Belatacept is used to prevent allograft rejection but fails to do so in a sizable minority of patients due to inadequate control of costimulation-resistant T cells. In this study, we report control of costimulation-resistant rejection when belatacept was combined with perioperative alemtuzumab-mediated lymphocyte depletion and rapamycin. To assess the means by which the alemtuzumab, belatacept and rapamycin (ABR) regimen controls belatacept-resistant rejection, we studied 20 ABR-treated patients and characterized peripheral lymphocyte phenotype and functional responses to donor, third-party and viral antigens using flow cytometry, intracellular cytokine staining and carboxyfluorescein succinimidyl ester-based lymphocyte proliferation. Compared with conventional immunosuppression in 10 patients, lymphocyte depletion evoked substantial homeostatic lymphocyte activation balanced by regulatory T and B cell phenotypes. The reconstituted T cell repertoire was enriched for CD28+ naïve cells, notably diminished in belatacept-resistant CD28- memory subsets and depleted of polyfunctional donor-specific T cells but able to respond to third-party and latent herpes viruses. B cell responses were similarly favorable, without alloantibody development and a reduction in memory subsets - changes not seen in conventionally treated patients. The ABR regimen uniquely altered the immune profile, producing a repertoire enriched for CD28+ T cells, hyporesponsive to donor alloantigen and competent in its protective immune capabilities. The resulting repertoire was permissive for control of rejection with belatacept monotherapy.
Recent evidence suggests that belatacept reduces the durability of preexisting antibodies to class I and class II human leukocyte antigens (HLAs). In this case series of 163 highly sensitized kidney transplant candidates whose calculated panel-reactive antibody (cPRA) activity was ≥98% to 100%, the impact of belatacept on preexisting HLA antibodies was assessed. Of the 163 candidates, 72 underwent transplantation between December 4, 2014 and April 15, 2017; 60 of these transplanted patients remained on belatacept consecutively for at least 6 months. We observed a decrease in the breadth and/or strength of HLA class I antibodies as assessed by FlowPRA in belatacept-treated patients compared to controls who did not receive belatacept. Specifically, significant HLA antibody reduction was evident for class I (P <.0009). Posttransplant belatacept-treated patients also had a clinically significant reduction in their cPRA compared to controls (P <.01). Collectively, these findings suggest belatacept can reduce HLA class I antibodies in a significant proportion of highly sensitized recipients and could be an option to improve pretransplant compatibility with organ donors.
Purpose: Muscle atrophy is a frequent complication of chronic kidney disease (CKD) and is associated with increased morbidity and mortality. The processes causing loss of muscle mass are also present in several catabolic conditions. Understanding the pathogenesis of CKD-induced muscle loss could lead to therapeutic interventions that prevent muscle wasting in CKD and potentially, other catabolic conditions.
Major findings: Insulin or IGF-1 resistance caused by CKD, acidosis, inflammation, glucocorticoids or cancer causes defects in insulin-stimulated intracellular signaling that suppresses IRS-1 activity leading to decreased phosphorylation of Akt (p-Akt). A low p-Akt activates caspase-3 which provides muscle proteins substrates of the ubiquitin-proteasome system (UPS). A low p-Akt also leads to decreased phosphorylation of forkhead transcription factors which enter the nucleus to stimulate the expression of atrogin-1/MAFbx and MuRF1,E3 ubiquitin ligases that can be associated with proteolysis of muscle cells by the UPS. Caspase-3 also stimulates proteasome-dependent proteolysis in muscle.
Summary: In CKD, diabetes, inflammatory conditions or in response to acidosis or excess glucocorticoids, insulin resistance develops, initiating reduced IRS-1/PI3K/Akt signaling. In CKD, this reduces p-Akt which stimulates muscle proteolysis by activating caspase-3 and the UPS. Second, caspase-3 cleaves actomyosin yielding substrates for the UPS and increased proteasome-mediated proteolysis. Third, p-Akt down-regulation suppresses myogenesis in CKD. Fourth, exercise in CKD stimulates insulin/IGF-1 signaling to reduce muscle atrophy. Lastly, there is evidence that microRNAs influence insulin signaling providing a potential opportunity to design therapeutic interventions. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.
