Connexins in endothelial cells as a therapeutic target for solid organ transplantation.

Connexins are a class of membrane proteins widely distributed throughout the body and have various functions based on their location and levels of expression. More specifically, connexin proteins expressed in endothelial cells (ECs) have unique roles in maintaining EC barrier integrity and function-a highly regulated process that is critical for pro-inflammatory and pro-coagulant reactions. In this minireview, we discuss the regulatory influence connexin proteins have in maintaining EC barrier integrity and their role in ischemia-reperfusion injury as it relates to organ transplantation. It is evident that certain isoforms of the connexin protein family are uniquely positioned to have far-reaching effects on preserving organ function; however, there is still much to be learned of their roles in transplant immunology and the application of this knowledge to the development of targeted therapeutics.

The conformational signature of ß-arrestin2 predicts its trafficking and signalling functions.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in ß-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of ß-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in ß-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average ß-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Costs and factors associated with heart failure following kidney transplantation - a single-center retrospective cohort study.

The number of adults with heart failure (HF) will increase by ~50% between 2012 and 2030. Among kidney transplant recipients, HF accounts for 16% of all post-transplant admissions. We describe the burden of HF and predictors of healthcare utilization following kidney transplantation. We retrospectively identified adults who underwent kidney transplantation at our institution (01/2007-12/2017). Data were acquired from electronic health records, with healthcare utilization obtained from a statewide database. The HF incidence rate and prevalence were estimated for each year, total charges for HF and non-HF patients were compared, and logistic regression was employed for a 3-year predictive model of healthcare utilization associated with HF. Among 1731 kidney transplant recipients, the post-transplant HF incidence rate ranged from 1.91 (year 3) to 6.80 (year 10) per 100 person-years, while the prevalence increased from 31.7% (year 1) to 48.1% (year 10). Median charges were $75 837 (HF) compared to $42 940 (non-HF) per person-year (P < 0.001). Pretransplant HF [odds ratio (OR) = 3.12] and an eGFR < 45 (OR = 4.73) were the strongest predictors of HF encounters (P < 0.05 for both). We observed a high and increasing prevalence of HF, which was associated with twice the costs. Kidney transplant recipients would benefit from interventions aimed at mitigating HF risk factors.

Hospital admissions and emergency department visits among kidney transplant recipients.

Reducing acute care utilization is a means of improving long-term patient outcomes. We sought to assess high inpatient (IP) admission and standalone emergency department (ED) utilization within a 9-month period post-kidney transplantation and to identify mutable factors to reduce utilization. In this ten-year retrospective study, 1599 adult kidney transplant recipients were identified. A previous transplant, graft loss, or death within 3 months post-transplantation excluded 319 patients. Comprehensive resource utilization data were obtained from a statewide database. Those with ≥2 IP admissions or standalone ED visits 4-12 months post-transplantation were classified as high utilizers. Multivariable logistic regression models were used for examining associations of predictors with high IP or ED utilization. Of 1280 kidney recipients, 209 and 183 were categorized as IP and ED high utilizers, respectively. Factors significantly associated with high IP utilization included valvular disease, body mass index ≥35, and IP or ED use <3 months post-transplantation; while factors associated with high ED utilization included IP or ED use <3 months post-transplantation, younger age, female, smoker, congestive heart failure, depression, and IP or ED use 1 year pre-transplantation. Inpatient and standalone ED utilization within a 9-month period after kidney transplantation is high and associated with sociodemographic factors, mutable comorbidities, and healthcare utilization.

Development and future deployment of a 5 years allograft survival model for kidney transplantation.

AIM: Identifying kidney transplant patients at highest risk for graft loss prior to loss may allow for effective interventions to improve 5 years survival. METHODS: We performed a 10 years retrospective cohort study of adult kidney transplant recipients (n = 1747). We acquired data from electronic health records, United Network of Organ Sharing, social determinants of health, natural language processing data extraction, and real-time capture of dynamically evolving clinical data obtained within 1 year of transplant; from which we developed a 5 years graft survival model. RESULTS: Total of 1439 met eligibility; 265 (18.4%) of them experienced graft loss by 5 years. Graft loss patients were characterized by: older age, being African-American, diabetic, unemployed, smokers, having marginal donor kidneys and cardiovascular comorbidities. Predictive dynamic variables included: low mean blood pressure, higher pulse pressures, higher heart rate, anaemia, lower estimated glomerular filtration rate peak, increased tacrolimus variability, rejection and readmissions. This Big Data analysis generated a 5 years graft loss model with an 82% predictive capacity, versus 66% using baseline United Network of Organ Sharing data alone. CONCLUSION: Our analysis yielded a 5 years graft loss model demonstrating superior predictive capacity compared with United Network of Organ Sharing data alone, allowing post-transplant individualized risk-assessed care prior to transitioning back to community care.

