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.
Acute lung injury leading to acute respiratory distress (ARDS) is a global health concern. ARDS patients have significant pulmonary inflammation leading to flooding of the pulmonary alveoli. This prevents normal gas exchange with consequent hypoxemia and causes mortality. A thin fluid layer in the alveoli is normal. The maintenance of this thin layer results from fluid movement out of the pulmonary capillaries into the alveolar interstitium driven by vascular hydrostatic pressure and then through alveolar tight junctions. This is then balanced by fluid reabsorption from the alveolar space mediated by transepithelial salt and water transport through alveolar cells. Reabsorption is a two-step process: first, sodium enters via sodium-permeable channels in the apical membranes of alveolar type 1 and 2 cells followed by active extrusion of sodium into the interstitium by the basolateral Na + , K + -ATPase. Anions follow the cationic charge gradien t and water follows the salt-induced osmotic gradient. The proximate cause of alveolar flooding is the result of a failure to reabsorb sufficient salt and water or a failure of the tight junctions to prevent excessive movement of fluid from the interstitium to alveolar lumen. Cytokine- and chemokine-induced inflammation can have a particularly profound effect on lung sodium transport since they can alter both ion channel and barrier function. Cytokines and chemokines affect alveolar amiloride-sensitive epithelial sodium channels (ENaCs), which play a crucial role in sodium transport and fluid reabsorption in the lung. This review discusses the regulation of ENaC via local and systemic cytokines during inflammatory disease and the effect on lung fluid balance.
Lymphocyte depletion has been shown to control costimulation blockade-resistant rejection but, in some settings, to exacerbate antibody-mediated rejection (AMR). We have used alemtuzumab, which depletes T and B cells, combined with belatacept and rapamycin and previously reported control of both costimulation blockade-resistant rejection and AMR. To evaluate this regimen's effect on B cell signatures, we investigated 40 patients undergoing this therapy. B cell counts and phenotypes were interrogated using flow cytometry, and serum was analyzed for total IgG, IgM, and donor-specific alloantibody (DSA). Alemtuzumab induction produced pan-lymphocyte depletion; B cells repopulated faster and more completely than T cells. Reconstituting B cells were predominantly naïve, and memory B cells were significantly reduced (P =.001) post repopulation. Two B cell populations with potential immunomodulatory effects—regulatory (CD38hiCD24hiIgMhiCD20hi) and transitional B cells (CD19+CD27−IgD+CD38hi)—were enriched posttransplant (P =.001). Total serum IgG decreased from baseline (P =.016) while IgM levels remained stable. Five patients developed DSAs within 36 months posttransplant, but none developed AMR. Baseline IgG levels in these patients were significantly higher than those in patients without DSAs. These findings suggest that belatacept and rapamycin together limit homeostatic B cell activation following B cell depletion and may lessen the risk of AMR. This regimen warrants prospective, comparative study. ClinicalTrials.gov NCT00565773.
BACKGROUND: Urea transporters (UTs) are important in urine concentration and in urea recycling, and UT-B has been implicated in both. In kidney, UT-B was originally localized to outer medullary descending vasa recta, and more recently detected in inner medullary descending vasa recta. Endogenously produced microRNAs (miRs) bind to the 3'UTR of genes and generally inhibit their translation, thus playing a pivotal role gene regulation. METHODS: Mice were dehydrated for 24 hours then sacrificed. Inner and outer medullas were analyzed by polymerase chain reaction (PCR) and quantitative PCR for miRNA expression and analyzed by western blotting for protein abundance. RESULTS: MiRNA sequencing analysis of mouse inner medullas showed a 40% increase in miRNA-200c in dehydrated mice compared with controls. An in silico analysis of the targets for miR-200c revealed that miRNA-200c could directly target the gene for UT-B. PCR confirmed that miR-200c is up-regulated in the inner medullas of dehydrated mice while western blot showed that UT-B protein abundance was down-regulated in the same portion of the kidney. However, in the outer medulla, miR-200c was reduced and UT-B protein was increased in dehydrated mice. CONCLUSIONS: This is the first indication that UT-B protein and miR-200c may each be differentially regulated by dehydration within the kidney outer and inner medulla. The inverse correlation between the direction of change in miR-200c and UT-B protein abundance in both the inner and outer medulla suggests that miR-200c may be associated with the change in UT-B protein in these 2 portions of the kidney medulla.
