Acute Kidney Injury and BK Polyomavirus in Urine Sediment Cells.

Polyomaviruses are widespread, with BK viruses being most common in humans who require immunosuppression due to allotransplantation. Infection with BK polyomavirus (BKV) may manifest as BK virus-associated nephropathy and hemorrhagic cystitis. Established diagnostic methods include the detection of polyomavirus in urine and blood by PCR and in tissue biopsies via immunohistochemistry. In this study, 79 patients with pathological renal retention parameters and acute kidney injury (AKI) were screened for BK polyomavirus replication by RNA extraction, reverse transcription, and virus-specific qPCR in urine sediment cells. A short fragment of the VP2 coding region was the target of qPCR amplification; patients with (n = 31) and without (n = 48) a history of renal transplantation were included. Urine sediment cell immunofluorescence staining for VP1 BK polyomavirus protein was performed using confocal microscopy. In 22 patients with acute renal injury, urinary sediment cells from 11 participants with kidney transplantation (KTX) and from 11 non-kidney transplanted patients (nonKTX) were positive for BK virus replication. BK virus copies were found more frequently in patients with AKI stage III (n = 14). Higher copy numbers were detected in KTX patients having experienced BK polyoma-nephropathy (BKPyVAN) in the past or diagnosed recently by histology (5.6 × 109-3.1 × 1010). One patient developed BK viremia following delayed graft function (DGF) with BK virus-positive urine sediment. In nonKTX patients with BK copies, decoy cells were absent; however, positive staining of cells was found with epithelial morphology. Decoy cells were only found in KTX patients with BKPyVAN. In AKI, damage to the tubular epithelium itself may render the epithelial cells more permissive for polyoma replication. This non-invasive diagnostic approach to assess BK polyomavirus replication in urine sediment cells has the potential to identify KTX patients at risk for viremia and BKPyVAN during AKI. This method might serve as a valuable screening tool for close monitoring and tailored immunosuppression decisions.

Renin-angiotensin system inhibitor discontinuation in COVID-19 did not modify systemic ACE2 in a randomized controlled trial.

Despite the similar clinical outcomes after renin-angiotensin system (RAS) inhibitor (RASi) continuation or withdrawal in COVID-19, the effects on angiotensin-converting enzyme 2 (ACE2) and RAS metabolites remain unclear. In a substudy of the randomized controlled Austrian Corona Virus Adaptive Clinical Trial (ACOVACT), patients with hypertension and COVID-19 were randomized 1:1 to either RASi continuation (n = 30) or switch to a non-RASi medication (n = 29). RAS metabolites were analyzed using a mixed linear regression model (n = 30). Time to a sustained clinical improvement was equal and ACE2 did not differ between the groups but increased over time in both. Overall ACE2 was higher with severe COVID-19. ACE-S and Ang II levels increased as expected with ACE inhibitor discontinuation. These data support the safety of RASi continuation in COVID-19, although RASi were frequently discontinued in our post hoc analysis. The study was not powered to draw definite conclusions on clinical outcomes using small sample sizes.

Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

Relating Molecular T Cell-mediated Rejection Activity in Kidney Transplant Biopsies to Time and to Histologic Tubulitis and Atrophy-fibrosis.

BACKGROUND: We studied the variation in molecular T cell-mediated rejection (TCMR) activity in kidney transplant indication biopsies and its relationship with histologic lesions (particularly tubulitis and atrophy-fibrosis) and time posttransplant. METHODS: We examined 175 kidney transplant biopsies with molecular TCMR as defined by archetypal analysis in the INTERCOMEX study ( ClinicalTrials.gov #NCT01299168). TCMR activity was defined by a molecular classifier. RESULTS: Archetypal analysis identified 2 TCMR classes, TCMR1 and TCMR2: TCMR1 had higher TCMR activity and more antibody-mediated rejection ("mixed") activity and arteritis but little hyalinosis, whereas TCMR2 had less TCMR activity but more atrophy-fibrosis. TCMR1 and TCMR2 had similar levels of molecular injury and tubulitis. Both TCMR1 and TCMR2 biopsies were uncommon after 2 y posttransplant and were rare after 10 y, particularly TCMR1. Within late TCMR biopsies, TCMR classifier activity and activity molecules such as IFNG fell progressively with time, but tubulitis and molecular injury were sustained. Atrophy-fibrosis was increased in TCMR biopsies, even in the first year posttransplant, and rose with time posttransplant. TCMR1 and TCMR2 both reduced graft survival, but in random forests, the strongest determinant of survival after biopsies with TCMR was molecular injury, not TCMR activity. CONCLUSIONS: TCMR varies in intensity but is always strongly related to molecular injury and atrophy-fibrosis, which ultimately explains its effect on survival. We hypothesize, based on the reciprocal relationship with hyalinosis, that the TCMR1-TCMR2 gradient reflects calcineurin inhibitor drug underexposure, whereas the time-dependent decline in TCMR activity and frequency after the first year reflects T-cell exhaustion.