Renal Function Variability: An Independent Risk Factor for Graft Loss and Death following Kidney Transplantation.

BACKGROUND: Several studies have been performed to evaluate surrogate markers of long-term allograft function in renal transplant recipients. These include serum creatinine, estimated glomerular filtration rate (eGFR), slope of eGFR, and more recently eGFR variability. The aim of this study was to measure eGFR slope while assessing the variability of this slope and if high variability occurring at any time post-transplant was predictive of poorer long-term outcomes in a large cohort of kidney transplant recipients. METHODS: Adult solitary kidney transplant recipients transplanted between July 1, 2005 and July 31, 2015 were included. The primary outcome was time to graft loss, defined as return to chronic dialysis, retransplant, or death. Secondary outcomes were death-censored graft loss and acute allograft rejection. Cox regression was utilized for primary and secondary outcomes. Multivariate logistic regression was used to determine baseline factors predictive of high eGFR variability. RESULTS: A total of 1,543 patients were included in the analysis. The percentage of patients who experienced an eGFR coefficient of variation of <30% was 79.6% (1,229/1,543), while 20.4% (314/1,543) patients had high eGFR variability (≥30%). Patients with high eGFR variability tended to be younger, African-American and female. Those with higher eGFR variability, accounting for confounding and other eGFR measures (peak and slope), had significantly lower overall patient and graft survival. CONCLUSION: This study provides a novel analysis of the utility of eGFR variability in a large cohort. The clinical use of the slope of eGFR and eGFR variability may aid in predicting long-term graft outcomes and facilitate early patient discussions to change the trajectory of allograft function.

A quantitative appraisal of African Americans' decisions to become registered organ donors at the driver's license office.

African American (AA) organ donation registration rates fall short of national objectives. The goal of the present study was to utilize data acquired from a quantitative telephone survey to provide information for a future Department of Motorized Vehicles (DMV) intervention to increase AA organ donor registration at the DMV. AAs (n = 20 177) that had visited an Alabama DMV office within a 3-month period were recruited via direct mailing to participate in a quantitative phone survey. Data from 155 respondents that participated in the survey were analyzed. Of those respondents deciding to become a registered organ donor (ROD; n = 122), one-third made that decision at the time of visiting the DMV. Of those who chose not to become a ROD (n = 33), one-third made the decision during the DMV visit. Almost 85% of all participants wanted to learn more about organ donation while waiting at the DMV, preferably via TV messaging (digital signage), with the messaging delivered from organ donors, transplant recipients, and healthcare experts. Altruism, accurate organ donation information, and encouragement from family and friends were the most important educational topics to support AAs becoming a ROD. These data provide a platform to inform future interventions designed to increase AAs becoming a ROD at the DMV.

Frizzled-5: a high affinity receptor for secreted frizzled-related protein-2 activation of nuclear factor of activated T-cells c3 signaling to promote angiogenesis.

Secreted frizzled-related protein 2 (SFRP2) is a pro-angiogenic factor expressed in the vasculature of a wide variety of human tumors, and modulates angiogenesis via the calcineurin-dependent nuclear factor of activated T-cells cytoplasmic 3 (NFATc3) pathway in endothelial cells. However, until now, SFRP2 receptor for this pathway was unknown. In the present study, we first used amino acid alignments and molecular modeling to demonstrate that SFRP2 interaction with frizzled-5 (FZD5) is typical of Wnt/FZD family members. To confirm this interaction, we performed co-immunofluorescence, co-immunoprecipitation, and ELISA binding assays, which demonstrated SFRP2/FZD5 binding. Functional knock-down studies further revealed that FZD5 is necessary for SFRP2-induced tube formation and intracellular calcium flux in endothelial cells. Using protein analysis on endothelial cell nuclear extracts, we also discovered that FZD5 is required for SFRP2-induced activation of NFATc3. Our novel findings reveal that FZD5 is a receptor for SFRP2 and mediates SFRP2-induced angiogenesis via calcineurin/NFATc3 pathway in endothelial cells.

