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Background: Among patients with COVID-19, kidney transplant recipients (KTRs) have poor outcomes compared with non-KTRs. To provide insight into management of immunosuppression during acute illness, we studied immune signatures from the peripheral blood during and after COVID-19 infection from a multicenter KTR cohort., Methods: We ascertained clinical data by chart review. A single sample of blood was collected for transcriptome analysis. Total RNA was poly-A selected and RNA was sequenced to evaluate transcriptome changes. We also measured cytokines and chemokines of serum samples collected during acute infection., Results: A total of 64 patients with COVID-19 in KTRs were enrolled, including 31 with acute COVID-19 (<4 weeks from diagnosis) and 33 with post-acute COVID-19 (>4 weeks postdiagnosis). In the blood transcriptome of acute cases, we identified genes in positive or negative association with COVID-19 severity scores. Functional enrichment analyses showed upregulation of neutrophil and innate immune pathways but downregulation of T cell and adaptive immune activation pathways. This finding was independent of lymphocyte count, despite reduced immunosuppressant use in most KTRs. Compared with acute cases, post-acute cases showed "normalization" of these enriched pathways after 4 weeks, suggesting recovery of adaptive immune system activation despite reinstitution of immunosuppression. Analysis of the non-KTR cohort with COVID-19 showed significant overlap with KTRs in these functions. Serum inflammatory cytokines followed an opposite trend ( i.e. , increased with disease severity), indicating that blood lymphocytes are not the primary source., Conclusions: The blood transcriptome of KTRs affected by COVID-19 shows decreases in T cell and adaptive immune activation pathways during acute disease that, despite reduced immunosuppressant use, associate with severity. These pathways show recovery after acute illness., (Copyright © 2022 by the American Society of Nephrology.)

Background: Kidney transplant recipients are at increased risk of severe outcomes during COVID-19. Antibodies against the virus are thought to offer protection, but a thorough characterization of anti-SARS-CoV-2 immune globulin isotypes in kidney transplant recipients following SARS-CoV-2 infection has not been reported., Methods: We performed a cross-sectional study of 49 kidney transplant recipients and 42 immunocompetent controls at early (≤14 days) or late (>14 days) time points after documented SARS-CoV-2 infection. Using a validated semiquantitative Luminex-based multiplex assay, we determined the abundances of IgM, IgG, IgG1-4, and IgA antibodies against five distinct viral epitopes., Results: Kidney transplant recipients showed lower levels of total IgG antitrimeric spike (S), S1, S2, and receptor binding domain (RBD) but not nucleocapsid (NC) at early versus late time points after SARS-CoV-2 infection. Early levels of IgG antispike protein epitopes were also lower than in immunocompetent controls. Anti-SARS-CoV-2 antibodies were predominantly IgG1 and IgG3, with modest class switching to IgG2 or IgG4 in either cohort. Later levels of IgG antispike, S1, S2, RBD, and NC did not significantly differ between cohorts. There was no significant difference in the kinetics of either IgM or IgA antispike, S1, RBD, or S2 on the basis of timing after diagnosis or transplant status., Conclusions: Kidney transplant recipients mount early anti-SARS-CoV-2 IgA and IgM responses, whereas IgG responses are delayed compared with immunocompetent individuals. These findings might explain the poor outcomes in transplant recipients with COVID-19., Podcast: This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2021&#95;11&#95;23&#95;briggsgriffin112321.mp3., (Copyright © 2021 by the American Society of Nephrology.)