Anti-interleukin-6 Antibody Clazakizumab in Antibody-mediated Renal Allograft Rejection: Accumulation of Antibody-neutralized Interleukin-6 Without Signs of Proinflammatory Rebound Phenomena.

BACKGROUND: Blockade of interleukin-6 (IL-6) has emerged as a promising therapeutic option for antibody-mediated rejection. Subtherapeutic anti-IL-6 antibody level or treatment cessation following prolonged cytokine neutralization may result in proinflammatory rebound phenomena via accumulation of IL-6 and/or modulated gene expression of major components of the IL-6/IL-6 receptor (IL-6R) axis. METHODS: We evaluated biologic material obtained from a randomized controlled, double-blind phase 2 trial designed to evaluate the safety and efficacy of the anti-IL-6 monoclonal antibody clazakizumab in late antibody-mediated rejection. Twenty kidney transplant recipients, allocated to clazakizumab or placebo, received 4-weekly doses over 12 wks, followed by a 40-wk extension where all recipients received clazakizumab. Serum proteins were detected using bead-based immunoassays and RNA transcripts using quantitative real-time polymerase chain reaction (peripheral blood) or microarray analysis (serial allograft biopsies). RESULTS: Clazakizumab treatment resulted in a substantial increase in median total (bound and unbound to drug) serum IL-6 level (1.4, 8015, and 13 600 pg/mL at 0, 12, and 52 wks), but median level of free (unbound to drug) IL-6 did not increase (3.0, 2.3, and 2.3 pg/mL, respectively). Neutralization of IL-6 did not boost soluble IL-6R or leukocyte or allograft expression of IL-6, IL-6R, and glycoprotein 130 mRNA. Cessation of treatment at the end of the trial did not result in a meaningful increase in C-reactive protein or accelerated progression of graft dysfunction during 12 mo of follow-up. CONCLUSION: Our results argue against clinically relevant rebound phenomena and modulation of major components of the IL-6/IL-6R axis following prolonged IL-6 neutralization with clazakizumab.

The systemic renin-angiotensin system in COVID-19.

SARS-CoV-2 gains cell entry via angiotensin-converting enzyme (ACE) 2, a membrane-bound enzyme of the "alternative" (alt) renin-angiotensin system (RAS). ACE2 counteracts angiotensin II by converting it to potentially protective angiotensin 1-7. Using mass spectrometry, we assessed key metabolites of the classical RAS (angiotensins I-II) and alt-RAS (angiotensins 1-7 and 1-5) pathways as well as ACE and ACE2 concentrations in 159 patients hospitalized with COVID-19, stratified by disease severity (severe, n = 76; non-severe: n = 83). Plasma renin activity (PRA-S) was calculated as the sum of RAS metabolites. We estimated ACE activity using the angiotensin II:I ratio (ACE-S) and estimated systemic alt-RAS activation using the ratio of alt-RAS axis metabolites to PRA-S (ALT-S). We applied mixed linear models to assess how PRA-S and ACE/ACE2 concentrations affected ALT-S, ACE-S, and angiotensins II and 1-7. Median angiotensin I and II levels were higher with severe versus non-severe COVID-19 (angiotensin I: 86 versus 30 pmol/L, p < 0.01; angiotensin II: 114 versus 58 pmol/L, p < 0.05), demonstrating activation of classical RAS. The difference disappeared with analysis limited to patients not taking a RAS inhibitor (angiotensin I: 40 versus 31 pmol/L, p = 0.251; angiotensin II: 76 versus 99 pmol/L, p = 0.833). ALT-S in severe COVID-19 increased with time (days 1-6: 0.12; days 11-16: 0.22) and correlated with ACE2 concentration (r = 0.831). ACE-S was lower in severe versus non-severe COVID-19 (1.6 versus 2.6; p < 0.001), but ACE concentrations were similar between groups and correlated weakly with ACE-S (r = 0.232). ACE2 and ACE-S trajectories in severe COVID-19, however, did not differ between survivors and non-survivors. Overall RAS alteration in severe COVID-19 resembled severity of disease-matched patients with influenza. In mixed linear models, renin activity most strongly predicted angiotensin II and 1-7 levels. ACE2 also predicted angiotensin 1-7 levels and ALT-S. No single factor or the combined model, however, could fully explain ACE-S. ACE2 and ACE-S trajectories in severe COVID-19 did not differ between survivors and non-survivors. In conclusion, angiotensin II was elevated in severe COVID-19 but was markedly influenced by RAS inhibitors and driven by overall RAS activation. ACE-S was significantly lower with severe COVID-19 and did not correlate with ACE concentrations. A shift to the alt-RAS axis because of increased ACE2 could partially explain the relative reduction in angiotensin II levels.