The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

Stimulation of Cyclooxygenase 2 Expression in Rat Peritoneal Mesothelial Cells.

Objective: Since peritoneal dialysis causes peritoneal fibrosis, we examined how glucose (osmotic factor), mannitol (osmotic control), and angiotensin II (AngII) regulate proinflammatory cyclooxygenase 2 (COX-2) in primary rat peritoneal mesothelial cells. Materials and Methods: For this study, we used the following material (n = 4-8 cell lines): cells, passages 1-2; 125I-AngII receptor surface binding (AT1R antagonist losartan, AT2R antagonist PD123319; both 10 µM); intracellular calcium probe calcium-5; COX-2 immunoblotting (ß-actin normalized); real-time PCR of COX-2 gene PTGS2, and NF-κB inhibitor Ro-1069920 (5 µM). Results: AngII surface receptors were predominantly AT1R (minimally AT2R). AngII and glucose increased COX-2 protein expression concentration dependently; mannitol also increased COX-2 expression. Maximal COX-2 protein expression was observed after 6 h (AngII) and 24 h (glucose, mannitol). The time course of increases in PTGS2 mRNA levels reflected that of COX-2 protein expression. At optimal exposure conditions (time/concentration), glucose was 5-fold more efficacious in stimulating COX-2 protein expression than AngII or mannitol. Losartan fully inhibited COX-2 protein responses to AngII and mannitol, but minimally inhibited responses to glucose. Ro-1069920 fully inhibited COX-2 protein responses to each effector. Conclusion: AngII, glucose, and osmotic stress (mannitol) activate COX-2; NF-κB may be an ideal site for COX-2 blockade, and COX-2 activation by osmotic stress requires AT1R, but activation by glucose is more robust and mechanistically complex. © 2014 S. Karger AG, Basel.

Angiotensin II activates NF-κB through AT1A receptor recruitment of ß-arrestin in cultured rat vascular smooth muscle cells.

Activation of the angiotensin type 1A receptor (AT1AR) in rat aorta vascular smooth muscle cells (RASMC) results in increased synthesis of the proinflammatory enzyme cyclooxygenase-2 (COX-2). We previously showed that nuclear localization of internalized AT1AR results in activation of transcription of the gene for COX-2, i.e., prostaglandin-endoperoxide synthase-2. Others have suggested that ANG II stimulation of COX-2 protein synthesis is mediated by NF-κB. The purpose of the present study was to examine the interrelationship between AT1AR activation, ß-arrestin recruitment, and NF-κB activation in the ability of ANG II to increase COX-2 protein synthesis in RASMC. In the present study we utilized RASMC, inhibitors of the NF-κB pathway, ß-arrestin knockdown, radioligand binding, immunoblotting, and immunofluorescence to characterize the roles of AT1AR internalization, NF-κB activation, and ß-arrestin in ANG II-induced COX-2 synthesis. Ro-106-9920 or parthenolide, agents that inhibit the initial steps of NF-κB activation, blocked ANG II-induced p65 NF-κB nuclear localization, COX-2 protein expression, ß-arrestin recruitment, and AT1AR internalization without inhibiting ANG II-induced p42/44 ERK activation. Curcumin, an inhibitor of NF-κB-induced transcription, blocked ANG II-induced COX-2 protein expression without altering AT1AR internalization, ANG II-induced p65 NF-κB nuclear localization, or p42/44 ERK activation. Small interfering RNA-induced knockdown of ß-arrestin-1 and -2 inhibited ANG II-induced p65 NF-κB nuclear localization. In vascular smooth muscle cells, internalization of the activated AT1AR mediated by ß-arrestins activates the NF-κB pathway, producing nuclear localization of the transcription factor and initiation of COX-2 protein synthesis, thereby linking internalization of the receptor with the NF-κB pathway.

Multilevel Intervention to Improve Racial Equity in Access to Kidney Transplant.