Adrenomedullin (ADM) is a vasodilator that causes natriuresis and diuresis. However, the direct effect of ADM on osmotic water permeability in the rat inner medullary collecting duct (IMCD) has not been tested. We investigated whether ADM and its ADM receptor components (CRLR, RAMP2, and 3) are expressed in rat inner medulla (IM) and whether ADM regulates osmotic water permeability in isolated perfused rat IMCDs. The mRNAs of ADM, CRLR, and RAMP2 and 3 were detected in rat IM. Abundant protein of CRLR and RAMP3 were also seen but RAMP2 protein level was extremely low. Adding ADM (100 nM) to the bath significantly decreased osmotic water permeability. ADM significantly decreased aquaporin-2 (AQP2) phosphorylation at Serine 256 (pS256) and increased it at Serine 261 (pS261). ADM significantly increased cAMP levels in IM. However, inhibition of cAMP by SQ22536 further decreased ADM-attenuated osmotic water permeability. Stimulation of cAMP by roflumilast increased ADM-attenuated osmotic water permeability. Previous studies show that ADM also stimulates phospholipase C (PLC) pathways including protein kinase C (PKC) and cGMP. We tested whether PLC pathways regulate ADM-attenuated osmotic water permeability. Blockade of either PLC by U73122 or PKC by rottlerin significantly augmented the ADM-attenuated osmotic water permeability and promoted pS256-AQP2 but did change pS261-AQP2. Inhibition of cGMP by L-NAME did not change AQP2 phosphorylation. In conclusion, ADM primarily binds to the CRLR-RAMP3 receptor to initiate signaling pathways in the IM. ADM reduced water reabsorption through a PLC-pathway involving PKC. ADM-attenuated water reabsorption may be related to decreased trafficking of AQP2 to the plasma membrane. cAMP is not involved in ADM-attenuated osmotic water permeability.
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Alexander G. Vandell;
Maximilian T. Lobmeyer;
Brian E. Gawronski;
Taimour Y. Langaee;
Yan Gong;
John G. Gums;
Amber L. Beitelshees;
Stephen T. Turner;
Arlene B Chapman;
Rhonda M. Cooper-DeHoff;
Kent R. Bailey;
Eric Boerwinkle;
Carl J. Pepine;
Stephen B. Liggett;
Julie A. Johnson
G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized that 3 nonsynonymous GRK4 single-nucleotide polymorphisms, R65L (rs2960306), A142V (rs1024323), and A486V (rs1801058), would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long-term cardiovascular outcomes (all-cause death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 single-nucleotide polymorphisms were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In whites and blacks, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (-9.1±6.8 versus -6.8±7.1 versus -5.3±6.4 mm Hg in participants with 0, 1, and 2 copies of 65L-142V, respectively; P=0.0088). One thousand four hundred sixty participants with hypertension and coronary artery disease from the INternational VErapamil SR/Trandolapril STudy (INVEST) were genotyped, and variant alleles of all 3 GRK4 single-nucleotide polymorphisms were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (odds ratio, 2.29 [1.48-3.55]; P=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest that the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R homozygotes, suggesting a potential interaction between these 2 genes.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
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Michael P. Madaio;
Istvan Czikora;
Nino Kvirkvelia;
Malgorzata McMenamin;
Qiang Yue;
Ting Liu;
Haroldo Toque;
Supriya Sridhar;
Katherine Covington;
Rabei Alaisami;
Paul O'Connor;
Robert W. Caldwell;
Jian-Kang Chen;
Matthias Claus;
Michael W. Brands;
Douglas Eaton;
Maritza Romero;
Rudolf Lucas
In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen.
The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.
Chronic kidney disease (CKD) accelerates muscle protein degradation by stimulating the ubiquitin proteasome system through activation of the E3 ligases, Atrogin-1/MAFbx and MuRF-1. Forkhead transcription factors (FoxOs) can control the expression of these E3 ligases, but the contribution of individual FoxOs to muscle wasting is unclear. To study this we created mice with a muscle-specific FoxO1 deletion. The absence of FoxO1 blocked 70% of the increase in E3 ligase induction by CKD as well as the proteolysis and loss of muscle mass. Thus, FoxO1 has a role in controlling ubiquitin proteasome system-related proteolysis. As microRNA (miR)-486 reportedly dampens FoxO1 expression and its activity, we transfected a miR-486 mimic into primary cultures of myotubes and found this blocked dexamethasone-stimulated protein degradation without influencing protein synthesis. It also decreased FoxO1 protein translation and increased FoxO1 phosphorylation by downregulation of PTEN phosphatase, a negative regulator of p-Akt. To test its efficacy in vivo, we electroporated miR-486 into muscles and found that the expression of the E3 ligases was suppressed and muscle mass increased despite CKD. Thus, FoxO1 is a dominant mediator of CKD-induced muscle wasting, and miR-486 coordinately decreases FoxO1 and PTEN to protect against this catabolic response.
