Tempol treatment normalizes membrane expression of epithelial transport proteins in the kidney of salt-loaded hypertensive diabetic db/db mice.

OBJECTIVE: Hypertension exacerbates the progression and severity of diabetic kidney disease. In this study, we addressed the hypothesis that tempol acts at multiple segments of the nephron to normalize the abundance of sodium coupled epithelial transport proteins in the luminal plasma membrane to mitigate high blood pressure in salt-loaded hypertensive diabetic db/db mice. METHODS: Soluble and membrane fractions from freshly homogenized kidney cortex tissue samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed for specific proteins by Western blotting. Western blotting for specific urinary extracellular vesicle (uEV) markers and nanoparticle tracking analysis was performed to characterize each uEV preparation from each group. A one-way ANOVA was performed to determine statistical significance between three groups (hypertensive diabetic db/db mice treated with vehicle, hypertensive diabetic db/db mice treated with tempol, and wild-type mice). RESULTS: Tempol treatment reduced systolic blood pressure in hypertensive diabetic db/db mice compared to db/db mice that received vehicle. We observed attenuated membrane protein expression of the sodium hydrogen exchanger 3 (NHE3), sodium potassium chloride co-transporter (NKCC2), sodium chloride cotransporter (NCC), and epithelial sodium channel (ENaC) in the kidney of salt-loaded hypertensive diabetic db/db mice infused with tempol by osmotic minipump for 5 days compared to hypertensive diabetic db/db mice infused with vehicle. Also, the infusion of tempol in hypertensive diabetic db/db mice reduced the augmented protein expression of protein kinase c (PKC) epsilon observed in the vehicle treated hypertensive diabetic db/db kidney when compared to the healthy wild-type kidney. The amount of uEV and their size profiles were comparable between the three groups. CONCLUSIONS: This study demonstrates that tempol down-regulates epithelial transport mechanisms in each segment of the nephron and normalizes salt-induced high blood pressure in diabetic animals presumably in a PKC dependent manner.

Adult Anomalous Left Coronary Artery Arising From the Pulmonary Artery (ALCAPA) Syndrome as First Presentation With Atrial Fibrillation in a Marathon Runner.

Anomalous left coronary artery arising from the pulmonary artery (ALCAPA) syndrome is a very rare congenital heart disease with an incidence of one in 300,000 and a high rate of mortality early in life if left untreated. Adult-type ALCAPA presents when significant collaterals develop from the right coronary artery (RCA) to the left coronary artery (LCA). Even with the collaterals, chronic sub-endocardial ischemia occurs in most cases, and patients die from sudden cardiac death. Here we present a case of a 38-year-old female who lived an active and healthy life and presented with chest pain and palpitations. Initial electrocardiography (EKG) showed atrial fibrillation with rapid ventricular response. Although initial cardiac enzymes were negative and there were no ischemic EKG changes, troponins became elevated over the course of the hospital stay and the patient underwent a left heart catheterization. Results revealed a dilated RCA extending to the left ventricle and an anomalous left main originating from the pulmonary artery with complete occlusion. The patient received medical management for acute coronary syndrome, including heparin infusion for 48 hours, aspirin, metoprolol, and atorvastatin. She was referred to a tertiary care facility for surgical correction of anomaly of the coronary arteries. The next day, the patient arrived in the emergency department with an acute onset of speech difficulty and left-sided weakness. A brain CT without contrast showed hematoma in the right frontal lobe. The patient underwent surgical evacuation of the hematoma with marked improvement of her weakness. The patient recovered after a successful surgical repair involving translocation of the left main coronary artery to the aorta. It has been reported that ALCAPA should be considered in a young adult with dilated cardiomyopathy and mitral regurgitation (MR). Other common presentations include acute myocardial infarction, angina, and dyspnea on exertion. Sudden cardiac death is not uncommon; however, it tends to decrease with age of diagnosis. Interestingly, our patient was known to have MR with regular follow-up at the cardiology clinic for years. Echocardiogram never showed any abnormalities other than MR. She never received further workup to address the reason of MR, although she has no underlying chronic conditions that can explain it. In relatively young patients with a healthy lifestyle presenting with chest pain, a broader look at etiologies should be considered. We would like to emphasize the importance of looking up for possible coronary artery disease, especially in young individuals.

Characteristics and outcomes of diabetic patients with acute exacerbation of COPD.