BACKGROUND: African Americans (AAs) have reduced access to kidney transplant (KTX). Our center undertook a multilevel quality improvement endeavor to address KTX access barriers, focused on vulnerable populations. This program included dialysis center patient/staff education, embedding telehealth services across South Carolina, partnering with community providers to facilitate testing/procedures, and increased use of high-risk donors. STUDY DESIGN: This was a time series analysis from 2017 to 2021 using autoregression to assess trends in equitable access to KTX for AAs. Equity was measured using a modified version of the Kidney Transplant Equity Index (KTEI), defined as the proportion of AAs in South Carolina with end-stage kidney disease (ESKD) vs the proportion of AAs initiating evaluation, completing evaluation, waitlisting, and undergoing KTX. A KTEI of 1.00 is considered complete equity; a KTEI of <1.00 is indicative of disparity. RESULTS: From January 2017 to September 2021, 11,487 ESKD patients (64.7% AA) were referred, 6,748 initiated an evaluation (62.8% AA), 4,109 completed evaluation (59.7% AA), 2,762 were waitlisted (60.0% AA), and 1,229 underwent KTX (55.3% AA). The KTEI for KTX demonstrated significant improvements in equity. The KTEI for initiated evaluations was 0.89 in 2017, improving to 1.00 in 2021 (p = 0.0045). Completed evaluation KTEI improved from 0.85 to 0.95 (p = 0.0230), while waitlist addition KTEI improved from 0.83 to 0.96 (p = 0.0072). The KTEI for KTX also improved from 0.76 to 0.91, which did not reach statistical significance (p = 0.0657). CONCLUSIONS: A multilevel intervention focused on improving access to vulnerable populations was significantly associated with reduced disparities for AAs.

Enzymatic processing of angiotensin peptides by human glomerular endothelial cells.

The intraglomerular renin-angiotensin system (RAS) is linked to the pathogenesis of progressive glomerular diseases. Glomerular podocytes and mesangial cells play distinct roles in the metabolism of angiotensin (ANG) peptides. However, our understanding of the RAS enzymatic capacity of glomerular endothelial cells (GEnCs) remains incomplete. We explored the mechanisms of endogenous cleavage of ANG substrates in cultured human GEnCs (hGEnCs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and isotope-labeled peptide quantification. Overall, hGEnCs metabolized ANG II at a significantly slower rate compared with podocytes, whereas the ANG I processing rate was comparable between glomerular cell types. ANG II was the most abundant fragment of ANG I, with lesser amount of ANG-(1-7) detected. Formation of ANG II from ANG I was largely abolished by an ANG-converting enzyme (ACE) inhibitor, whereas ANG-(1-7) formation was decreased by a prolylendopeptidase (PEP) inhibitor, but not by a neprilysin inhibitor. Cleavage of ANG II resulted in partial conversion to ANG-(1-7), a process that was attenuated by an ACE2 inhibitor, as well as by an inhibitor of PEP and prolylcarboxypeptidase. Further fragmentation of ANG-(1-7) to ANG-(1-5) was mediated by ACE. In addition, evidence of aminopeptidase N activity (APN) was demonstrated by detecting amelioration of conversion of ANG III to ANG IV by an APN inhibitor. While we failed to find expression or activity of aminopeptidase A, a modest activity attributable to aspartyl aminopeptidase was detected. Messenger RNA and gene expression of the implicated enzymes were confirmed. These results indicate that hGEnCs possess prominent ACE activity, but modest ANG II-metabolizing activity compared with that of podocytes. PEP, ACE2, prolylcarboxypeptidase, APN, and aspartyl aminopeptidase are also enzymes contained in hGEnCs that participate in membrane-bound ANG peptide cleavage. Injury to specific cell types within the glomeruli may alter the intrarenal RAS balance.

A Dialysis Center Educational Video Intervention Increases Patient Self-Efficacy and Kidney Transplant Evaluations.