Background: Although high-affinity IgG auto- and alloantibodies are important drivers of kidney inflammation that can result in ESKD, therapeutic approaches that effectively reduce such pathogenic antibodies remain elusive. Erythropoietin (EPO) has immunomodulatory functions, but its effects on antibody production are unknown., Methods: We assessed the effect and underlying mechanisms of EPO/EPO receptor (EPOR) signaling on primary and secondary, T cell-dependent and T-independent antibody formation using in vitro culture systems, murine models of organ transplantation and lupus nephritis, and mice conditionally deficient for the EPOR expressed on T cells or B cells., Results: In wild-type mice, recombinant EPO inhibited primary, T cell-dependent humoral immunity to model antigens and strong, polyclonal stimuli, but did not alter T-independent humoral immune responses. EPO also significantly impaired secondary humoral immunity in a potent allogeneic organ transplant model system. The effects required T cell, but not B cell, expression of the EPOR and resulted in diminished frequencies of germinal center (GC) B cells and T follicular helper cells (T FH ). In vitro and in vivo experiments showed that EPO directly prevented T FH differentiation and function via a STAT5-dependent mechanism that reduces CD4 + T cell expression of Bcl6 . In lupus models, EPO reduced T FH , GC B cells, and autoantibody production, and abrogated autoimmune glomerulonephritis, demonstrating clinical relevance. In vitro studies verified that EPO prevents differentiation of human T FH cells., Conclusions: Our findings newly demonstrate that EPO inhibits T FH -dependent antibody formation, an observation with potential implications for treating antibody-mediated diseases, including those of the kidney., (Copyright © 2021 by the American Society of Nephrology.)

Background: The chimeric anti-CD20 monoclonal antibody rituximab is effective in steroid-dependent and calcineurin inhibitor-dependent forms of nephrotic syndrome, but many patients relapse at 1 year. Because ofatumumab, a fully human anti-CD20 monoclonal antibody, has a more extended binding site and higher affinity to CD20 compared with rituximab, it might offer superior efficacy in these patients., Methods: We designed a single-center randomized clinical trial to compare the long-term efficacy of ofatumumab versus rituximab in children and young adults with nephrotic syndrome maintained in remission with prednisone and calcineurin inhibitors. We randomized 140 children and young adults (aged 2-24 years) to receive intravenous ofatumumab (1.50 mg/1.73 m 2 ) or rituximab (375 mg/m 2 ). After infusions, oral drugs were tapered and withdrawn within 60 days. The primary outcome was relapse at 1 year, which was analyzed following the intent-to-treat principle. The secondary endpoint was relapse within 24 months from infusion, on the basis of urine dipstick and confirmed by a urine protein-to-creatinine ratio <200., Results: At 12 months, 37 of 70 (53%) participants who received ofatumumab experienced relapse versus 36 of 70 (51%) who received rituximab (odds ratio [OR], 1.06; 95% confidence interval [95% CI], 0.55 to 2.06). At 24 months, 53 of 70 (76%) participants who received ofatumumab experienced relapse, versus 46 of 70 (66%) who received rituximab (OR, 1.6; 95% CI, 0.8 to 3.3). The two groups exhibited comparable B cell subpopulation reconstitution and did not differ in adverse events., Conclusions: A single dose of ofatumumab was not superior to a single dose of rituximab in maintaining remission in children with steroid-dependent and calcineurin inhibitor-dependent nephrotic syndrome., Clinical Trial Registration Numbers: ClinicalTrials.gov (NCT02394119) and https://www.clinicaltrialsregister.eu/ctr-search/search (2015-000624-28)., (Copyright © 2021 by the American Society of Nephrology.)

Background: In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies., Methods: We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort., Results: Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss., Conclusions: Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients., (Copyright © 2019 by the American Society of Nephrology.)

Background: We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin-binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development., Methods: We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli., Results: Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3-binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration., Conclusions: We demonstrate a novel mechanism that may explain SHROOM3's dichotomous associations in glomerular versus nonglomerular compartments in CKD., (Copyright © 2018 by the American Society of Nephrology.)

