Chronic-active T cell-mediated rejection (caTCMR) remains debated, and molecular diagnostics might aid risk-stratification. This retrospective two-center observational study analyzed 26 kidney allograft biopsies with caTCMR, comparing them to 40 borderline T cell-mediated rejection (bTCMR)/acute T cell-mediated rejection (aTCMR) cases (without caTCMR) and 308 subthreshold/negative controls after excluding microvascular inflammation at/above threshold and overlapping pathologies. All biopsies received transcriptomic evaluation through microarray-based gene expression profiling. caTCMR cases with aTCMR (n = 8) showed higher molecular T cell-mediated rejection (TCMR) activity (median probability of molecular TCMR [TCMRprob] 0.54 [0.30-0.83]) than "pure" caTCMR (n = 11; TCMRprob 0.03 [0.01-0.28], P = .012) or caTCMR with bTCMR (n = 7; TCMRprob 0.01 [0.01-0.77], P = .036). TCMRprob was low in subthreshold/negative controls but elevated in aTCMR, BK-virus nephropathy and pyelonephritis cases (side cohort). Molecular sign-outs classified 4 of 11 (36%) "pure" caTCMR cases as molecular TCMR. Within the TCMR continuum (26 caTCMR plus 40 bTCMR/aTCMR), interstitial inflammation, tubulitis, and total inflammation-lesions were associated in univariable analyses with TCMRprob >0.2, while interstitial inflammation in areas of interstitial fibrosis and tubular atrophy/tubulitis in areas of interstitial fibrosis and tubular atrophy correlated with molecular chronicity. Seven of 26 (27%) caTCMR and 7 of 40 (18%) bTCMR/aTCMR cases showed mixed molecular rejection activity above thresholds. In conclusion, significant molecular TCMR activity was present in a subset of caTCMR cases and was associated with higher inflammation/tubulitis/total inflammation scores and, at times, molecular antibody-mediated rejection signals, even with microvascular inflammation <2. To confirm these preliminary observations, future studies and external validation are needed.

Introduction:Although genetic testing is increasingly used in evaluating living kidney donor (LKD) candidates and recipients, objective data on genetic testing practices are limited. This study aimed to describe current genetic testing practices in LKD evaluation using data from the international living donor genetic registry. Methods:A research electronic data capture (REDCap) registry was developed to collect cross-sectional, deidentified information on LKD candidates and genetic test results. Participating centers registered candidates meeting at least 1 of the following criteria: (i) underwent genetic testing; (ii) family history of genetic kidney disease; and/or (iii) were evaluated to donate to a biologically related recipient with kidney disease of unknown etiology. Data were collected between June 1, 2023 and November 10, 2025. Results:Among 1259 LKD evaluations from 24 centers (10 US, 14 international), genetic testing was performed in 295 (23.4%). US donor candidates were younger (median 39 vs. 48 years, P < 0.001). Testing strategies differed by region as follows: international centers predominantly used recipient-first testing (92.1%), whereas US centers more often performed direct LKD testing (61.5%; P < 0.001). These differences persisted after excluding apolipoprotein L-1 (APOL1) testing, with direct LKD candidate testing remaining more common in US (34.0% vs. 7.9%; P < 0.001). Among tested LKD candidates, 20.7% (61/295) were not approved for donation, with 7.8% (23/295) attributed to genetic findings. In multivariable analysis, younger donor age was independently associated with LKD nonacceptance (adjusted odds ratio [OR]: 0.89 per-year; 95% confidence interval [CI]: 0.83-0.95; P < 0.001). Conclusion:Genetic testing practices vary substantially across regions, with US centers favoring direct LKD testing and international centers using recipient-first approaches. Younger donor candidate age was independently associated with nonacceptance.

Because older donor age is a major concern when considering kidneys for potential transplantation, we explored the actual effect of donor age on the features of kidneys that have been transplanted. We studied the correlations of donor age with molecular injury and rejection scores in 4,502 kidney transplant biopsies assessed by microarrays as well as function and postbiopsy survival. We used multivariable analyses to correct for the correlations of donor age with other predictive variables: recipient age, time of biopsy after transplant, and deceased versus living donors. Older donor age correlated with lower glomerular filtration rate (GFR) and increased acute and chronic injury transcripts but had no effect on rejection, which was anticorrelated with recipient age. Acute injury transcripts peaked immediately after transplant and regressed. Older donor age had little effect on acute molecular injury immediately after transplant but strongly increased molecular injury scores at later times, peaking about 1-year after transplant, indicating that older age does not increase molecular injury but increases failed repair after injury. As expected, older donor age correlated with increased chronic injury and lower GFR, evident from the earliest time after transplant, pretransplant aging. However, despite substantial age-related effects, the quantitative contribution of donor aging to molecular injury, function, and survival was very small.

