies have shown that pharmacological activation of calcium-sensing receptor (CaSR) attenuates renal fibrosis in some animal models beyond modification of bone and mineral homeostasis; however, its underlying mechanisms remain largely unknown. Since excessive collagen deposition is the key feature of fibrosis, the present study aimed to examine whether CaSR was involved in the regulation of collagen expression in rats with adenine diet-induced renal fibrosis and in profibrotic transforming growth factor (TGF)-ß1-treated renal proximal tubular epithelial cells (PTECs). The results showed that the CaSR agonist cinacalcet significantly attenuated renal collagen accumulation and tubular injury in adenine diet-fed rats. Additionally, the in vitro experiment showed that profibrotic TGF-ß1 significantly increased the expression of collagen and decreased CaSR expression at the mRNA and protein levels in a concentration- and time-dependent manner. Furthermore, the CaSR CRISPR activation plasmid and cinacalcet partially abrogated the upregulation of collagen induced by TGF-ß1 treatment. Blockade of CaSR by the CRISPR/Cas9 KO plasmid or the pharmacological antagonist Calhex231 further enhanced TGF-ß1-induced collagen expression. Mechanistic experiments found that Smad2 phosphorylation and Snail expression were markedly increased in PTECs treated with TGF-ß1, whereas the CaSR CRISPR activation plasmid and cinacalcet substantially suppressed this induction. In summary, this study provides evidence for a direct renal tubular epithelial protective effect of CaSR activation in renal fibrosis, possibly through suppression of collagen expression in PTECs.

A Practical Electronic Health Record-Based Dry Weight Supervision Model for Hemodialysis Patients.

Objective: Dry Weight (DW) is a typical hemodialysis (HD) prescription for End-Stage Renal Disease (ESRD) patients. However, an accurate DW assessment is difficult due to the complication of body components and individual variations. Our objective is to model a clinically practicable DW estimator. Method: We proposed a time series-based regression method to evaluate the weight fluctuation of HD patients according to Electronic Health Record (EHR). A total of 34 patients with 5100 HD sessions data were selected and partitioned into three groups; in HD-stabilized, HD-intolerant, and near-death. Each group's most recent 150 HD sessions data were adopted to evaluate the proposed model. Results: Within a 0.5 kg absolute error margin, our model achieved 95.44%, 91.95%, and 83.12% post-dialysis weight prediction accuracies for the HD-stabilized, HD-intolerant, and near-death groups, respectively. Within a 1%relative error margin, the proposed method achieved 97.99%, 95.36%, and 66.38% accuracies. For HD-stabilized patients, the Mean Absolute Error (MAE) of the proposed method was 0.17 kg ± 0.04 kg. In the model comparison experiment, the performance test showed that the quality of the proposed model was superior to those of the state-of-the-art models. Conclusion: The outcome of this research indicates that the proposed model could potentially automate the clinical weight management for HD patients. Clinical Impact: This work can aid physicians to monitor and estimate DW. It can also be a health risk indicator for HD patients.

Prostaglandin E2 receptor EP2 mediates the effect of cyclooxygenase 2 on secondary parathyroid hyperplasia in end-stage renal disease.

BACKGROUND: Secondary hyperparathyroidism (SHPT) in patients with end-stage renal disease (ESRD) is characterized by hyperplasia of the parathyroid glands (PTGs), while the underlying mechanism is not completely understood. Previously we demonstrated a relationship between cyclooxygenase 2 (COX2) overexpression and parathyroid hyperplasia and here we investigate the role of COX2 downstream metabolic product prostaglandin E2 (PGE2) and its receptor EP2 in the pathogenesis of SHPT. METHODS: PTGs isolated from ESRD patients with advanced SHPT were used to test the expression of COX2-microsomal prostaglandin E synthase-1 (mPGES-1)-EP2 pathway. A diffuse proliferative section of the PTGs was used for tissue culture and treated with high phosphate (HPi) medium, COX2-PGE2-EP2 pathway inhibitors or agonists. EP2 short hairpin RNA (shRNA) lentivirus was locally applied to treat an SHPT rat model. RESULTS: In PTGs isolated from ESRD patients, enhanced immunoactivities of COX2, mPGES-1 and EP2 were observed. In primary cultured PTG tissues, HPi induced intact parathyroid hormone (iPTH) secretion, proliferating cell nuclear antigen (PCNA) expression and COX2 activity, while COX2 and EP2 inhibitors attenuated hyperparathyroidism promoted by HPi. Furthermore, PGE2 or EP2 agonist (butaprost) directly stimulated hyperparathyroidism, whereas EP2 receptor antagonist or cyclic adenosine monophosphate inhibitor attenuated the hyperparathyroidism promoted by PGE2 or butaprost. EP2 shRNA treatment significantly reduced excessive expressions of EP2 and PCNA in the PTGs of nephrectomy rats fed an HPi diet, diminished the size of PTGs and downregulated serum iPTH levels. CONCLUSIONS: The COX2 downstream PGE2 and its receptor EP2 may play an important role in HPi-induced parathyroid hyperplasia and may serve as a potential therapeutic target for SHPT in ESRD.

Role of nab-paclitaxel in metastatic breast cancer: a meta-analysis of randomized clinical trials.

Whether nab-paclitaxel and conventional taxanes are equally effective for metastatic breast cancer (MBC) remains unclear. We conducted meta-analysis of trials that compared nab-paclitaxel-based chemotherapy with solvent-based paclitaxel (sb-paclitaxel) and docetaxel-based chemotherapy. A literature search was performed to identify articles that compared nab-paclitaxel-based chemotherapy with sb-paclitaxel or docetaxel-based chemotherapy for MBC. Four randomized controlled trials (1,506 patients) were identified from 1,268 reports. We detected equivalent overall response, overall survival, and survival probability (one-year, two-year). Grade 3 to 4 hematological and non-hematological toxicities were also comparable except that sensory neuropathy was more prominent for nab-paclitaxel-based chemotherapy (16.9% vs. 10%, odds ratio = 1.89, 95% confidence interval = 1.36-2.61, P < 0.001). No significant publication bias was detected. Consistent results stratified by treatment arm, study phase, treatment line, and study location were observed, except that overall response rate to nab-paclitaxel-based chemotherapy was significantly higher in the subgroup of randomized phase II trials, non-first-line treatment, and East Asian population. This meta-analysis failed to demonstrate advantages of nab-paclitaxel compared with sb-paclitaxel and docetaxel in patients with MBC. The newer agent was associated with increased sensory neuropathy, equivalent survival, and possibly increased overall response for some specific patients.