Mass cytometry reveals single-cell kinetics of cytotoxic lymphocyte evolution in CMV-infected renal transplant patients.

Cytomegalovirus (CMV) infection is associated with graft rejection in renal transplantation. Memory-like natural killer (NK) cells expressing NKG2C and lacking FcεRIγ are established during CMV infection. Additionally, CD8+ T cells expressing NKG2C have been observed in some CMV-seropositive patients. However, in vivo kinetics detailing the development and differentiation of these lymphocyte subsets during CMV infection remain limited. Here, we interrogated the in vivo kinetics of lymphocytes in CMV-infected renal transplant patients using longitudinal samples compared with those of nonviremic (NV) patients. Recipient CMV-seropositive (R+) patients had preexisting memory-like NK cells (NKG2C+CD57+FcεRIγ-) at baseline, which decreased in the periphery immediately after transplantation in both viremic and NV patients. We identified a subset of prememory-like NK cells (NKG2C+CD57+FcεRIγlow-dim) that increased during viremia in R+ viremic patients. These cells showed a higher cytotoxic profile than preexisting memory-like NK cells with transient up-regulation of FcεRIγ and Ki67 expression at the acute phase, with the subsequent accumulation of new memory-like NK cells at later phases of viremia. Furthermore, cytotoxic NKG2C+CD8+ T cells and γδ T cells significantly increased in viremic patients but not in NV patients. These three different cytotoxic cells combinatorially responded to viremia, showing a relatively early response in R+ viremic patients compared with recipient CMV-seronegative viremic patients. All viremic patients, except one, overcame viremia and did not experience graft rejection. These data provide insights into the in vivo dynamics and interplay of cytotoxic lymphocytes responding to CMV viremia, which are potentially linked with control of CMV viremia to prevent graft rejection.

Multiplexed droplet single-cell sequencing (Mux-Seq) of normal and transplant kidney.

Maintenance of systemic homeostasis by kidney requires the coordinated response of diverse cell types. The use of single-cell RNA sequencing (scRNAseq) for patient tissue samples remains fraught with difficulties with cell isolation, purity, and experimental bias. The ability to characterize immune and parenchymal cells during transplant rejection will be invaluable in defining transplant pathology where tissue availability is restricted to needle biopsy fragments. Herein, we present feasibility data for multiplexing approach for droplet scRNAseq (Mux-Seq). Mux-Seq has the potential to minimize experimental batch bias and variation even with very small sample input. In this first proof-of-concept study for this approach, explant tissues from six normal and two transplant recipients after multiple early post-transplant rejection episodes leading to nephrectomy due to aggressive antibody mediated rejection, were pooled for Mux-Seq. A computational tool, Demuxlet was applied for demultiplexing the individual cells from the pooled experiment. Each sample was also applied individually in a single microfluidic run (singleplex) to correlate results with the pooled data from the same sample. Our applied protocol demonstrated that data from Mux-Seq correlated highly with singleplex (Pearson coefficient 0.982) sequencing results, with the ability to identify many known and novel kidney cell types including different infiltrating immune cells. Trajectory analysis of proximal tubule and endothelial cells demonstrated separation between healthy and injured kidney from transplant explant suggesting evolving stages of cell- specific differentiation in alloimmune injury. This study provides the technical groundwork for understanding the pathogenesis of alloimmune injury and host tissue response in transplant rejection and normal human kidney and provides a protocol for optimized processing precious and low input human kidney biopsy tissue for larger scale studies.

Perturbations in podocyte transcriptome and biological pathways induced by FSGS associated circulating factors.

Background: Focal segmental glomerulosclerosis (FSGS) is frequently associated with heavy proteinuria and progressive renal failure requiring dialysis or kidney transplantation. However, primary FSGS also has a ~40% risk of recurrence of disease in the transplanted kidney (rFSGS). Multiple circulating factors have been identified to contribute to the pathogenesis of primary and rFSGS including soluble urokinase-type plasminogen activator receptor (suPAR) and patient-derived CD40 autoantibody (CD40autoAb). However, the downstream effector pathways specific to individual factors require further study. The tumor necrosis factor, TNF pathway activation by one or more circulating factors present in the sera of patients with FSGS has been supported by multiple studies. Methods: A human in vitro model was used to study podocyte injury measured as the loss of actin stress fibers. Anti-CD40 autoantibody was isolated from FSGS patients (recurrent and non-recurrent) and control patients with ESRD due to non-FSGS related causes. Two novel human antibodies-anti-uPAR (2G10) and anti-CD40 antibody (Bristol Meyer Squibb, 986090) were tested for their ability to rescue podocyte injury. Podocytes treated with patient derived antibody were transcriptionally profiled using whole human genome microarray. Results: Here we show that podocyte injury caused by sera from FSGS patients is mediated by CD40 and suPAR and can be blocked by human anti-uPAR and anti-CD40 antibodies. Transcriptomic studies to compare the molecules and pathways activated in response to CD40 autoantibody from rFSGS patients (rFSGS/CD40autoAb) and suPAR, identified unique inflammatory pathways associated with FSGS injury. Conclusions: We identified several novel and previously described genes associated with FSGS progression. Targeted blockade of suPAR and CD40 pathways with novel human antibodies showed inhibition of podocyte injury in FSGS.