Anti-interleukin-6 Antibody Clazakizumab in Antibody-mediated Kidney Transplant Rejection: Effect on Donor-derived Cell-free DNA and C-X-C Motif Chemokine Ligand 10.

Targeting interleukin-6 (IL-6) was shown to counteract donor-specific antibody production and antibody-mediated rejection (AMR) activity. It is not known whether, or to what extent, IL-6 antagonism modulates biomarkers indicative of tissue damage (donor-derived cell-free DNA [dd-cfDNA]) and parenchymal inflammation (C-X-C motif chemokine ligand [CXCL] 10). Methods: We report a secondary endpoint analysis of a phase 2 trial of anti-IL-6 antibody clazakizumab in late AMR (ClinicalTrials.gov, NCT03444103). Twenty kidney transplant recipients were randomized to treatment with clazakizumab or placebo over 12 wk (part A), followed by an extension in which all recipients received clazakizumab through week 52 (part B). Biomarkers were evaluated at day 0 and after 12 and 52 wk, respectively. Results: Fractional dd-cfDNA (dd-cfDNA[%]) did not significantly change under clazakizumab, with no differences between study arms (clazakizumab versus placebo) at week 12 (1.65% [median; interquartile range: 0.91%-2.78%] versus 0.97% [0.56%-2.30%]; P = 0.25) and no significant decrease from weeks 12 to 52 (1.15% [0.70%-2.38%] versus 1.0% [0.61%-1.70%]; P = 0.25). Similarly, urine CXCL10 was not different between groups at week 12 (55.7 [41.0-91.4] versus 60.2 [48.8-208.7.0] pg/mg creatinine; P = 0.44) and did not change over part B (CXCL10 [pg/mg creatinine]: from 58 [46.3-93.1] to 67.4 [41.5-132.0] pg/mL creatinine; P = 0.95). Similar results were obtained for serum CXCL10. There was no association between biomarker levels and resolution of molecular and morphologic AMR activity. Conclusions: Our results suggest that IL-6 blockade does not significantly affect levels of dd-cfDNA[%] and CXCL10. Subtle responses to this therapeutic principle may be overlooked by early biomarker surveillance.

Micellar Curcumin: Pharmacokinetics and Effects on Inflammation Markers and PCSK-9 Concentrations in Healthy Subjects in a Double-Blind, Randomized, Active-Controlled, Crossover Trial.

SCOPE: Preclinical models have demonstrated the anti-inflammatory and lipid-lowering effects of curcumin. Innovative formulations have been developed to overcome the poor bioavailability of native curcumin. The study hypothesizes that the bioavailability of micellar curcumin is superior to native curcumin and investigates the potential anti-inflammatory and proprotein convertase subtilisin/kexin type 9 (PCSK9) concentration lowering effects. METHODS AND RESULTS: In this double-blind, randomized, crossover trial, 15 healthy volunteers receive micellar or native curcumin (105 mg day-1 ) for 7 days with a ≥7 days washout period. Curcumin and metabolite concentrations are quantified by high-performance liquid chromatography with fluorescence detection (HPLC-FD), and pharmacokinetics are calculated. To analyze anti-inflammatory effects, blood samples (baseline, 2 h, 7 days) are stimulated with 50 ng mL-1 lipopolysaccharides (LPS). Interleukin (IL)-6, tumor-necrosis factor (TNF-α), and PCSK9 concentrations are quantified. Micellar curcumin demonstrates improved bioavailability (≈39-fold higher maximum concentrations, ≈14-fold higher area-under-the-time-concentration curve, p < 0.001) but does not reduce pro-inflammatory cytokines in the chosen model. Subjects receiving micellar curcumin have significantly lower PCSK9 concentrations (≈10% reduction) after 7 days compared to baseline (p = 0.038). CONCLUSION: Micellar curcumin demonstrates an improved oral bioavailability but does not show anti-inflammatory effects in this model. Potential effects on PCSK9 concentrations warrant further investigation.