RATIONALE AIMS AND OBJECTIVES: Patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and diabetes mellitus form a special population due to an increased risk of hyperglycemia from the use of corticosteroids. There is limited data regarding specific outcomes in diabetic patients with AECOPD. METHODS: A retrospective data analysis of adult patients admitted to North Florida Division of the Hospital Corporation of America (HCA Healthcare) with a primary or secondary diagnosis of AECOPD from January 1, 2018, to December 31, 2018. We excluded patients who needed intensive care unit (ICU) care on day 0. Outcomes assessed included length of stay, mortality, and need for ICU transfer after 48 h from admission. Characteristics included age, sex, and race, comorbidities such as diabetes mellitus, chronic kidney disease, acute kidney injury, congestive heart failure, and anemia were analyzed. Comparisons were analyzed via binary and multivariate logistic regression models. RESULTS: A total of 3788 patients admitted for AECOPD were included; amongst them, 1356 patients (~36%) had diabetes mellitus. This subset of patients had higher rates of comorbidities. A significant portion of diabetic patients (72%) received intravenous rather than oral steroids, similar to non-diabetic patients. In addition, diabetic patients were more likely to develop acute kidney injury (14.2% vs 8.0%, p < 0.004) and decompensated heart failure (9.2% vs 4.6%, p < 0.001). Diabetic patients had higher length of stay and increased need for ICU transfer. However, diabetes itself did not independently affect length of stay (CI -0.028, 0.479, p = 0.081) when adjusted to comorbidities and patient's characteristics. Moreover, diabetes was independently associated with an increased need for transfer to ICU (Odds ratio 1.9, p = 0.031). The oral route of steroid use was associated with decreased LOS (ß coefficient - 0.9, p < 0.001). CONCLUSION: Diabetes mellitus is independently associated with increased ICU transfers amongst patients hospitalized with AECOPD. The use of oral steroids rather than intravenous steroids was independently associated with decreased length of stay in diabetic and non-diabetic patients. Despite no difference in intravenous vs. oral corticosteroids demonstrated in previous COPD trials, a significant portion of diabetic patients continue to receive intravenous corticosteroids. Further investigation is required to explore these findings.

Seizure as an Initial Presentation for Posterior Reversible Encephalopathy Syndrome in Undiagnosed Systemic Lupus Erythematosus and Lupus Nephritis: A Case Report.

Posterior reversible encephalopathy syndrome (PRES) is a syndrome presenting with neurological manifestations including headaches, seizures, and notable changes in brain imaging. It is typically associated with an acute increase in blood pressure, metabolic abnormalities, and/or medication effects. PRES is challenging to diagnose due to its variable presentation and low incidence. Herein we describe a compelling case of PRES syndrome secondary to uncontrolled hypertension in the setting of systemic lupus erythematosus (SLE) and lupus nephritis.

Activation of multiple receptors stimulates extracellular vesicle release from trophoblast cells.

Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G-protein-coupled receptors (GPCRs) expressed by a single cell-type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or -BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines-JAR choriocarcinoma cells and HTR-8/SVneo cells derived from first-trimester human villous trophoblast-to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell-culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)-ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91-, 2.79-, 1.87-, and 3.11-fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor-mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63-fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10-fold below that required to increase intracellular calcium. CCK activation of HTR-8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06-fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor-stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell-type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.

Lipidomic analysis of urinary exosomes from hereditary α-tryptasemia patients and healthy volunteers.

Exosomes are nano-sized vesicles that are involved in various biological processes including cell differentiation, proliferation, signaling, and intercellular communication. Urinary exosomes were isolated from a cohort of hereditary α-tryptasemia (HαT) patients and from healthy volunteers. There was a greater number of exosomes isolated from the urine in the HαT group compared to the control volunteers. Here, we investigated the differences in both lipid classes and lipid species within urinary exosomes of the two groups. Lipids were extracted from urinary exosomes and subjected to liquid chromatography mass spectrometry using a targeted approach. Various molecular species of glycerophospholipids, glycerolipids, and sterols were significantly reduced in HαT patients. Out of a possible 1127 lipids, 521 lipid species were detected, and relative quantities were calculated. Sixty-four lipids were significantly reduced in urinary exosomes of HαT patients compared to controls. All significantly reduced sphingolipids and most of the phospholipids were saturated or mono-unsaturated lipids. These results suggest exosome secretion is augmented in HαT patients and the lipids within these exosomes may be involved in various biological processes. The unique lipid composition of urinary exosomes from HαT patients will contribute to our understanding of the biochemistry of this disease.