Introduction: The optimal treatment for end-stage kidney disease is renal transplant. However, only 1 in 5 (21.5%) patients nationwide receiving dialysis are on a transplant waitlist. Factors associated with patients not initiating a transplant evaluation are complex and include patient specific factors such as transplant knowledge and self-efficacy. Research Question: Can a dialysis center-based educational video intervention increase dialysis patients' transplant knowledge, self-efficacy, and transplant evaluations initiated? Design: Dialysis patients who had not yet completed a transplant evaluation were provided a transplant educational video while receiving hemodialysis. Patients' transplant knowledge, self-efficacy to initiate an evaluation, and dialysis center rates of transplant referral and evaluation were assessed before and after this intervention. Results: Of 340 patients approached at 14 centers, 252 (74%) completed the intervention. The intervention increased transplant knowledge (Likert scale 1 to 5: 2.53 [0.10] vs 4.62 [0.05], P < .001) and transplant self-efficacy (2.55 [0.10] to 4.33 [0.07], P < .001. The incidence rate per 100 patient years of transplant evaluations increased 85% (IRR 1.85 [95% CI: 1.02, 3.35], P = .0422) following the intervention. The incidence rates of referrals also increased 56% (IRR 1.56 [95% CI: 1.03, 2.37], P = .0352), while there was a nonsignificant 47% increase in incidence rates of waitlist entries (IRR 1.47 [95% CI: 0.45, 4.74], P = .5210). Conclusion: This dialysis center-based video intervention provides promising preliminary evidence to conduct a large-scale randomized controlled trial to test its effectiveness in increasing self-efficacy of dialysis patients to initiate a transplant evaluation.

Essential role of c-Cbl in amphiregulin-induced recycling and signaling of the endogenous epidermal growth factor receptor.

The intracellular processing of the epidermal growth factor receptor (EGFR) induced by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) has been studied meticulously, with the former resulting in EGFR degradation and the latter in EGFR recycling to the plasma membrane. However, little is known about how other EGF family growth factors affect the trafficking of the EGFR. Additionally, although both EGF and TGF-alpha have been shown to effectively induce initial c-Cbl (ubiquitin ligase)-mediated ubiquitination of the EGFR, limited information is available regarding the role of c-Cblin the trafficking and signaling of recycling EGFR. Thus, in this study, we investigated the roles of c-Cblin endogenous EGFR trafficking and signaling after stimulation with amphiregulin (AR). We demonstrated that a physiological concentration of AR induced recycling of the endogenous EGFR to the plasma membrane, which correlated closely with transient association of the EGFR with c-Cbl and transient EGFR ubiquitination. Most importantly, we used c-Cbl small interfering RNA (siRNA) duplexes and ac-Cbl dominant negative mutant to show that c-Cbl is critical for the efficient transition of the EGFR from early endosomes to a recycling pathway and that c-Cbl regulates the duration of extracellular signal regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) phosphorylation. These data support novel functions of c-Cbl in mediating recycling of EGF receptors to the plasma membrane, as well as in mediating the duration of activation (transient vs sustained) of ERK1/2 MAPK phosphorylation.

COX-2 expression stimulated by Angiotensin II depends upon AT1 receptor internalization in vascular smooth muscle cells.

Previously, we demonstrated that nuclear localization of the Angiotensin II AT1A receptor was associated with the activation of transcription for the COX-2 gene, PTGS-2. The hypothesis of the present study is that AT1AR internalization from the plasma membrane is a first step in the nuclear localization of the endogenous AT1AR of rat aortic vascular smooth muscle cells and the resultant increase of COX-2 protein expression. Angiotensin II produced both a time- and concentration-dependent increase in COX-2 protein expression in these cells. Treatment with sucrose or phenylarsine oxide, inhibitors of receptor internalization, significantly inhibited AT1AR internalization and abolished the increase in COX-2 protein produced by Angiotensin II without affecting COX-2 expression on its own. Sucrose pre-treatment of rat aortic vascular smooth muscle cells resulted in an increase in p42/44 and p38 activation, while phenylarsine oxide pre-treatment activated only p38 kinase without inhibiting activation of p42/44 produced by Angiotensin II. These results demonstrate that inhibiting the internalization of the AT1AR results in a loss of ability of Angiotensin II to increase the protein expression of COX-2, thus supporting previous work showing a relationship between AT1AR nuclear localization and activation of COX-2 gene expression. Surprisingly, in contrast to other studies, the data also indicates that activation of p42/44 and/or p38 does not correlate with the increased expression of COX-2.

Angiotensin II-induced cyclooxygenase 2 expression in rat aorta vascular smooth muscle cells does not require heterotrimeric G protein activation.