Although spontaneous kidney transplant acceptance/tolerance occurs in mice and occasionally in humans, mechanisms remain unclear. Herein we test the hypothesis that EPO, a hormone predominantly produced by the adult kidney, has immunomodulating properties that are required for spontaneous kidney graft acceptance. In vitro , in a manner dependent on the EPO receptor and CD131 on antigen-presenting cells, EPO induced the secretion of active TGF β by antigen-presenting cells, which in turn converted naïve CD4 + T cells into functional Foxp3 + regulatory T cells (Treg). In murine transplant models, pharmacologic downregulation of kidney-derived EPO prevented spontaneous Treg generation. In a controlled, prospective cohort clinical study, EPO administration at doses used to correct anemia augmented the frequency of peripheral CD4 + CD25 + CD127 lo T cells in humans with CKD. Furthermore, EPO directly inhibited conventional T cell proliferation in vitro via tyrosine phosphatase SHP-1-dependent uncoupling of IL-2R β signaling. Conversely, EPO-initiated signals facilitated Treg proliferation by augmenting IL-2R γ signaling and maintaining constitutively quenched IL-2R β signaling. In additional murine transplant models, recombinant EPO administration prolonged heart allograft survival, whereas pharmacologic downregulation of kidney-derived EPO reduced the expression of TGF β mRNA and abrogated kidney allograft acceptance. Together, our findings delineate the protolerogenic properties of EPO in inhibiting conventional T cells while simultaneously promoting Treg induction, and suggest that manipulating the EPO/EPO receptor signaling axis could be exploited to prevent and/or treat T cell-mediated pathologies, including transplant rejection., (Copyright © 2017 by the American Society of Nephrology.)

Correction of anemia with erythropoietin (EPO) is associated with improved kidney transplant outcomes. Emerging evidence, predominantly from animal models, indicates that these observations may be erythropoiesis-independent and that EPO exhibits immunosuppressive properties. We examined the effects of EPO on human T-cell alloimmunity by first documenting that CD4(+) and CD8(+) T cells express EPO receptor (EPO-R) on their surfaces. In mixed lymphocyte reactions, EPO induced a dose-dependent decrease in allogeneic CD4(+) T-cell proliferation (EPO 1000 U/ml: 44.6%±22.9% of vehicle, P<0.05; 2000 U/ml: 11.1%±4% of vehicle, P<0.001) without inducing cell death. The effects required signals transmitted directly through the EPO-R expressed on T cells, resulting in diminished Th1 differentiation without effects on regulatory T-cell induction. Mechanistic studies revealed that EPO prevented IL-2-induced proliferation by uncoupling IL-2 receptor signaling, inhibiting phosphorylation of the intracellular intermediaries AKT and extracellular signal-regulated kinase that are known to mediate T-cell expansion. EPO treatment reduced expansion of human naïve CD4(+) T cells after adoptive transfer into NOD scid γc(null) mouse recipients, verifying the effects in vivo. Although activated T cells expressed CD131, an alternative EPO receptor, addition of a specific CD131 agonist peptide, ARA290, did not alter T-cell proliferation or cytokine production. Our findings link EPO-R signaling on T cells to inhibition of T-cell immunity, providing one mechanism that could explain the observed protective effects of EPO in kidney transplant recipients., (Copyright © 2014 by the American Society of Nephrology.)