Perturbations of the T-cell immune repertoire in kidney transplant rejection.

In this cross-sectional and longitudinal analysis of mapping the T-cell repertoire in kidney transplant recipients, we have investigated and validated T-cell clonality, immune repertoire chronology at rejection, and contemporaneous allograft biopsy quantitative tissue injury, to better understand the pathobiology of acute T-cell fraction, T-cell repertoire and antibody-mediated kidney transplant rejection. To follow the dynamic evolution of T-cell repertoire changes before and after engraftment and during biopsy-confirmed acute rejection, we sequenced 323 peripheral blood samples from 200 unique kidney transplant recipients, with (n=100) and without (n=100) biopsy-confirmed acute rejection. We report that patients who develop acute allograft rejection, have lower (p=0.01) T-cell fraction even before transplantation, followed by its rise after transplantation and at the time of acute rejection accompanied by high TCR repertoire turnover (p=0.004). Acute rejection episodes occurring after the first 6 months post-transplantation, and those with a component of antibody-mediated rejection, had the highest turnover; p=0.0016) of their T-cell repertoire. In conclusion, we validated that detecting repertoire changes in kidney transplantation correlates with post-transplant rejection episodes suggesting that T-cell receptor sequencing may provide recipient pre-transplant and post-transplant predictors of rejection risk.

A urine score for noninvasive accurate diagnosis and prediction of kidney transplant rejection.

Accurate and noninvasive monitoring of renal allograft posttransplant is essential for early detection of acute rejection (AR) and to affect the long-term survival of the transplant. We present the development and validation of a noninvasive, spot urine-based diagnostic assay based on measurements of six urinary DNA, protein, and metabolic biomarkers. The performance of this assay for detecting kidney injury in both native kidneys and renal allografts is presented on a cohort of 601 distinct urine samples. The urinary composite score enables diagnosis of AR, with a receiver-operator characteristic curve area under the curve of 0.99 and an accuracy of 96%. In addition, we demonstrate the clinical utility of this assay for predicting AR before a rise in the serum creatinine, enabling earlier detection of rejection than currently possible by standard of care tests. This noninvasive, sensitive, and quantitative approach is a robust and informative method for the rapid and routine monitoring of renal allografts.

Corrigendum: Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.

[This corrects the article DOI: 10.3389/fmed.2020.00499.].

Near-Single-Cell Proteomics Profiling of the Proximal Tubular and Glomerulus of the Normal Human Kidney.

Molecular assessments at the single cell level can accelerate biological research by providing detailed assessments of cellular organization and tissue heterogeneity in both disease and health. The human kidney has complex multi-cellular states with varying functionality, much of which can now be completely harnessed with recent technological advances in tissue proteomics at a near single-cell level. We discuss the foundational steps in the first application of this mass spectrometry (MS) based proteomics method for analysis of sub-sections of the normal human kidney, as part of the Kidney Precision Medicine Project (KPMP). Using ~30-40 laser captured micro-dissected kidney cells, we identified more than 2,500 human proteins, with specificity to the proximal tubular (PT; n = 25 proteins) and glomerular (Glom; n = 67 proteins) regions of the kidney and their unique metabolic functions. This pilot study provides the roadmap for application of our near-single-cell proteomics workflow for analysis of other renal micro-compartments, on a larger scale, to unravel perturbations of renal sub-cellular function in the normal kidney as well as different etiologies of acute and chronic kidney disease.

Use of the Tissue Common Rejection Module Score in Kidney Transplant as an Objective Measure of Allograft Inflammation.