Podocytes, dynamic polarized cells wrapped around glomerular capillaries, are an essential component of the glomerular filtration barrier. BK channels consist of one of the slit diaphragm (SD) proteins in podocytes, interact with the actin cytoskeleton, and play vital roles in glomerular filtration. Mechanistic target of rapamycin (mTOR) complexes regulate expression of SD proteins, as well as cytoskeleton structure, in podocytes. However, whether mTOR complexes regulate podocyte BK channels is still unclear. Here, we investigated the mechanism of mTOR complex regulation of BK channels via real-time PCR, western blot, immunofluorescence, and patch clamping. Inhibiting mTORC1 with rapamycin or downregulating Raptor had no significant effect on BK channel mRNA and protein levels and bioactivity. However, the dual inhibitor of mTORC1 and mTORC2 AZD8055 and short hairpin RNA targeting Rictor downregulated BK channel mRNA and protein levels and bioactivity. In addition, MK2206, GF109203X, and GSK650394, which are inhibitors of Akt, PKCα, and SGK1, respectively, were employed to test the downstream signaling pathway of mTORC2. MK2206 and GF109203X had no effect on BK channel protein levels. MK2206 caused an obvious decrease in the current density of the BK channels. Moreover, GSK650394 downregulated the BK channel protein and mRNA levels. These results indicate mTORC2 not only regulates the distribution of BK channels through Akt, but also modulates BK channel protein expression via SGK1 in podocytes.
by
George W Burke;
Jayanthi Chandar;
Junichiro Sageshima;
Mariella Ortigosa-Goggins;
Pooja Amarapurkar;
Alla Mitrofanova;
Marissa J Defreitas;
Chryso P Katsoufis;
Wacharee Seeherunvong;
Alexandra Centeno;
Javier Pagan;
Lumen A Mendez-Castaner;
Adela D Mattiazzi;
Warren L Kupin;
Giselle Guerra;
Linda J Chen;
Mahmoud Morsi;
Jose MG Figueiro;
Rodrigo Vianna;
Carolyn L Abitbol;
David Roth;
Alessia Fornoni;
Phillip Ruiz;
Gaetano Ciancio;
Eduardo H Garin
Background: Primary FSGS manifests with nephrotic syndrome and may recur following KT. Failure to respond to conventional therapy after recurrence results in poor outcomes. Evaluation of podocyte B7-1 expression and treatment with abatacept (a B7-1 antagonist) has shown promise but remains controversial. Methods: From 2012 to 2020, twelve patients developed post-KT FSGS with nephrotic range proteinuria, failed conventional therapy, and were treated with abatacept. Nine/twelve (< 21 years old) experienced recurrent FSGS; three adults developed de novo FSGS, occurring from immediately, up to 8 years after KT. KT biopsies were stained for B7-1. Results: Nine KTRs (75%) responded to abatacept. Seven of nine KTRs were B7-1 positive and responded with improvement/resolution of proteinuria. Two patients with rFSGS without biopsies resolved proteinuria after abatacept. Pre-treatment UPCR was 27.0 ± 20.4 (median 13, range 8–56); follow-up UPCR was 0.8 ± 1.3 (median 0.2, range 0.07–3.9, p < 0.004). Two patients who were B7-1 negative on multiple KT biopsies did not respond to abatacept and lost graft function. One patient developed proteinuria while receiving belatacept, stained B7-1 positive, but did not respond to abatacept. Conclusions: Podocyte B7-1 staining in biopsies of KTRs with post-transplant FSGS identifies a subset of patients who may benefit from abatacept. Graphical abstract: A higher resolution version of the Graphical abstract is available as Supplementary information [Figure not available: see fulltext.]
Uremic cardiomyopathy and muscle atrophy are associated with insulin resistance and contribute to chronic kidney disease (CKD)-induced morbidity and mortality. We hypothesized that restoration of miR-26a levels would enhance exosome-mediated microRNA transfer to improve muscle wasting and cardiomyopathy that occur in CKD.
Methods: Using next generation sequencing and qPCR, we found that CKD mice had a decreased level of miR-26a in heart and skeletal muscle. We engineered an exosome vector that contained Lamp2b, an exosomal membrane protein gene fused with a muscle-specific surface peptide that targets muscle delivery. We transfected this vector into muscle satellite cells and then transduced these cells with adenovirus that expresses miR-26a to produce exosomes encapsulated miR-26a (Exo/miR-26a). Exo/miR-26a was injected once per week for 8 weeks into the tibialis anterior (TA) muscle of 5/6 nephrectomized CKD mice. Results: Treatment with Exo/miR-26a resulted in increased expression of miR-26a in skeletal muscle and heart. Overexpression of miR-26a increased the skeletal muscle cross-sectional area, decreased the upregulation of FBXO32/atrogin-1 and TRIM63/MuRF1 and depressed cardiac fibrosis lesions. In the hearts of CKD mice, FoxO1 was activated, and connective tissue growth factor, fibronectin and collagen type I alpha 1 were increased. These responses were blunted by injection of Exo/miR-26a. Echocardiograms showed that cardiac function was improved in CKD mice treated with Exo/miR-26a.
Conclusion: Overexpression of miR-26a in muscle prevented CKD-induced muscle wasting and attenuated cardiomyopathy via exosome-mediated miR-26a transfer. These results suggest possible therapeutic strategies for using exosome delivery of miR-26a to treat complications of CKD.
The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-ßgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.