The outcome of steroid-dependent or frequently relapsing nephrotic syndrome of minimal change disease (MCD), mesangial proliferative GN (MesGN), or FSGS may be poor and with major treatment toxicity. This academic, multicenter, off-on trial (ClinicalTrials.gov #NCT00981838) primarily evaluated the effects of rituximab therapy followed by immunosuppression withdrawal on disease recurrence in 10 children and 20 adults with MCD/MesGN (n=22) or FSGS who had suffered ≥2 recurrences over the previous year and were in steroid-induced remission for ≥1 month. Participants received one dose (n=28) or two doses of rituximab (375 mg/m(2) intravenously). At 1 year, all patients were in remission: 18 were treatment-free and 15 never relapsed. Compared with the year before rituximab treatment, total relapses decreased from 88 to 22 and the per-patient median number of relapses decreased from 2.5 (interquartile range [IQR], 2-4) to 0.5 (IQR, 0-1; P<0.001) during 1 year of follow-up. Reduction was significant across subgroups (children, adults, MCD/MesGN, and FSGS; P<0.01). After rituximab, the per-patient steroid maintenance median dose decreased from 0.27 mg/kg (IQR, 0.19-0.60) to 0 mg/kg (IQR, 0-0.23) (P<0.001), and the median cumulative dose to achieve relapse remission decreased from 19.5 mg/kg (IQR, 13.0-29.2) to 0.5 mg/kg (IQR, 0-9.4) (P<0.001). Furthermore, the mean estimated GFR increased from 111.3±25.7 to 121.8±29.2 ml/min per 1.73 m(2) (P=0.01), with the largest increases in children and in FSGS subgroups. The mean height z score slope stabilized in children (P<0.01). Treatment was well tolerated. Rituximab effectively and safely prevented recurrences and reduced the need for immunosuppression in steroid-dependent or frequently relapsing nephrotic syndrome, and halted disease-associated growth deficit in children.

As CKD continues to increase worldwide, along with the demand for related life-saving therapies, the financial burden of CKD will place an increasing drain on health care systems. Experimental studies showed that glomerular capillary hypertension and impaired sieving function with consequent protein overload play a pathogenic role in the progression of CKD. Consistently, human studies show that proteinuria is an independent predictor of progression and that its reduction is renoprotective. At comparable BP control, inhibitors of the renin-angiotensin system (RAS), including angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs), more effectively than non-RAS inhibitor therapy reduce proteinuria, slow progression to ESRD, and even improve the kidney function achieving disease regression in some cases. In participants with diabetes, RAS inhibitors delay the onset of microalbuminuria and its progression to macroalbuminuria, and ACE inhibitors may reduce the excess cardiovascular mortality associated with diabetic renal disease. In addition to RAS inhibitors, however, multimodal approaches including lifestyle modifications and multidrug therapy will be required in most cases to optimize control of the several risk factors for CKD and related cardiovascular morbidity. Whether novel medications may help further improve the cost-effectiveness of renoprotective interventions is a matter of investigation.

Selective depletion of B cells with the mAb rituximab may benefit the autoimmune glomerular disease idiopathic membranous nephropathy (IMN). Here, we describe our experience treating 100 consecutive IMN patients with persistent nephrotic syndrome with rituximab. We defined complete remission as persistent proteinuria <0.3 g/24 h and partial remission as persistent proteinuria <3 g/24 h, each also having >50% reduction in proteinuria from baseline. During a median follow-up of 29 months after rituximab administration, 65 patients achieved complete or partial remission. The median time to remission was 7.1 months. All 24 patients who had at least 4 years of follow-up achieved complete or partial remission. Rates of remission were similar between patients with or without previous immunosuppressive treatment. Four patients died and four progressed to ESRD. Measured GFR increased by a mean 13.2 (SD 19.6) ml/min per 1.73 m(2) among those who achieved complete remission. Serum albumin significantly increased and albumin fractional clearance decreased among those achieving complete or partial remission. Proteinuria at baseline and the follow-up duration each independently predicted the decline of proteinuria. Furthermore, the magnitude of proteinuria reduction significantly correlated with slower GFR decline (P=0.0001). No treatment-related serious adverse events occurred. In summary, rituximab achieved disease remission and stabilized or improved renal function in a large cohort of high-risk patients with IMN.