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-µm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were read by both pathologists, a total of 40 mismatches in the diagnosis were present. The median tCRM scores for AR, BL, and stable diagnoses were 4.87, 1.85, and 1.27, respectively, with an overall significant difference among all histologic groups (Kruskal-Wallis, p < 0.0001). There were significant differences in tCRM scores between pathologists both finding inflammation vs. disagreement (p = 0.003), and both finding inflammation vs. both finding no inflammation (p < 0.001), along with overall significance between all scores (Kruskal-Wallis, p < 0.001). A logistic regression model predicting graft inflammation using various clinical predictor variables and tCRM revealed the tCRM score as the only significant predictor of graft inflammation (OR: 1.90, 95% CI: 1.40-2.68, p < 0.0001). Accurate, quantitative, and unbiased assessment of rejection of the clinical sample is critical. Given the discrepant diagnoses between pathologists on the same samples, individuals could utilize the tCRM score as a tiebreaker in unclear situations. We propose that the tCRM quantitative score can provide unbiased quantification of graft inflammation, and its rapid evaluation by PCR on the FFPE shave can become a critical adjunct to help drive clinical decision making and immunosuppression delivery.

A urinary Common Rejection Module (uCRM) score for non-invasive kidney transplant monitoring.

A Common Rejection Module (CRM) consisting of 11 genes expressed in allograft biopsies was previously reported to serve as a biomarker for acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. We investigated the use of this gene panel on the urine cell pellet of kidney transplant patients. Urinary cell sediments collected from patients with biopsy-confirmed acute rejection, borderline AR (bAR), BK virus nephropathy (BKVN), and stable kidney grafts with normal protocol biopsies (STA) were analyzed for expression of these 11 genes using quantitative polymerase chain reaction (qPCR). We assessed these 11 CRM genes for their abundance, autocorrelation, and individual expression levels. Expression of 10/11 genes were elevated in AR when compared to STA. Psmb9 and Cxcl10could classify AR versus STA as accurately as the 11-gene model (sensitivity = 93.6%, specificity = 97.6%). A uCRM score, based on the geometric mean of the expression levels, could distinguish AR from STA with high accuracy (AUC = 0.9886) and correlated specifically with histologic measures of tubulitis and interstitial inflammation rather than tubular atrophy, glomerulosclerosis, intimal proliferation, tubular vacuolization or acute glomerulitis. This urine gene expression-based score may enable the non-invasive and quantitative monitoring of AR.

Cell-Free DNA and CXCL10 Derived from Bronchoalveolar Lavage Predict Lung Transplant Survival.

Standard methods for detecting chronic lung allograft dysfunction (CLAD) and rejection have poor sensitivity and specificity and have conventionally required bronchoscopies and biopsies. Plasma cell-free DNA (cfDNA) has been shown to be increased in various types of allograft injury in transplant recipients and CXCL10 has been reported to be increased in the lung tissue of patients undergoing CLAD. This study used a novel cfDNA and CXCL10 assay to evaluate the noninvasive assessment of CLAD phenotype and prediction of survival from bronchoalveolar lavage (BAL) fluid. A total of 60 BAL samples (20 with bronchiolitis obliterans (BOS), 20 with restrictive allograft syndrome (RAS), and 20 with stable allografts (STA)) were collected from 60 unique lung transplant patients; cfDNA and CXCL10 were measured by the ELISA-based KIT assay. Median cfDNA was significantly higher in BOS patients (6739 genomic equivalents (GE)/mL) versus STA (2920 GE/mL) and RAS (4174 GE/mL) (p < 0.01 all comparisons). Likelihood ratio tests revealed a significant association of overall survival with cfDNA (p = 0.0083), CXCL10 (p = 0.0146), and the interaction of cfDNA and CXCL10 (p = 0.023) based on multivariate Cox proportional hazards regression. Dichotomizing patients based on the median cfDNA level controlled for the mean level of CXCL10 revealed an over two-fold longer median overall survival time in patients with low levels of cfDNA. The KIT assay could predict allograft survival with superior performance compared with traditional biomarkers. These data support the pursuit of larger prospective studies to evaluate the predictive performance of cfDNA and CXCL10 prior to lung allograft failure.

A Novel Multi-Biomarker Assay for Non-Invasive Quantitative Monitoring of Kidney Injury.

