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.

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.

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.

Association of High Burden of End-stage Kidney Disease With Decreased Kidney Transplant Rates With the Updated US Kidney Allocation Policy.

Importance: The Organ Procurement and Transplantation Network (OPTN) approved changes to the US kidney allocation system in 2019. The potential effects of this policy change using transplant rates normalized to end-stage kidney disease (ESKD) incidence have not been investigated. Objective: To estimate how the OPTN kidney allocation policy will affect areas of the US currently demonstrating low rates of kidney transplant, when accounting for the regional burden of ESKD. Design, Setting, and Participants: This cross-sectional population-based economic evaluation analyzed access of patients with ESKD to kidney transplant in the US. Participants included patients with incident ESKD, those on the kidney transplant wait list, and those who received a kidney transplant. Data were collected from January 1 to December 31, 2017, and were analyzed in 2019. Main Outcomes and Measures: The probability of a patient with ESKD being placed on the transplant wait list or receiving a deceased donor kidney transplant. States and donor service areas (DSAs) were compared for gains and losses in rates of transplanted kidneys under the new allocation system. Transplant rates were normalized for ESKD burden. Results: A total of 122 659 patients had incident ESKD in the US in 2017 (58.2% men; mean [SD] age, 62.8 [15.1] years). The probability of a patient with ESKD receiving a deceased donor kidney transplant varied 3-fold across the US (from 6.36% in West Virginia to 18.68% in the District of Columbia). Modeling of the OPTN demonstrates that DSAs from New York (124%), Georgia (65%), and Illinois (56%) are estimated to experience the largest increases in deceased donor kidney allocation. Other than Georgia, these states have kidney transplant rates per incident ESKD cases above the mean (of 50 states plus the District of Columbia, New York is 16th and Illinois is 24th). In contrast, DSAs from Nevada (-74%), Ohio (-67%), and North Carolina (-61%)-each of which has a transplant rate per incident ESKD cases significantly below the mean-are estimated to experience the largest decreases in deceased donor allocation (of 50 states plus the District of Columbia, North Carolina is 34th, Ohio is 38th, and Nevada is 47th). Conclusions and Relevance: The new OPTN-approved kidney allocation policy may result in worsening geographic disparities in access to transplants when measured against the burden of ESKD within a particular region of the US.

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.

A Video Intervention to Increase Organ Donor Registration at the Department of Motorized Vehicles.

BACKGROUND: The goal of this study was to increase organ donor registrations at the Department of Motorized Vehicles (DMV) via utilization of a sustainable, low-cost, African American-centric organ donation educational video. Results from previous studies provided a framework to generate a 10-minute video that featured registered organ donors, deceased donor families, and transplant recipients. METHODS: The video was presented via an interrupted time series design (repeating on 2 mo, off 2 mo) on televisions placed in 6 regional DMVs. During the 12-month study, 162 387 patrons visited the DMVs. RESULTS: Increases in organ donor registration were consistently observed in each DMV while the video was on compared with off (mean = +2.3% [range +1.98% to +3.35%]; P < 0.0001). Multivariable analysis demonstrated that females (odds ratio [OR], 1.29; 95% confidence interval [CI], 1.26-1.31), younger age (OR, 0.982/y; 95% CI, 0.982-0.983), and the video intervention (OR, 1.09; 95% CI, 1.07-1.12) were significantly associated with increased registration; while compared with Caucasian race, African American race was not (OR, 0.22; 95% CI, 0.22-0.23). There was no video-dependent effect on registration between Caucasians and African Americans (P = 0.62). Exit interviews demonstrated only 16% of patrons could identify the key message in the video (becoming a registered organ donor). CONCLUSIONS: An educational video promoting organ donation resulted in increased organ donor registration at the DMV. The intervention was equally effective in African Americans and Caucasians. Future efforts should focus upon target-specific messaging and patron consumption of the educational video.

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.

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.

The impact of time-varying clinical surrogates on disparities in African-American kidney transplant recipients - a retrospective longitudinal cohort study.

An improved understanding of the impact of clinical surrogates on disparities in African-American (AA) kidney transplantation (KTX) is needed. We conducted a 10-year retrospective longitudinal cohort study of electronically abstracted clinical data assessing the impact of surrogates on disparities in KTX. Clinical surrogates were assessed by posttransplant year (1, 2, 3 or 4) and defined as acute rejection (Banff ≥1A), mean SBP >140 mmHg, tacrolimus variability (CV) >40%, mean glucose >160 mg/dl and mean hemoglobin <10 g/dl. We utilized landmark methodology to minimize immortal time bias and logistic and survival regression to assess outcomes; 1610 KTX were assessed (54.2% AAs), with 1000, 468, 368 and 303 included in the year 1, 2, 3 and 4 complete case analyses, respectively. AAs had significantly higher odds of developing a clinical surrogate, which increased in posttransplant years three and four [OR year 1 1.99 (1.38-2.88), year 2 1.77 (1.20-2.62), year 3 2.35 (1.49-3.71), year 4 2.85 (1.72-4.70)]. Adjusting for the five clinical surrogates in survival models explained a significant portion of the higher risks of graft loss in AAs in post-transplant years three and four. Results suggest focusing efforts on improving late clinical surrogate management within AAs may help mitigate racial disparities in KTX.

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.

A Novel CLCN5 Mutation Associated With Focal Segmental Glomerulosclerosis and Podocyte Injury.

INTRODUCTION: Tubular dysfunction is characteristic of Dent's disease; however, focal segmental glomerulosclerosis (FSGS) can also be present. Glomerulosclerosis could be secondary to tubular injury, but it remains uncertain whether the CLCN5 gene, which encodes an endosomal chloride and/or hydrogen exchanger, plays a role in podocyte biology. Here, we implicate a role for CLCN5 in podocyte function and pathophysiology. METHODS: Whole exome capture and sequencing of the proband and 5 maternally-related family members was conducted to identify X-linked mutations associated with biopsy-proven FSGS. Human podocyte cultures were used to characterize the mutant phenotype on podocyte function. RESULTS: We identified a novel mutation (L521F) in CLCN5 in 2 members of a Hispanic family who presented with a histologic diagnosis of FSGS and low-molecular-weight proteinuria without hypercalciuria. Presence of CLCN5 was confirmed in cultured human podocytes. Podocytes transfected with the wild-type or the mutant (L521F) CLCN5 constructs showed differential localization. CLCN5 knockdown in podocytes resulted in defective transferrin endocytosis and was associated with decreased cell proliferation and increased cell migration, which are hallmarks of podocyte injury. CONCLUSIONS: The CLCN5 mutation, which causes Dent's disease, may be associated with FSGS without hyercalcuria and nepthrolithiasis. The present findings supported the hypothesis that CLCN5 participates in protein trafficking in podocytes and plays a critical role in organizing the components of the podocyte slit diaphragm to help maintain normal cell physiology and a functional filtration barrier. In addition to tubular dysfunction, mutations in CLCN5 may also lead to podocyte dysfunction, which results in a histologic picture of FSGS that may be a primary event and not a consequence of tubular damage.

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.

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.

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.

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.

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.

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.

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.

Lack of renoprotective effect of chronic intravenous angiotensin-(1-7) or angiotensin-(2-10) in a rat model of focal segmental glomerulosclerosis.

Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8-12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100-400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.

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.