Heavy proteinuria is a major determinant of progression to ESRD for patients with chronic nephropathies and reducing proteinuria should be a key target for renoprotective therapy. In the Remission Clinic, we applied a multimodal intervention to target urinary proteins in 56 consecutive patients who had >3 g proteinuria/d despite angiotensin-converting enzyme inhibitor therapy. We compared the rate of GFR decline and incidence of ESRD in this cohort with 56 matched historical reference subjects who had received conventional therapy titrated to a target BP. During a median follow-up of 4 yr, the monthly rate of GFR decline was significantly lower in the Remission Clinic cohort (median -0.17 versus -0.56 ml/min per 1.73 m2; P < 0.0001), and ESRD events were significantly reduced (3.6 versus 30.4% reached ESRD). Follow-up BP, cholesterol, and proteinuria were lower in Remission Clinic patients than in reference subjects, such that disease remission or regression was achieved in up to 50% of patients who would have been otherwise expected to progress rapidly to ESRD on conventional therapy. Proteinuria reduction independently predicted a slower rate of GFR decline and ESRD incidence, but response to treatment differed depending on the underlying disease. Regarding safety, no patient was with drawn because of hyperkalemia. In summary, multidrug treatment titrated to urinary protein level can be safely and effectively applied to normalize proteinuria and to slow the loss of renal function significantly,especially among patients without type 2 diabetes and with otherwise rapidly progressing chronic nephropathies.

The Mycophenolate Steroids Sparing (MYSS) study found that in renal transplant recipients who were on immunosuppressive therapy with the cyclosporine microemulsion Neoral, mycophenolate mofetil (MMF) was not better than azathioprine in preventing acute rejection at 21 mo after transplantation and was 15 times more expensive. The MYSS Follow-up Study, an extension of MYSS, was aimed at comparing long-term outcome of 248 MYSS patients according to their original randomization to MMF (1 g twice daily) or azathioprine (75 to 100 mg/d). Primary outcome was estimated GFR at 5 yr after transplantation. Mean 5-yr GFR difference between azathioprine and mycophenolate was 4.67 ml/min per 1.73 m(2) (95% confidence interval [CI] -0.43 to 9.77 ml/min per 1.73 m(2); P = 0.07). GFR from month 6 (mean +/- SEM: 54.3 +/- 1.6 versus 53.9 +/- 1.5 ml/min per 1.73 m(2); P = 0.83) to month 72 after transplantation (49.5 +/- 2.2 versus 47.3 +/- 2.4 ml/min per 1.73 m(2); P = 0.50); GFR slopes (mean +/- SEM: -1.10 +/- 0.56 versus -1.23 +/- 0.31 ml/min per 1.73 m(2) per year; P = 0.83); and 72-mo patient mortality (4.0 versus 4.0% [P = 0.95]; HR 0.96; 95% CI 0.28 to 3.31; P = 0.95), graft loss (6.8 versus 6.1% [P = 0.82]; HR 0.89; 95% CI 0.32 to 2.46; P = 0.83), incidence of persistent proteinuria (25.0 versus 27.4%; P = 0.72), late (>6 mo after transplantation) rejections (25.3 versus 21.2%; P = 0.53), and adverse events were similar on azathioprine (n = 124) and MMF (n = 124), respectively. Outcomes in the two groups were comparable also among patients with or without steroid therapy, considered separately. In kidney transplantation, the long-term risk/benefit profile of MMF and azathioprine therapy in combination with cyclosporine Neoral is similar. In view of the cost, standard immunosuppression regimens for kidney transplantation should perhaps include azathioprine rather than MMF.

Allogeneic immune responses are modulated by a subset of host T cells with regulatory function (Treg) contained within the CD4(+)CD25(high) subset. Evidence exists that Treg expand after peritransplantation lymphopenia, inhibit graft rejection, and induce and maintain tolerance. Little, however, is known about the role of Treg in the clinical setting. IL-2 and activation by T cell receptor engagement are instrumental to generate and maintain Treg, but the influence of immunosuppressants on Treg homeostasis in humans in vivo has not been investigated. This study monitored Treg phenotype and function during immune reconstitution in renal transplant recipients who underwent profound T cell depletion with Campath-1H and received sirolimus or cyclosporine (CsA) as part of their maintenance immunosuppressive therapy. CD4(+)CD25(high) cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus- but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4(+)CD25(high) Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.