Introduction:Biopsy-based transcriptomics (BBT) supports antibody-mediated rejection (AMR) diagnostics, but Banff 2022 probable AMR and donor-specific antibodies (DSA)-/C4d-microvascular inflammation (MVI) remain unvalidated by BBT. Methods:We analyzed 562 kidney allograft biopsies from Prague and Zurich by histology and Molecular Microscope Diagnostic System (MMDx). Biopsies were classified into the following: (i) active AMR-spectrum lesions without transplant glomerulopathy (cg) (active AMR, probable AMR, MVI, DSA-/C4-); (ii) combined active-chronic lesions with cg (chronic-active AMR, probable AMR with cg, MVI, DSA-/C4d- with cg); (iii) chronic AMR, defined by cg without active lesions; and (iv) controls without rejection. Results:Molecular AMR was detected in 24% of probable AMR and 23% of probable AMR with cg biopsies, 43% of MVI, DSA-/C4d-, 51% of active AMR, 56% of MVI, DSA-/C4d- with cg, and 63% of chronic-active AMR, but was rare in chronic AMR (6%). AMR probability classifier (AMRprob) scores were higher in probable AMR than in controls and similar between chronic AMR and controls. AMRprob scores were comparable among active AMR, chronic-active AMR, and MVI, DSA-/C4d- groups, all of which exhibited elevated T cell-mediated rejection (TCMR) prob scores compared with probable AMR and controls.Least absolute shrinkage and selection operator-penalized Cox regression predicting 3-years graft survival trained in the Zurich cohort showed similar performance of molecular (cgprob, ctprob, tprob, acute kidney injury [AKI]score) and histologic model (Banff cg, interstitial inflammation [ci], tubular atrophy [ct], and tubulitis [t]) (concordance 0.84 vs. 0.82), with the external validation in the Prague cohort (0.78 vs. 0.75). Conclusion:BBT primarily reflects MVI rather than DSA and is particularly informative in probable AMR and MVI-positive biopsies.

KEY POINTS:Single-molecule real-time sequencing with the PacMUC1 script resolved exact MUC1 variable tandem repeat structure and full allelic variation. In 300 individuals, the protocol identified 215 distinct MUC1 tandem repeat alleles with 80 repeat units and nine frameshift mutation types. Probe extension assay identified 90% of families with frameshift mutations, detection of frameshifted mucin-1 aided genetically unresolved cases. BACKGROUND:ADTKD- MUC1 is caused by frameshift mutations in the MUC1 gene, producing a frameshifted neoprotein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with approximately 80% guanine/cytosine content, has made it largely inaccessible to standard short-read sequencing, leaving the reference sequence and natural variation poorly defined and complicating mutation detection. METHODS:Using single-molecule real-time (SMRT) sequencing, we characterized MUC1 VNTR in 300 individuals, including 279 from 143 families suspected of having ADTKD- MUC1 , assessing VNTR length, repeat structure, and frameshift mutations. Results were compared with the Clinical Laboratory Improvement Amendments-approved probe-extension assay, detecting the prevalent 59dupC mutation, and with MUC1fs immunohistochemistry, which detects the pathogenic protein independent of the underlying genomic change. RESULTS:We identified 215 unique VNTR alleles composed of 80 distinct repeat units, 46 (58%) of which were novel, and nine distinct frameshift mutations present on 52 mutated alleles. Overall, MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD- MUC1 , comprising 135 affected individuals (48%). The SMRT assay outperformed the probe-extension assay by identifying frameshift mutations in two families with previously inconclusive results and in eight additional families whose mutations were undetectable by the probe-extension design. When successful, SMRT assay showed 100% concordance with probe-extension assay at the family level and 98% at the individual level, with discordance attributable to allelic dropout inherent to both long-range PCR amplification and long-read sequencing. Analysis of the mutational spectrum confirmed 59dupC as the most prevalent mutation, affecting approximately 90% of families, while the other eight mutation types occurred at most twice. CONCLUSIONS:The SMRT assay outperformed the Clinical Laboratory Improvement Amendments-approved probe-extension assay by detecting essentially all VNTR-associated frameshift mutations. The probe-extension assay identified approximately 90% of affected families. MUC1fs immunohistochemistry added diagnostic value in genetically unresolved cases by detecting the pathogenic protein independent of the underlying mutation.