Mal protein stabilizes luminal membrane PLC-ß3 and negatively regulates ENaC in mouse cortical collecting duct cells.

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl- cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-ß3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-ß3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-ß3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

Chitosan-coated black sesame (Sesamum indicum L.) seed pulp as a novel candidate adsorbent for Cr(VI) elimination.

This study evaluates the application of Cr(VI) adsorption from the prepared synthetic solution by black sesame (Sesamum indicum L.) seed pulp (BSSP) and chitosan (Cts)-coated black sesame seed pulp beads (Cts-BSSP). BSSP and Cts-BSSP were used as an adsorbent without any chemical or physical treatment to remove Cr(VI) from an aqueous medium. The results indicated that the Cr(VI) removal was pH-dependent and reached an optimum at pH 2.0. It has been observed that the percentage of adsorption increased from 62% to 95% when the amount of Cts-BSSP increased from 0.0125 g to 0.0250 g. The required adsorbent amount for the maximum removal was 0.05 g and 0.1 g for Cst-BSSP and BSSP, respectively. The contact time for the adsorption was 120 min and 90 min for BSSP and Cst-BSSP, respectively. Scanning electron microscopy and Fourier transform infrared spectroscopy were used to explore the possible adsorption mechanism for Cr(VI). The equilibrium data for the BSSP and Cts-BSSP were used with the Langmuir and Freundlich adsorption isotherm models to assess the adsorption capacity and relevant mechanism. The adsorption capacity of the Cts-BSSP for Cr(VI) is relatively high compared to BSSP. The monolayer maximum adsorption capacities for Cr(VI) ions were 31.44 and 18.32 mg/g for Cts-BSSP and BSSP, respectively.

Modulation of calreticulin expression reveals a novel exosome-mediated mechanism of Z variant α1-antitrypsin disposal.

α1-Antitrypsin deficiency (AATD) is an inherited disease characterized by emphysema and liver disease. AATD is most often caused by a single amino acid substitution at position 342 in the mature protein, resulting in the Z mutation of the AAT gene (ZAAT). This substitution is associated with misfolding and accumulation of ZAAT in the endoplasmic reticulum (ER) of hepatocytes, causing a toxic gain of function. ERdj3 is an ER luminal DnaJ homologue, which, along with calreticulin, directly interacts with misfolded ZAAT. We hypothesize that depletion of each of these chaperones will change the fate of ZAAT polymers. Our study demonstrates that calreticulin modulation reveals a novel ZAAT degradation mechanism mediated by exosomes. Using human PiZZ hepatocytes and K42, a mouse calreticulin-deficient fibroblast cell line, our results show ERdj3 and calreticulin directly interact with ZAAT in PiZZ hepatocytes. Silencing calreticulin induces calcium independent ZAAT-ERdj3 secretion through the exosome pathway. This co-secretion decreases ZAAT aggregates within the ER of hepatocytes. We demonstrate that calreticulin has an inhibitory effect on exosome-mediated ZAAT-ERdj3 secretion. This is a novel ZAAT degradation process that involves a DnaJ homologue chaperone bound to ZAAT. In this context, calreticulin modulation may eliminate the toxic gain of function associated with aggregation of ZAAT in lung and liver, thus providing a potential new therapeutic approach to the treatment of AATD-related liver disease.

Follicular fluid exosomes act on the bovine oocyte to improve oocyte competence to support development and survival to heat shock.

Addition of follicular fluid to oocyte maturation medium can affect cumulus cell function, increase competence of the oocytes to be fertilised and develop to the blastocyst stage and protect the oocyte from heat shock. Here, it was tested whether exosomes in follicular fluid are responsible for the effects of follicular fluid on the function of the cumulus-oocyte complex (COC). This was accomplished by culturing COCs during oocyte maturation at 38.5°C (body temperature of the cow) or 41°C (heat shock) with follicular fluid or exosomes derived from follicular fluid and evaluating various aspects of function of the oocyte and the embryo derived from it. Negative effects of heat shock on cleavage and blastocyst development, but not cumulus expansion, were reduced by follicular fluid and exosomes. The results support the idea that exosomes in follicular fluid play important roles during oocyte maturation to enhance oocyte function and protect it from stress.