Levels of donor-derived cell-free DNA and chemokines in BK polyomavirus-associated nephropathy.

BACKGROUND: BK polyomavirus-associated nephropathy (BKPyVAN) carries a risk of irreversible allograft injury. While detection of BK viremia and biopsy assessment are the current diagnostic gold standard, the diagnostic value of biomarkers reflecting tissue injury (donor-derived cell-free DNA [dd-cfDNA]) or immune activation (C-X-C motif chemokine ligand [CXCL]9 and CXCL10) remains poorly defined. METHODS: For this retrospective study, 19 cases of BKPyVAN were selected from the Vienna transplant cohort (biopsies performed between 2012 and 2019). Eight patients with T cell-mediated rejection (TCMR), 17 with antibody-mediated rejection (ABMR) and 10 patients without polyomavirus nephropathy or rejection served as controls. Fractions of dd-cfDNA were quantified using next-generation sequencing and CXCL9 and CXCL10 were detected using multiplex immunoassays. RESULTS: BKPyVAN was associated with a slight increase in dd-cfDNA (median; interquartile range: .38% [.27%-1.2%] vs. .21% [.12%-.34%] in non-rejecting control patients; p = .005). Levels were far lower than in ABMR (1.2% [.82%-2.5%]; p = .004]), but not different from TCMR (.54% [.26%-3.56%]; p = .52). Within the BKPyVAN cohort, we found no relationship between dd-cfDNA levels and the extent of tubulo-interstitial infiltrates, BKPyVAN class and BK viremia/viruria, respectively. In some contrast to dd-cfDNA, concentrations of urinary CXCL9 and CXCL10 exceeded those detected in ABMR, but similar increases were also found in TCMR. CONCLUSION: BKPyVAN can induce moderate increases in dd-cfDNA and concomitant high urinary excretion of chemokines, but this pattern may be indistinguishable from that of TCMR. Our results argue against a significant value of these biomarkers to reliably distinguish BKPyVAN from rejection.

Stopping of Mycophenolic Acid in Kidney Transplant Recipients for 2 Weeks Peri-Vaccination Does Not Increase Response to SARS-CoV-2 Vaccination-A Non-randomized, Controlled Pilot Study.

Introduction: Kidney transplant recipients (KTR) are at high risk of developing severe COVID-19. However, vaccine response in this population is severely impaired with humoral response rates of 36-54 and 55-69% after two or three doses of SARS-COV-2 vaccines, respectively. Triple immunosuppression and specifically the use of anti-proliferative agents such as mycophenolic acid (MPA) or azathioprine (AZA) have been identified as risk factors for vaccine hypo-responsiveness. Methods: We hypothesized that in vaccine non-responders to at least three previous vaccine doses, pausing of MPA or AZA for 1 week before and 1 week after an additional vaccination would improve humoral response rates. We conducted an open-label, non-randomized controlled pilot study including 40 KTR with no detectable humoral response after three or four previous vaccine doses. Primary endpoint was seroconversion following SARS-CoV-2 vaccination. MPA and AZA was paused in 18 patients 1 week before until 1 week after an additional vaccine dose while immunosuppression was continued in 22 patients. Results: There was no difference in the humoral response rate between the MPA/AZA pause group and the control group (29 vs. 32%, p > 0.99). Absolute antibody levels were also not statistically significantly different between the two groups (p = 0.716).Renal function in the MPA/AZA pause group remained stable and there was no detection of new onset donor-specific antibodies or an increase of donor-derived cell-free DNA serving as a marker of allograft damage throughout the study period. Conclusion: Pausing of MPA/AZA for 2 weeks peri-vaccination did not increase the rate of seroconversion in kidney transplant. However, one in three KTR without humoral immune response to at least three previous vaccinations developed antibodies after an additional vaccine dose supporting continued vaccination in non-responders.