Donor-derived cell-free DNA (dd-cfDNA) is an emerging tool for noninvasive kidney allograft pathology diagnostics. However, its utility in anti-rejection treatment follow-up remains to be established. We measured dd-cfDNA in 41 kidney transplant recipients (KTRs) at biopsy demonstrating rejection and then weekly for 3 weeks (155 samples). Biopsies were assessed by a pathologist (Banff 2022) and by Molecular Microscope Diagnostic System (MMDx), all KTRs received antirejection therapy. We found that dd-cfDNA fraction decreased in most KTRs after treatment. However, absolute dd-cfDNA decreased significantly only in those treated for T cell-mediated rejection (77 copies/mL [cp/mL] at biopsy, 35 cp/mL at week 3), while there was no significant decrease (93 cp/mL at biopsy, 73 cp/mL at week 3) in patients with antibody-mediated rejection (ABMR). Furthermore, we found that absolute dd-cfDNA did not decrease in KTRs treated with plasma exchange. Finally, there was an unanticipated increase of total cell-free DNA (cfDNA) in all KTRs after treatment. In conclusion, dd-cfDNA follow-up assessments may be confounded by treatment-induced increase of total cfDNA. Thus, absolute dd-cfDNA concentrations, in addition to fraction, might provide more complete information about the response to anti-rejection treatment.

Human leukocyte antigen-incompatible (HLA-i) kidney transplantation enables access to transplantation for highly sensitized patients with preformed donor-specific antibodies (DSA) but remains associated with frequent early DSA rebound and alloimmune endothelial injury, resulting in a high incidence of antibody-mediated rejection (AMR) despite current desensitization and induction protocols that include imlifidase. Kidney allograft biopsy remains the diagnostic gold standard for detecting overt and subclinical AMR, yet conventional histology does not always capture early or subclinical antibody-mediated injury, particularly in sensitized recipients. Tissue-based transcriptomics, Molecular Microscope Diagnostic system (MMDx) and Banff Human Organ Transplant gene panel (B-HOT), provide enhanced sensitivity for detecting molecular AMR phenotypes that often precede morphologic changes and may be particularly informative in the early post-transplant period. Plasma donor-derived cell-free DNA (dd-cfDNA) as non-invasive biomarker correlates with molecular rejection activity and may support longitudinal monitoring in sensitized high-risk patients, although interpretation is limited in the immediate post-transplant period. The integration of histology, DSA monitoring, transcriptomics, and emerging biomarkers enables a multimodal approach to AMR phenotyping, which is particularly critical in HLA-i transplantation to detect subclinical injury early, guide timely intervention, and prevent the development of chronic and largely untreatable transplant glomerulopathy.

Deceased donors with diabetes have been considered suitable for kidney transplant recipients without diabetes. The aim of this study was to identify transcriptomic signatures associated with donor diabetes in zero-hour kidney biopsies, and their changes after transplantation into non-diabetic recipients. Between September 2021 and November 2022, 288 deceased donor zero-hour kidney biopsies were prospectively collected. Biopsies from diabetic donors (Dd+, n = 43) were compared with propensity score–matched biopsies from non-diabetic donors (Dd−, n = 86) using microarray to identify diabetes-associated transcripts. Subsequently, paired analyses of corresponding zero-hour and 3-month protocol biopsies (diabetic donor/non-diabetic recipient pairs (Dd+/Rd−, n = 13)) were performed to study evolution of diabetes-associated transcripts after transplantation and as controls in biopsies from non-diabetic donors transplanted into non-diabetic (Dd−/Rd−, n = 26) or diabetic recipients (Dd−/Rd+, n = 11). Among the 47 upregulated transcripts discriminating diabetic donor kidneys from non-diabetic ones, aldo-ketoreductases (AKR1C2, AKR1B10 and AKR1B15) and fibrosis associated transcripts (VNN1, VCAN, CXCL6 and IGHM, IGHA1, JCHAIN) were prevalent. Kallikrein-kinin system (KLK1, KNG1, and SERPINF2) were among 11 downregulated transcripts. Assessment of transcriptome evolution at 3-months protocol biopsy in Dd+/Rd- revealed decrease in expression of aldo-ketoreductases (AKR1C2, AKR1B10) and NQO1 and increase of SERPINF2, without changes in the expression of these transcripts in controls. We identified four transcripts associated with donor diabetes whose expression attenuated after transplantation in non-diabetic recipients.

The aim of this prospective study was to assess the duration of culture-viable SARS-CoV-2 and to monitor the emergence of mutations in a cohort of 23 kidney transplant recipients (KTRs) from June 2022 to June 2023. Combined nares/oropharyngeal swabs were collected weekly starting as soon as possible after symptom onset. The time from symptom onset to a negative culture was 11 days (interquar tile range, 8-14), while the time to negative reverse transcriptase quantitative polymerase chain reaction was 18 days (inter quartile range, 15-30). Beyond the first swab, 21.7% had a positive culture, and 8.7% replicated viable virus for longer than 30 days. T cell depletion (rate ratio, 2.5; 95% confidence interval [95% CI], 1.9-3.3; P <.001) and time from transplantation (rate ratio, 0.93; 95% CI, 0.90-0.97; P 1/4.006) were associated with the time of viable virus shedding. A cycle threshold value of 24.2 demonstrated a 91.3% negative predictive value of viability (95% credible interval [95% CrI], 76-100). The odds of viability decreased by 69% per week of infection (odds ratio, 0.31; 95% CrI, 0.12-0.76). Overall, ribonucleic acid sequencing did not show accelerated molecular evolution though mutation rate could be increased in molnupiravir-treated KTRs. In conclusion, viable SARS-CoV-2 is eliminated rapidly, the risk of virus evolution is low, and prolonged self-isolation is generally unnecessary for most KTRs.

Background:Biopsy-based transcriptomics (BBT) was implemented in Central Europe in 2022 to improve kidney transplant diagnostics. Differences in diagnostic practices across transplant centers remain underexplored. Methods:This retrospective multicenter study analyzed 474 kidney graft biopsies from 10 transplant centers between August 2022 and May 2024, where, besides routine histology, BBT using the Molecular Microscope Diagnostic System (MMDx) was performed. Differences in BBT indications and discrepancies between histology assessment by Banff 2022 and MMDx sign-outs among transplant centers were evaluated. Results:Most centers used BBT in only 12%-31% of all performed biopsies, relying on histology alone for most diagnostic decisions. BBT indications varied across centers: 3 focused on histological no-rejection with clinical discrepancy (44%, 45%, and 70%), 2 on chronic or chronic-active antibody-mediated rejection (AMR; clinical discrepancy 44% and 48%), and 2 on borderline changes (clinical discrepancy 30% and 33%). BBT showed moderate agreement with histology (κ = 0.49), with similar discrepancy rates between high- and low-volume centers. Molecular AMR was found in 44% of probable AMR, 63% of active AMR, 63% of microvascular inflammation, C4d- and donor-specific antibody (DSA)-, 77% of chronic-active AMR, and 21% chronic AMR. Molecular T cell-mediated rejection (TCMR) was confirmed in 26% of histologically active TCMR, in 9% of chronic TCMR, and in 16% of borderline changes. In histologic no-rejection cases, molecular AMR was present in 10% of DSA- and 34% of DSA+ biopsies. Conclusions:A moderate discrepancy between histology and MMDx sign-outs was found regardless of the center volume. BBT indications notably varied among centers. Standardized indications should be defined to improve the integration of molecular diagnostics into routine care.

It has been previously reported that Caucasian population has superior kidney transplantation outcomes compared to other racial groups, especially in the US. Data from the Czech Republic are lacking. In this single centre retrospective cohort study, we analysed 2315 kidney transplant recipients between years 2014–2024 and identified 24 ethnic minority patients (Asians n = 20, African Europeans n = 4). A control group consisting of Caucasian population was established by propensity score matching in a 3:1 ratio considering recipient and donor age, recipient sex, body mass index (BMI), maximum panel-reactive antibody (PRA max), number of HLA mismatches, living donor, time on dialysis and order of transplantation. The primary endpoint was a composite of poor graft function (defined as a persistent decline in eGFR <40 ml/min/1.73 m2), graft failure, rejection and all-cause mortality at 1- and 5- years post-transplant. Secondary outcomes were individual components of primary endpoint, eGFR at 1- year post-transplant and incidence of delayed graft function. Furthermore, we compared the differences in time on dialysis prior to transplantation, time on the waiting list prior to transplantation and time from starting dialysis to being wait-listed between the racial minority group and the Caucasian population. There was no difference in the incidence of primary composite endpoint between the racial minority group and the Caucasian control group at 1-year (16.7 % vs. 19.4 %, respectively; P = 0.84) and 5-years post-transplant (20.8 % vs. 26.4%, respectively; P = 0.751). There was also no difference in the incidence of secondary outcomes. However, we found that the racial minority group spent longer time on dialysis before being wait-listed compared to the Caucasian population [median 1.2 years (IQR 0.9–1.9) vs. 0.8 years (IQR 0.26–1.64), respectively; P = 0.030]. Furthermore, there was also an evident trend towards longer time on dialysis prior to transplantation in the racial minority group compared to the Caucasian group [median 2.4 years IQR (1.4–4.3) vs. 1.75 years (IQR 0.74–3.3), respectively; P = 0.051] and on the waiting list prior to transplantation [median 1.2 years IQR (0.34–2.7) in racial minority group vs. median 0.56 years IQR (0.16–1.56) in Caucasian population; P = 0.094]. In this study, kidney transplant outcomes were similar between the racial minority group and a well-matched Caucasian control group. As the Czech Republic has universal healthcare coverage, these results suggest that the previously reported discrepancies of transplantation outcomes in racial minority groups likely stem from socioeconomic factors, different healthcare coverage across countries, potential discrimination and language barrier influencing access to healthcare rather than biologic and genetic factors. However, racial minority groups are still disadvantaged by longer time on dialysis before reaching transplantation. Further study of underlying factors is necessary.