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Vicente E. Torres;
Olivier Devuyst;
Arlene Chapman;
Ron T. Gansevoort;
Ronald D. Perrone;
John Ouyang;
Jaime D. Blais;
Frank S. Czerwiec;
Olga Sergeyeva
Background: In TEMPO 3:4, the vasopressin V2-receptor antagonist tolvaptan slowed kidney growth and function decline in autosomal dominant polycystic kidney disease (ADPKD) patients with relatively preserved kidney function. Methods: Prospective, phase 3b, multi-center, randomized-withdrawal, placebo-controlled, double-blind trial of tolvaptan in ADPKD patients with late stage 2 to early stage 4 chronic kidney disease (CKD). The primary endpoint was estimated glomerular filtration rate (eGFR) change from pre-treatment baseline to post-treatment follow-up. Secondary endpoints included annualized eGFR slope, incidence of ADPKD complications, and overall and hepatic safety profiles. Participants were 18-55 year-old ADPKD patients with baseline eGFR ≥25 and ≤65 mL/min/1.73 m2 or 56-65 year-old with eGFR ≥25 and ≤44 mL/min/1.73 m2 and evidence of eGFR decline >2.0 mL/min/1.73 m2 per year. Daily split doses of tolvaptan were titrated to tolerance (30/15, 45/15, 60/30, or 90/30 mg) and maintained for 12 months, after an 8-week pre-randomization period to screen out subjects unable to tolerate at least 60/30 mg for 3 weeks. Results: Of 1,495 subjects who entered the tolvaptan titration period, 125 (8.4%) discontinued the study before randomization. One thousand three hundred seventy subjects (684 tolvaptan, 686 placebo) from 213 centers across 21 countries were randomized. Baseline demographics were well balanced across treatment arms. Information collected during the study included eGFR, survey scores (PKD history and outcome), adverse events, vital signs, hematology, urinalysis, and serum chemistry tests. Conclusion: Replicating Evidence of Preserved Renal Function: An Investigation of Tolvaptan Safety and Efficacy (REPRISE) determines whether tolvaptan administered over 1 year exhibits disease-modifying properties in ADPKD patients with late stage 2 to early stage 4 CKD, which provides an important therapeutic advancement for this difficult-to-treat disease.
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Juling Zhou;
Xiaosen Ouyang;
Xiangqin Cui;
Trenton R. Schoeb;
Lesley E. Smythies;
Martin R. Johnson;
Lisa M. Guay-Woodford;
Arlene Chapman;
Michal Mrug
Monocyte and macrophage markers are among the most highly overexpressed genes in cpk mouse kidneys with severely progressive renal cystic disease. We show here that one of these markers, CD14, is abnormally transcribed, activated and shed in cystic kidneys. However, these abnormalities were not associated with an increased number of interstitial CD14-positive mononuclear cells. Instead, we found that most non-cystic and cystic renal tubular epithelia were CD14-positive; even distal nephron-derived principal cells. Cd14 was significantly overexpressed in the kidneys of 5-day-old cpk mice and further increased as the disease progressed. In the cpk model with variable rates of cystic kidney enlargement (due to an intercross of two distinct genetic backgrounds), Cd14 expression positively correlated with kidney volume, exceeding the correlation with MCP-1, an established marker of autosomal-dominant polycystic kidney disease (ADPKD). In 16 patients with ADPKD, the baseline urinary CD14 level showed some tendency to correlate with the 2-year change in total kidney volume; however, the tendency was not statistically significant. But the association was significant when the analysis was confined to males. Clearly more studies need to be done to evaluate the utility of CD14 as a marker for outcomes in ADPKD.