Early Estimated Glomerular Filtration Rate Trajectories After Kidney Transplant Biopsy as a Surrogate Endpoint for Graft Survival in Late Antibody-Mediated Rejection.

Background: Late antibody-mediated rejection (ABMR) after kidney transplantation is a major cause of long-term allograft loss with currently no proven treatment strategy. Design for trials testing treatment for late ABMR poses a major challenge as hard clinical endpoints require large sample sizes. We performed a retrospective cohort study applying commonly used selection criteria to evaluate the slope of the estimated glomerular filtration rate (eGFR) within an early and short timeframe after biopsy as a surrogate of future allograft loss for clinical trials addressing late ABMR. Methods: Study subjects were identified upon screening of the Vienna transplant biopsy database. Main inclusion criteria were (i) a solitary kidney transplant between 2000 and 2013, (ii) diagnosis of ABMR according to the Banff 2015 scheme at >12 months post-transplantation, (iii) age 15-75 years at ABMR diagnosis, (iv) an eGFR > 25 mL/min/1.73 m2 at ABMR diagnosis, and (v) a follow-up for at least 36 months after ABMR diagnosis. The primary outcome variable was death-censored graft survival. A mixed effects model with linear splines was used for eGFR slope modeling and association of graft failure and eGFR slope was assessed applying a multivariate competing risk analysis with landmarks set at 12 and 24 months after index biopsy. Results: A total of 70 allografts from 68 patients were included. An eGFR loss of 1 ml/min/1.73 m2 per year significantly increased the risk for allograft failure, when eGFR slopes were modeled over 12 months [HR 1.1 (95% CI: 1.01-1.3), p = 0.020] or over 24 months [HR 1.3 (95% CI: 1.1-1.4), p = 0.001] after diagnosis of ABMR with landmarks set at both time points. Covariables influencing graft loss in all models were histologic evidence of glomerulonephritis concurring with ABMR as well as the administration of anti-thymocyte globulin (ATG) at the time of transplantation. Conclusion: Our study supports the use of the eGFR slope modeled for at least 12 months after biopsy-proven diagnosis of late ABMR, as a surrogate parameter for future allograft loss. The simultaneous occurrence of glomerulonephritis together with ABMR at index biopsy and the use of ATG at the time of transplantation-likely representing a confounder in pre-sensitized recipients-were strongly associated with worse transplant outcomes.

Molecular diagnosis of ABMR with or without donor-specific antibody in kidney transplant biopsies: Differences in timing and intensity but similar mechanisms and outcomes.

We studied the clinical, histologic, and molecular features distinguishing DSA-negative from DSA-positive molecularly defined antibody-mediated rejection (mABMR). We analyzed mABMR biopsies with available DSA assessments from the INTERCOMEX study: 148 DSA-negative versus 248 DSA-positive, compared with 864 no rejection (excluding TCMR and Mixed). DSA-positivity varied with mABMR stage: early-stage (EABMR) 56%; fully developed (FABMR) 70%; and late-stage (LABMR) 58%. DSA-negative patients with mABMR were usually sensitized, 60% being HLA antibody-positive. Compared with DSA-positive mABMR, DSA-negative mABMR was more often C4d-negative; earlier by 1.5 years (average 2.4 vs. 3.9 years); and had lower ABMR activity and earlier stage in molecular and histology features. However, the top ABMR-associated transcripts were identical in DSA-negative versus DSA-positive mABMR, for example, NK-associated (e.g., KLRD1 and GZMB) and IFNG-inducible (e.g., PLA1A). Genome-wide class comparison between DSA-negative and DSA-positive mABMR showed no significant differences in transcript expression except those related to lower intensity and earlier time of DSA-negative ABMR. Three-year graft loss in DSA-negative mABMR was the same as DSA-positive mABMR, even after adjusting for ABMR stage. Thus, compared with DSA-positive mABMR, DSA-negative mABMR is on average earlier, less active, and more often C4d-negative but has similar graft loss, and genome-wide analysis suggests that it involves the same mechanisms. SUMMARY SENTENCE: In 398 kidney transplant biopsies with molecular antibody-mediated rejection, the 150 DSA-negative cases are earlier, less intense, and mostly C4d-negative, but use identical molecular mechanisms and have the same risk of graft loss as the 248 DSA-positive cases.