Angiotensin II (AngII) initiates cellular effects via its G protein-coupled angiotensin 1 (AT(1)) receptor (AT(1)R). Previously, we showed that AngII-induced expression of the prostanoid-producing enzyme cyclooxygenase 2 (COX-2) was dependent upon nuclear trafficking of activated AT(1)R. In the present study, mastoparan (an activator of G proteins), suramin (an inhibitor of G proteins), 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122; a specific inhibitor of phospholipase C), and sarcosine(1)-Ile(4)-Ile(8)-AngII (SII-AngII; a G protein-independent AT(1)R agonist) were used to determine the involvement of G proteins and AT(1A)R trafficking in AngII-stimulated COX-2 protein expression in human embryonic kidney-293 cells stably expressing AT(1A)/green fluorescent protein receptors and cultured vascular smooth muscle cells, respectively. Mastoparan alone stimulated release of intracellular calcium and increased COX-2 expression. Preincubation with mastoparan inhibited AngII-induced calcium signaling without altering AngII-induced AT(1A)R trafficking, p42/44 extracellular signal-regulated kinase (ERK) activation, or COX-2 expression. Suramin or U73122 had no significant effect on their own; they did not inhibit AngII-induced AT(1A)R trafficking, p42/44 ERK activation, or COX-2 expression; but they did inhibit AngII-induced calcium responses. SII-AngII stimulated AT(1A)R trafficking and increased COX-2 protein expression without activating intracellular calcium release. These data suggest that G protein activation results in increased COX-2 protein expression, but AngII-induced COX-2 expression seems to occur independently of G protein activation.

An African American Perspective on Familial Notification of Becoming a Registered Organ Donor.

INTRODUCTION: Studies demonstrate that family notification is much less frequent in African Americans than in Caucasians. Familial notification of one's decision to become a registered organ donor (ROD) is important to ensure adherence to the decedent's donation decision and to disseminate prodonation attitudes. The purpose of this study was to explore the experiences of familial notification among recent African American RODs and to identify intervention strategies to overcome potential barriers to the notification process. METHODS/APPROACH: The study used a qualitative focus group approach. An inductive thematic analysis identified common categories and themes in the recorded and transcribed discussions. FINDINGS: The focus groups consisted of 50 African American participants who had recently visited Alabama Department of Motorized Vehicles and made the voluntary decision (yes or no) about becoming an organ donor. Three major themes describing the African American experiences with notifying their family members about their decision to become a ROD emerged. These themes were as follows: motivation for the notification, notification conversation, and promoting familial notification. Specific discussions centered upon the importance of and barriers to familial notification, information, and strategies needed for successful notification. Strategies identified were use of media and social networks to provide enhanced knowledge on the notification process and the importance of health-care, community-provided knowledge about the donation process. DISCUSSION: Findings from this study provide a framework for future interventions designed to assist African American RODs in notifying family members of their status.

Angiotensin II receptors and peritoneal dialysis-induced peritoneal fibrosis.

The vasoactive hormone angiotensin II initiates its major hemodynamic effects through interaction with AT1 receptors, a member of the class of G protein-coupled receptors. Acting through its AT1R, angiotensin II regulates blood pressure and renal salt and water balance. Recent evidence points to additional pathological influences of activation of AT1R, in particular inflammation, fibrosis and atherosclerosis. The transcription factor nuclear factor κB, a key mediator in inflammation and atherosclerosis, can be activated by angiotensin II through a mechanism that may involve arrestin-dependent AT1 receptor internalization. Peritoneal dialysis is a therapeutic modality for treating patients with end-stage kidney disease. The effectiveness of peritoneal dialysis at removing waste from the circulation is compromised over time as a consequence of peritoneal dialysis-induced peritoneal fibrosis. The non-physiological dialysis solution used in peritoneal dialysis, i.e. highly concentrated, hyperosmotic glucose, acidic pH as well as large volumes infused into the peritoneal cavity, contributes to the development of fibrosis. Numerous trials have been conducted altering certain components of the peritoneal dialysis fluid in hopes of preventing or delaying the fibrotic response with limited success. We hypothesize that structural activation of AT1R by hyperosmotic peritoneal dialysis fluid activates the internalization process and subsequent signaling through the transcription factor nuclear factor κB, resulting in the generation of pro-fibrotic/pro-inflammatory mediators producing peritoneal fibrosis.

Clathrin-dependent internalization of the angiotensin II AT1A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells.

The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by ß-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through ß-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors.