A universal urinary cell gene signature of acute rejection in kidney allografts.

INTRODUCTION: Acute rejection (AR) undermines the life-extending benefits of kidney transplantation and is diagnosed using the invasive biopsy procedure. T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (Mixed Rejection [MR]) are the three major types of AR. Development of noninvasive biomarkers diagnostic of AR due to any of the three types is a useful addition to the diagnostic armamentarium. METHODS: We developed customized RT-qPCR assays and measured urinary cell mRNA copy numbers in 145 biopsy-matched urine samples from 126 kidney allograft recipients. We determined whether the urinary cell three-gene signature diagnostic of TCMR (Suthanthiran et al., 2013) discriminates patients with no rejection biopsies (NR, n = 50) from those with ABMR (n = 28) or MR (n = 20) biopsies. RESULTS: The urinary cell three-gene signature discriminated all three types of rejection biopsies from NR biopsies (P < 0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded P < 0.0001 for NR vs. TCMR; P < 0.001 for NR vs. ABMR; and P < 0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (P < 0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (P < 0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (P < 0.0001). All three rejection categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons P > 0.05). CONCLUSION: The urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients.

Transcriptomic signatures of chronic active antibody-mediated rejection deciphered by RNA sequencing of human kidney allografts.

Natural killer (NK) cells mediate spontaneous cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. This dual functionality could enable their participation in chronic active antibody-mediated rejection (CA-ABMR). Earlier microarray profiling studies have not subcategorized antibody-mediated rejection into CA-ABMR and active-ABMR, and the gene expression pattern of CA-ABMR has not been compared with that of T cell-mediated rejection (TCMR). To fill these gaps, we RNA sequenced human kidney allograft biopsies categorized as CA-ABMR, active-ABMR, TCMR, or No Rejection (NR). Among the 15,910 genes identified in the biopsies, 60, 114, and 231 genes were uniquely overexpressed in CA-ABMR, TCMR, and active-ABMR, respectively; compared to NR, 50 genes were shared between CA-ABMR and active-ABMR, and 164 genes between CA-ABMR and TCMR. The overexpressed genes were annotated to NK cells and T cells in CA-ABMR and TCMR, and to neutrophils and monocytes in active-ABMR. The NK cell cytotoxicity and allograft rejection pathways were enriched in CA-ABMR. Genes encoding perforin, granzymes, and death receptor were overexpressed in CA-ABMR versus active-ABMR but not compared to TCMR. NK cell cytotoxicity pathway gene set variation analysis score was higher in CA-ABMR compared to active-ABMR but not in TCMR. Principal component analysis of the deconvolved immune cellular transcriptomes separated CA-ABMR and TCMR from active-ABMR and NR. Immunohistochemistry of kidney allograft biopsies validated a higher proportion of CD56+ NK cells in CA-ABMR than in active-ABMR. Thus, CA-ABMR was exemplified by the overexpression of the NK cell cytotoxicity pathway gene set and, surprisingly, molecularly more like TCMR than active-ABMR.

Urinary cell mRNA profiling of kidney allograft recipients: Development of a portable protocol for noninvasive diagnosis of T cell mediated rejection and BK virus nephropathy.

BACKGROUND: We developed urinary cell mRNA profiling for noninvasive diagnosis of acute T cell mediated rejection (TCMR) and BK virus nephropathy (BKVN), two significant post-transplant complications. Our profiling protocol for the multicenter Clinical Trial of Transplantation-04 (CTOT-04) study consisted of centrifugation of urine to prepare cell pellets, washes, addition of an RNA preservative, storage at 800C and shipment in cold containers to our Gene Expression Monitoring (GEM) Core for RNA isolation and quantification of mRNA in RT-qPCR assays. To simplify profiling, we developed a filter-based protocol (ZFBP) that eliminated the need for centrifugation, RNA preservative, storage at 800C, and shipment in cold containers for mRNA profiling. Furthermore, we trained kidney allograft recipients to perform the filtration of urine at home using the filter and post the urinary cell lysate containing the RNA at ambient temperature to our GEM Core for profiling. Here, we report our refinement of ZFBP and investigation of its diagnostic performance characteristics. METHODS: Total RNA was isolated from kidney allograft biopsy-matched urines using a filter-based protocol complemented by a silica-membrane-based cartridge for mRNA enrichment, the Weill Cornell Hybrid Protocol (WCHP). Absolute copy numbers of CD3ε mRNA, CXCL10 mRNA, and 18S rRNA, components of the CTOT-04 three-gene TCMR diagnostic signature, and urinary cell BKV VP 1 mRNA copy number were measured using RT-qPCR assays. Mann-Whitney test, Fischer exact test, and receiver operating characteristic (ROC) curve analysis were used for data analyses. RESULTS: Urinary cell three-gene TCMR diagnostic signature scores in urines processed using the WCHP discriminated kidney allograft recipients with TCMR (12 TCMR biopsies from 11 patients) from those without TCMR or BKVN (29 No TCMR/No BKVN biopsies from 29 patients). The median (25th and 75th percentiles) score of the CTOT-04 three-gene TCMR diagnostic signature was -0.448 (-1.664, 0.204) in the TCMR group and - 2.542 (-3.267, -1.365) in the No TCMR/ No BKVN group (P = 0.0005, Mann-Whitney test). ROC curve analysis discriminated the TCMR group from the No TCMR/ No BKVN group; the area under the ROC curve (AUROC) was 0.84 (95% Confidence Intervals [CI], 0.69 to 0.98) (P < 0.001), and TCMR was diagnosed with a sensitivity of 67% (95% CI, 35 to 89) at a specificity of 86% (95% CI, 67 to 95) using the CTOT-04 validated cutpoint of -1.213 (P = 0.0016, Fisher exact test). BKV VP1 mRNA copy number in urines processed using the WCHP discriminated patients with BKVN (n = 7) from patients without TCMR or BKVN (n = 29) and the AUROC was 1.0 (95% CI, 1.00 to 1.00) (P < 0.0001) and BKVN was diagnosed with a sensitivity of 86% (95% CI, 42 to 99) at a specificity of 100% (95% CI, 85 to 100) with the previously validated cutpoint of 6.5 × 108 BKV-VP1 mRNA copies per microgram of RNA (P < 0.0001, Fisher exact test). CONCLUSION: Urine processed using the WCHP predicted TCMR and BKVN in kidney allograft recipients. WCHP represents not only a significant advance toward the portability of urinary cell mRNA profiling but also improved patient management by minimizing their visits for urine collection.

Urinary Cell Gene Signature of Acute Rejection in Kidney Allografts.

Introduction: A kidney allograft biopsy may display acute T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (MR). Development of noninvasive biomarkers diagnostic of all three types of acute rejection is a useful addition to the diagnostic armamentarium. Methods: We developed customized RT-qPCR assays and measured urinary cell mRNA copy number in 145 biopsy-matched urine samples from 126 kidney allograft recipients and calculated urinary cell three-gene signature score from log 10 -transformed values for the 18S-normalized CD3E mRNA, 18S-normalized CXCL10 mRNA and 18S rRNA. We determined whether the signature score in biopsy-matched urine specimens discriminates biopsies without rejection (NR, n=50) from biopsies displaying TCMR (n=47), ABMR (n=28) or MR (n=20). Results: Urinary cell three-gene signature discriminated TCMR, ABMR or MR biopsies from NR biopsies (P <0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded P<0.0001 for NR vs. TCMR; P <0.001 for NR vs. ABMR; and P <0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (P<0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (P<0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (P<0.0001). All three rejection biopsy categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons P>0.05). Conclusion: Urinary cell three-gene signature score may serve as a universal diagnostic signature of acute rejection due to TCMR, ABMR or MR in human kidney allografts with similar performance characteristics.

Development of a Bak gene based standard curve for absolute quantification of BK virus in real time quantitative PCR assay and noninvasive diagnosis of BK virus nephropathy in kidney allograft recipients.

BACKGROUND: BK virus nephropathy (BKVN) is a frequent and serious post-transplant complication and undermines realization of the full benefits of kidney transplantation. We developed a Bak amplicon-based standard curve for absolute quantification of BKV VP1 mRNA copy number in the real time quantitative PCR (RT-qPCR) assay and investigated the performance characteristics of this novel assay. METHODS: We determined analytical specificity, sensitivity, and precision of our 73 bp mouse Bak amplicon based standard curve for absolute quantification of BKV VP1 mRNA in RT-qPCR assays. The diagnostic accuracy of the Bak standard curve in the RT-qPCR assay for the noninvasive diagnosis of BKVN in human kidney allograft recipients was investigated by quantification of BKV VP1 mRNA copy number in 192 urine samples matched to 192 kidney allograft biopsies from 155 unique kidney allograft recipients. Intraclass correlation coefficients (ICC) were calculated for the threshold cycles (Ct) and BKV VP1 mRNA copy number observed in the RT-qPCR assay with the Bak standard curve or the BKV standard curve. RESULTS: Performance characteristics of the Bak amplicon-based RT-qPCR assay were exceptional with a slope of -3.291, Y-intercept of 38.60, R2 value of 1.00, efficiency of 101% and error of 0.014. Amplification was specific for the Bak amplicon. Intra assay standard deviation (SD) was 0.08 or less and inter assay SD was 0.11 or less for 31 cycles or less of amplification of the Bak amplicon. Receiver operating characteristic (ROC) curve analysis of BKV VP1 mRNA copy number in 192 biopsy matched urines yielded an area under the ROC of 0.982 (95% CI, 0.964 to 0.999, P < 0.0001) for discriminating patients with BKVN biopsies from patients without BKVN biopsies. The striking identity in the measurement of BKV VP1 mRNA copy numbers in the Bak amplicon-based RT-qPCR assay and in the BKV amplicon-based RT-qPCR assay was shown by an ICC of 1.00 when the Cts were compared, and an ICC of 0.99 when the log10 BKV VP1 mRNA copy numbers were compared. CONCLUSIONS: Bak standard curve for absolute quantification of BKV VP1 mRNA copy number in the RT-qPCR assay demonstrated high efficiency, short and long-term precision and analytical specificity. BKVN was diagnosed with high accuracy. Our new findings, viewed in the light of our earlier demonstration that absolute quantification of a panel of mRNAs encoding immunoregulatory proteins is feasible with the Bak amplicon-based RT-qPCR assays, suggest that the Bak standard curve could serve as a universal calibrator for absolute quantification of transcripts in RT-qPCR assays and help reduce the workload, costs and eliminate contamination of genes of interest by repeated amplification of gene specific standard curves.

Complications of rabbit anti-thymocyte globulin induction immunosuppression in HIV-infected kidney transplant recipients.

Background: Kidney transplantation in HIV-infected individuals with end-stage kidney disease is associated with improved survival compared to dialysis. Rabbit anti-thymocyte globulin (rATG) induction in HIV-infected kidney transplant recipients has been associated with a lower risk of acute rejection, but data on the rates of de novo malignancy and BK viremia in these patients is lacking. Methods: We performed a single-center retrospective cohort study of adult HIV-infected individuals who underwent kidney transplantation with rATG induction between January 2006 and December 2016. The primary outcome was the development of de novo malignancy. Secondary outcomes included the development of BK viremia, infections requiring hospitalization, HIV progression, biopsy-proven acute rejection, and patient and allograft survival. Results: Twenty-seven HIV-infected individuals with end-stage kidney disease received deceased (n=23) or living (n=4) donor kidney transplants. The cumulative rate of malignancy at five years was 29%, of whom 29% died because of advanced malignancy. BK viremia was detected in six participants (22%), of whom one had biopsy-proven BK virus-associated nephropathy and all of whom cleared the BK viremia. Five-year acute rejection rates, patient survival and death-censored allograft survival were 17%, 85% and 80% respectively. Conclusion: rATG induction in HIV-infected kidney transplant recipients was associated with a low risk of acute rejection, but a potentially higher risk of de novo malignancies and BK viremia in this cohort. Screening strategies to closely monitor for BK virus infection and malignancy post-transplantation may improve outcomes in HIV-infected kidney transplant recipients receiving rATG induction.

Urinary cell mRNA profiling of kidney allograft recipients: A systematic investigation of a filtration based protocol for the simplification of urine processing.