Archetypal Analysis of Injury in Kidney Transplant Biopsies Identifies Two Classes of Early AKI.

All transplanted kidneys are subjected to some degree of injury as a result of the donation-implantation process and various post-transplant stresses such as rejection. Because transplants are frequently biopsied, they present an opportunity to explore the full spectrum of kidney response-to-wounding from all causes. Defining parenchymal damage in transplanted organs is important for clinical management because it determines function and survival. In this study, we classified the scenarios associated with parenchymal injury in genome-wide microarray results from 1,526 kidney transplant indication biopsies collected during the INTERCOMEX study. We defined injury groups by using archetypal analysis (AA) of scores for gene sets and classifiers previously identified in various injury states. Six groups and their characteristics were defined in this population: No injury, minor injury, two classes of acute kidney injury ("AKI," AKI1, and AKI2), chronic kidney disease (CKD), and CKD combined with AKI. We compared the two classes of AKI, namely, AKI1 and AKI2. AKI1 had a poor function and increased parenchymal dedifferentiation but minimal response-to-injury and inflammation, instead having increased expression of PARD3, a gene previously characterized as being related to epithelial polarity and adherens junctions. In contrast, AKI2 had a poor function and increased response-to-injury, significant inflammation, and increased macrophage activity. In random forest analysis, the most important predictors of function (estimated glomerular filtration rate) and graft loss were injury-based molecular scores, not rejection scores. AKI1 and AKI2 differed in 3-year graft survival, with better survival in the AKI2 group. Thus, injury archetype analysis of injury-induced gene expression shows new heterogeneity in kidney response-to-wounding, revealing AKI1, a class of early transplants with a poor function but minimal inflammation or response to injury, a deviant response characterized as PC3, and an increased risk of failure. Given the relationship between parenchymal injury and kidney survival, further characterization of the injury phenotypes in kidney transplants will be important for an improved understanding that could have implications for understanding native kidney diseases (ClinicalTrials.gov #NCT01299168).

Safety, tolerability, and efficacy of monoclonal CD38 antibody felzartamab in late antibody-mediated renal allograft rejection: study protocol for a phase 2 trial.

BACKGROUND: Antibody-mediated rejection (ABMR) is a cardinal cause of renal allograft loss. This rejection type, which may occur at any time after transplantation, commonly presents as a continuum of microvascular inflammation (MVI) culminating in chronic tissue injury. While the clinical relevance of ABMR is well recognized, its treatment, particularly a long time after transplantation, has remained a big challenge. A promising strategy to counteract ABMR may be the use of CD38-directed treatment to deplete alloantibody-producing plasma cells (PC) and natural killer (NK) cells. METHODS: This investigator-initiated trial is planned as a randomized, placebo-controlled, double-blind, parallel-group, multi-center phase 2 trial designed to assess the safety and tolerability (primary endpoint), pharmacokinetics, immunogenicity, and efficacy of the fully human CD38 monoclonal antibody felzartamab (MOR202) in late ABMR. The trial will include 20 anti-HLA donor-specific antibody (DSA)-positive renal allograft recipients diagnosed with active or chronic active ABMR ≥ 180 days post-transplantation. Subjects will be randomized 1:1 to receive felzartamab (16 mg/kg per infusion) or placebo for a period of 6 months (intravenous administration on day 0, and after 1, 2, 3, 4, 8, 12, 16, and 20 weeks). Two follow-up allograft biopsies will be performed at weeks 24 and 52. Secondary endpoints (preliminary assessment) will include morphologic and molecular rejection activity in renal biopsies, immunologic biomarkers in the blood and urine, and surrogate parameters predicting the progression to allograft failure (slope of renal function; iBOX prediction score). DISCUSSION: Based on the hypothesis that felzartamab is able to halt the progression of ABMR via targeting antibody-producing PC and NK cells, we believe that our trial could potentially provide the first proof of concept of a new treatment in ABMR based on a prospective randomized clinical trial. TRIAL REGISTRATION: EU Clinical Trials Register (EudraCT) 2021-000545-40 . Registered on 23 June 2021. CLINICALTRIALS: gov NCT05021484 . Registered on 25 August 2021.