BACKGROUND: Kidney transplantation is a life-restorative therapy, but immune rejection undermines allograft survival. Urinary cell mRNA profiles offer a noninvasive means of diagnosing kidney allograft rejection, but urine processing protocols have logistical constraints. We aimed to determine whether the centrifugation-based method for urinary cell mRNA profiling could be replaced with a simpler filtration-based method without undermining quality. METHODS: We isolated RNA from urine collected from kidney allograft recipients using the Cornell centrifugation-based protocol (CCBP) or the Zymo filter-based protocol (ZFBP) and compared RNA purity and yield using a spectrophotometer or a fluorometer and measured absolute copy number of transcripts using customized real-time quantitative PCR assays. We investigated the performance characteristics of RNA isolated using ZFBP and stored either at -80 °C or at ambient temperature for 2 to 4 days and also when shipped to our Gene Expression Monitoring (GEM) Core at ambient temperature. We examined the feasibility of initial processing of urine samples by kidney allograft recipients trained by the GEM Core staff and the diagnostic utility for acute rejection, of urine processed using the ZFBP. RESULTS: RNA purity (P = 0.0007, Wilcoxon matched paired signed-ranks test) and yield (P < 0.0001) were higher with ZFBP vs. CCBP, and absolute copy number of 18S rRNA was similar (P = 0.79) following normalization of RNA yield by reverse transcribing a constant amount of RNA isolated using either protocol. RNA purity, yield, and absolute copy numbers of 18S rRNA, TGF-ß1 mRNA and microRNA-26a were not different (P > 0.05) in the filtrates containing RNA stored either at -80 °C or at ambient temperature for 2 to 4 days or shipped overnight at ambient temperature. RNA purity, yield, and absolute copy numbers of 18S rRNA and TGF-ß1 mRNA were also not different (P > 0.05) between home processed and laboratory processed urine filtrates. Urinary cell levels of mRNA for granzyme B (P = 0.01) and perforin (P = 0.0002) in the filtrates were diagnostic of acute rejection in human kidney allografts. CONCLUSIONS: Urinary cell mRNA profiling was simplified using the ZFBP without undermining RNA quality or diagnostic utility. Home processing by the kidney allograft recipients, the stability of RNA containing filtrates at ambient temperature, and the elimination of the need for centrifuges and freezers represent some of the advantages of ZFBP over the CCBP for urinary cell mRNA profiling.

Urinary Cell mRNA Profiles Predictive of Human Kidney Allograft Status.

Immune monitoring of kidney allograft recipients and personalized therapeutics may help reach the aspirational goal of "one transplant for life." The invasive kidney biopsy procedure, the diagnostic tool of choice, has become safer and the biopsy classification more refined. Nevertheless, biopsy-associated complications, interobserver variability in biopsy specimen scoring, and costs continue to be significant concerns. The dynamics of the immune repertoire make frequent assessments of allograft status necessary, but repeat biopsies of the kidney are neither practical nor safe. To address the existing challenges, we developed urinary cell mRNA profiling and investigated the diagnostic, prognostic, and predictive accuracy of absolute levels of a hypothesis-based panel of mRNAs encoding immunoregulatory proteins. Enabled by our refinements of the PCR assay and by investigating mechanistic hypotheses, our single-center studies identified urinary cell mRNAs associated with T cell-mediated rejection, antibody-mediated rejection, interstitial fibrosis and tubular atrophy, and BK virus nephropathy. In the multicenter National Institutes of Health Clinical Trials in Organ Transplantation-04, we discovered and validated a urinary cell three-gene signature of T-cell CD3 ε chain mRNA, interferon gamma inducible protein 10 (IP-10) mRNA, and 18s ribosomal RNA that is diagnostic of subclinical acute cellular rejection and acute cellular rejection and prognostic of acute cellular rejection and graft function. The trajectory of the signature score remained flat and below the diagnostic threshold for acute cellular rejection in the patients with no rejection biopsy specimens, whereas a sharp rise was observed during the weeks before the biopsy specimen that showed acute cellular rejection. Our RNA sequencing and bioinformatics identified kidney allograft biopsy specimen gene signatures of acute rejection to be enriched in urinary cells matched to acute rejection biopsy specimens. The urinary cellular landscape was more diverse and more enriched for immune cell types compared with kidney allograft biopsy specimens. Urinary cell mRNA profile-guided clinical trials are needed to evaluate their value compared with current standard of care.