Deletion of the Natural Killer Cell Receptor NKG2C Encoding KLR2C Gene and Kidney Transplant Outcome.

Natural killer (NK) cells may contribute to antibody-mediated rejection (ABMR) of renal allografts. The role of distinct NK cell subsets in this specific context, such as NK cells expressing the activating receptor NKG2C, is unknown. Our aim was to investigate whether KLRC2 gene deletion variants which determine NKG2C expression affect the pathogenicity of donor-specific antibodies (DSA) and, if so, influence long-term graft survival. We genotyped the KLRC2wt/del variants for two distinct kidney transplant cohorts, (i) a cross-sectional cohort of 86 recipients who, on the basis of a positive post-transplant DSA result, all underwent allograft biopsies, and (ii) 1,860 recipients of a deceased donor renal allograft randomly selected from the Collaborative Transplant Study (CTS) database. In the DSA+ patient cohort, KLRC2wt/wt (80%) was associated with antibody-mediated rejection (ABMR; 65% versus 29% among KLRC2wt/del subjects; P=0.012), microvascular inflammation [MVI; median g+ptc score: 2 (interquartile range: 0-4) versus 0 (0-1), P=0.002], a molecular classifier of ABMR [0.41 (0.14-0.72) versus 0.10 (0.07-0.27), P=0.001], and elevated NK cell-related transcripts (P=0.017). In combined analyses of KLRC2 variants and a functional polymorphism in the Fc gamma receptor IIIA gene (FCGR3A-V/F158), ABMR rates and activity gradually increased with the number of risk genotypes. In DSA+ and CTS cohorts, however, the KLRC2wt/wt variant did not impact long-term death-censored graft survival, also when combined with the FCGR3A-V158 risk variant. KLRC2wt/wt may be associated with DSA-triggered MVI and ABMR-associated gene expression patterns, but the findings observed in a highly selected cohort of DSA+ patients did not translate into meaningful graft survival differences in a large multicenter kidney transplant cohort not selected for HLA sensitization.

Corticosteroid Treatment Prevents Lipopolysaccharide-Induced Increase of ACE2 and Reduces Fibrin Degradation Products in Bronchoalveolar Lavage Fluid.

The assessment of systemic corticosteroid effects on intrapulmonary disease biomarkers is challenging. This retrospective evaluation of a human endotoxemia model quantified ACE2 and fibrin degradation product (FDP) concentrations in bronchoalveolar lavage fluid (BALF) samples from a randomized, double-blind, placebo-controlled study (NCT01714427). Twenty-four healthy volunteers received either 2 × 40 mg intravenous dexamethasone or placebo. These doses were administered 12 h apart prior to bronchoscopy-guided intrabronchial lipopolysaccharide (LPS) stimulation (control: saline into the contralateral lung segment). We quantified ACE2 concentration, the Angiotensin-II-to-Angiotensin-1-7 conversion rate as well as FDP in BALF 6 h after LPS instillation. In placebo-treated subjects, LPS instillation increased ACE2 concentrations compared to unstimulated lung segments [1,481 (IQR: 736-1,965) vs. 546 (413-988) pg/mL; p = 0.016]. Dexamethasone abolished the increase in ACE2 concentrations (p=0.13). Accordingly, LPS instillation increased the Angiotensin-II-to-Angiotensin-1-7 conversion capacity significantly in the placebo cohort, indicating increased enzymatic activity (p = 0.012). FDP increased following LPS-instillation [8.9 (2.7-12.2) vs. 6.6 (0.9-9.6) ng/mL, p = 0.025] in the placebo group, while dexamethasone caused a shut-down of fibrinolysis in both lung segments. LPS instillation increased ACE2 concentration, its enzymatic activity and FDP, which was mitigated by systemic dexamethasone treatment. Our results strengthen previously published findings regarding the efficiency of corticosteroids for the treatment of COVID-19-induced acute lung injury.

Comparison of SARS-CoV-2 Antibody Response 4 Weeks After Homologous vs Heterologous Third Vaccine Dose in Kidney Transplant Recipients: A Randomized Clinical Trial.