COVID-19 outcomes in patients waitlisted for kidney transplantation and kidney transplant recipients.

The COVID-19 pandemic has brought unprecedented challenges to the transplant community. The reduction in transplantation volume during this time is partly due to concerns over potentially increased susceptibility and worsened outcomes of COVID-19 in immunosuppressed recipients. The consequences of COVID-19 on patients waitlisted for kidney transplantation, however, have not previously been characterized. We studied 56 waitlisted patients and 80 kidney transplant recipients diagnosed with COVID-19 between March 13 and May 20, 2020. Despite similar demographics and burden of comorbidities between waitlisted and transplant patients, waitlisted patients were more likely to require hospitalization (82% vs. 65%, P = .03) and were at a higher risk of mortality (34% vs. 16%, P = .02). Intubation was required in one third of hospitalized patients in each group, and portended a very poor prognosis. The vast majority of patients who died were male (84% waitlist, 100% transplant). Multivariate analysis demonstrated waitlist status, age, and male sex were independently associated with mortality. COVID-19 has had a dramatic impact on waitlisted patients, decreasing their opportunities for transplantation and posing significant mortality risk. Understanding the impact of COVID-19 on waitlist patients in comparison to transplant recipients may aid centers in weighing the risks and benefits of transplantation in the setting of ongoing COVID-19.

Kidney allograft recipients, immunosuppression, and coronavirus disease-2019: a report of consecutive cases from a New York City transplant center.

BACKGROUND: Kidney graft recipients receiving immunosuppressive therapy may be at heightened risk for coronavirus disease 2019 (Covid-19) and adverse outcomes. It is therefore important to characterize the clinical course and outcome of Covid-19 in this population and identify safe therapeutic strategies. METHODS: We performed a retrospective chart review of 73 adult kidney graft recipients evaluated for Covid-19 from 13 March to 20 April 2020. Primary outcomes included recovery from symptoms, acute kidney injury, graft failure and case fatality rate. RESULTS: Of the 73 patients screened, 54 tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-39 with moderate to severe symptoms requiring hospital admission and 15 with mild symptoms managed in the ambulatory setting. Hospitalized patients were more likely to be male, of Hispanic ethnicity and to have cardiovascular disease. In the hospitalized group, tacrolimus dosage was reduced in 46% of patients and mycophenolate mofetil (MMF) therapy was stopped in 61% of patients. None of the ambulatory patients had tacrolimus reduction or discontinuation of MMF. Azithromycin or doxycycline was prescribed at a similar rate among hospitalized and ambulatory patients (38% versus 40%). Hydroxychloroquine was prescribed in 79% of hospitalized patients. Graft failure requiring hemodialysis occurred in 3 of 39 hospitalized patients (8%) and 7 patients died, resulting in a case fatality rate of 13% among Covid-19-positive patients and 18% among hospitalized Covid-19-positive patients. CONCLUSIONS: Data from our study suggest that a strategy of systematic triage to outpatient or inpatient care, early management of concurrent bacterial infections and judicious adjustment of immunosuppressive drugs rather than cessation is feasible in kidney transplant recipients with Covid-19.

Correction: Co-Evolution of Mitochondrial tRNA Import and Codon Usage Determines Translational Efficiency in the Green Alga Chlamydomonas.

[This corrects the article DOI: 10.1371/journal.pgen.1002946.].

A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association.

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear-encoded and imported into the mitochondria through post-translational or co-translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome-scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'-UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.

In vitro RNA uptake studies in plant mitochondria.

During evolution, most of the ancestral genes from the endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. To allow the comeback of proteins and RNAs [in particular transfer RNA (tRNAs)] into the organelle, macromolecule import systems were universally established. While protein import processes have been studied into details, much less is known about tRNA mitochondrial import. In plants, part of the knowledge on the tRNA import process into mitochondria has been acquired thanks to in vitro import assays. Furthermore, the development of in vitro RNA import strategies allowed the study of plant mitochondrial gene expression. The purpose of this chapter is to provide detailed protocols to perform in vitro RNA uptake into potato (Solanum tuberosum) or Arabidopsis (Arabidopsis thaliana) mitochondria as well as approaches to analyze them.