Importance: Fewer than 50% of kidney transplant recipients (KTRs) develop antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Preliminary data suggest that a heterologous vaccination, combining mRNA and viral vector vaccines, may increase immunogenicity. Objective: To assess the effectiveness of a third dose of an mRNA vs a vector vaccine in KTRs who did not have antibodies against the SARS-CoV-2 spike protein after 2 doses of an mRNA vaccine. Design, Setting, and Participants: This was a single center, single-blinded, 1:1 randomized clinical trial of a third dose of vaccine against SARS-CoV-2, conducted from June 15 to August 16, 2021, in 201 KTRs who had not developed SARS-CoV-2 spike protein antibodies after 2 doses of an mRNA vaccine. Data analyses were performed from August 17 to August 31, 2021. Interventions: mRNA (BNT162b2 or mRNA-1273) or vector (Ad26COVS1) as a third dose of a SARS-CoV-2 vaccine. Main Outcomes and Measures: The primary study end point was seroconversion after 4 weeks (29-42 days) following the third vaccine dose. Secondary end points included neutralizing antibodies and T-cell response assessed by interferon-γ release assays (IGRA). In addition, the association of patient characteristics and vaccine response was assessed using logistic regression, and the reactogenicity of the vaccines was compared. Results: Among the study population of 197 kidney transplant recipients (mean [SD] age, 61.2 [12.4] years; 82 [42%] women), 39% developed SARS-CoV-2 antibodies after the third vaccine. There was no statistically significant difference between groups, with an antibody response rate of 35% and 42% for the mRNA and vector vaccines, respectively. Only 22% of seroconverted patients had neutralizing antibodies. Similarly, T-cell response assessed by IGRA was low with only 17 patients showing a positive response after the third vaccination. Receiving nontriple immunosuppression (odds ratio [OR], 3.59; 95% CI, 1.33-10.75), longer time after kidney transplant (OR, 1.44; 95% CI, 1.15-1.83, per doubling of years), and torque teno virus plasma levels (OR, 0.92; 95% CI, 0.88-0.96, per doubling of levels) were associated with vaccine response. The third dose of an mRNA vaccine was associated with a higher frequency of local pain at the injection site compared with the vector vaccine, while systemic symptoms were comparable between groups. Conclusions and Relevance: This randomized clinical trial found that 39% of KTRs without an immune response against SARS-CoV-2 after 2 doses of an mRNA vaccine developed antibodies against the SARS-CoV-2 spike protein 4 weeks after a third dose of an mRNA or a vector vaccine. The heterologous vaccination strategy with a vector-based vaccine was well tolerated and safe but not significantly better than the homologous mRNA-based strategy. Trial Registration: EudraCT Identifier: 2021-002927-39.

Diagnostic value of donor-derived cell-free DNA to predict antibody-mediated rejection in donor-specific antibody-positive renal allograft recipients.

Circulating donor-specific antibodies (DSA) do not necessarily indicate antibody-mediated rejection (ABMR). Here, we evaluated the diagnostic value of donor-derived cell-free DNA (dd-cfDNA) as an add-on to DSA detection. The study included two independent cohorts of DSA+ kidney allograft recipients, 45 subclinical cases identified by cross-sectional antibody screening (cohort 1), and 30 recipients subjected to indication biopsies (cohort 2). About 50% of the DSA+ recipients had ABMR and displayed higher dd-cfDNA levels than DSA+ ABMR- recipients (cohort 1: 1.90% [median; IQR: 0.78-3.90%] vs. 0.52% [0.35-0.72%]; P < 0.001); (cohort 2: 1.20% [0.82-2.50%] vs. 0.59% [0.28-2.05%]; P = 0.086). Receiver operating characteristic (ROC) analysis revealed an area under the curve (AUC) of 0.89 and 0.69 for dd-cfDNA, and 0.88 and 0.77 for DSA mean fluorescence intensity (MFI), respectively. In combined models, adding dd-cfDNA to DSA-MFI or vice versa significantly improved the diagnostic accuracy. Limited diagnostic performance of dd-cfDNA in cohort 2 was related to the frequent finding of other types of graft injury among ABMR- recipients, like T cell-mediated rejection or glomerulonephritis. For dd-cfDNA in relation to injury of any cause an AUC of 0.97 was calculated. Monitoring of dd-cfDNA in DSA+ patients may be a useful tool to detect ABMR and other types of injury.