The current standard of care measures for kidney function, proteinuria, and serum creatinine (SCr) are poor predictors of early-stage kidney disease. Measures that can detect chronic kidney disease in its earlier stages are needed to enable therapeutic intervention and reduce adverse outcomes of chronic kidney disease. We have developed the Kidney Injury Test (KIT) and a novel KIT Score based on the composite measurement and validation of multiple biomarkers across a unique set of 397 urine samples. The test is performed on urine samples that require no processing at the site of collection and without target sequencing or amplification. We sought to verify that the pre-defined KIT test, KIT Score, and clinical thresholds correlate with established chronic kidney disease (CKD) and may provide predictive information on early kidney injury status above and beyond proteinuria and renal function measurements alone. Statistical analyses across six DNA, protein, and metabolite markers were performed on a subset of residual spot urine samples with CKD that met assay performance quality controls from patients attending the clinical labs at the University of California, San Francisco (UCSF) as part of an ongoing IRB-approved prospective study. Inclusion criteria included selection of patients with confirmed CKD and normal healthy controls; exclusion criteria included incomplete or missing information for sample classification, logistical delays in transport/processing of urine samples or low sample volume, and acute kidney injury. Multivariate logistic regression of kidney injury status and likelihood ratio statistics were used to assess the contribution of the KIT Score for prediction of kidney injury status and stage of CKD as well as assess the potential contribution of the KIT Score for detection of early-stage CKD above and beyond traditional measures of renal function. Urine samples were processed by a proprietary immunoprobe for measuring cell-free DNA (cfDNA), methylated cfDNA, clusterin, CXCL10, total protein, and creatinine. The KIT Score and stratified KIT Score Risk Group (high versus low) had a sensitivity and specificity for detection of kidney injury status (healthy or CKD) of 97.3% (95% CI: 94.6-99.3%) and 94.1% (95% CI: 82.3-100%). In addition, in patients with normal renal function (estimated glomerular filtration rate (eGFR) ≥ 90), the KIT Score clearly identifies those with predisposing risk factors for CKD, which could not be detected by eGFR or proteinuria (p < 0.001). The KIT Score uncovers a burden of kidney injury that may yet be incompletely recognized, opening the door for earlier detection, intervention and preservation of renal function.

The common rejection module in chronic rejection post lung transplantation.

RATIONALE: Recent studies suggest that similar injury mechanisms are in place across different solid organ transplants, resulting in the identification of a common rejection module (CRM), consisting of 11 genes that are overexpressed during acute and, to a lesser extent, chronic allograft rejection. OBJECTIVES: We wanted to evaluate the usefulness of the CRM module in identifying acute rejection (AR) and different phenotypes of chronic lung transplant rejection (CLAD), i.e., bronchiolitis obliterans syndrome (BOS) and restrictive allograft syndrome (RAS), using transbronchial brushings, broncho-alveolar lavage (BAL) samples, and explant tissue. METHODS: Gene expression measurements for the 11 CRM genes (CD6, TAP1, CXCL10, CXCL9, INPP5D, ISG20, LCK, NKG7, PSMB9, RUNX3, and BASP1) were performed via qRT-PCR in 14 transbronchial brushings (AR, n = 4; no AR, n = 10), 32 BAL samples (stable, n = 13; AR, n = 8; BOS, n = 9; RAS, n = 10), and 44 tissue specimens (unused donor lungs, n = 15; BOS, n = 13; RAS, n = 16). A geometric mean score was calculated to quantitate overall burden of immune injury and a new computational model was built for the most significant genes in lung transplant injury. RESULTS: Acute rejection showed a significant difference in almost every gene analysed, validating previous observations from microarray analysis. RAS tissue demonstrated a higher geometric mean score (6.35) compared to donor tissue (4.09, p = 0.018). Analysis of individual CRM genes showed an increased expression of ISG20, CXCL10 and CXCL9 in RAS. In BAL samples, no differences were detected in gene expression or geometric mean scores between the various groups (stable, 5.15; AR, 5.81; BOS, 5.62; RAS, 7.31). A newly modelled 2-gene tissue CRM score did not demonstrate any difference between BOS and RAS (p>0.05). However, the model was able to discriminate RAS from BOS tissue (AUC = 0.75, 95% CI = 0.55-0.94, p = 0.025). CONCLUSION: Transcriptional tissue analysis for CRM genes in CLAD can identify acute rejection and distinguish RAS from BOS. The immune activation in RAS seems similar to acute rejection after kidney/liver/heart transplantation.

Optimizing Detection of Kidney Transplant Injury by Assessment of Donor-Derived Cell-Free DNA via Massively Multiplex PCR.

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7⁻99.8%) and 72.6% specificity (95% CI, 65.4⁻79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.

Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system.

CONTEXT: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult. OBJECTIVE: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years. METHODS: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system. RESULTS: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012). CONCLUSIONS: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.

A Computational Gene Expression Score for Predicting Immune Injury in Renal Allografts.

BACKGROUND: Whole genome microarray meta-analyses of 1030 kidney, heart, lung and liver allograft biopsies identified a common immune response module (CRM) of 11 genes that define acute rejection (AR) across different engrafted tissues. We evaluated if the CRM genes can provide a molecular microscope to quantify graft injury in acute rejection (AR) and predict risk of progressive interstitial fibrosis and tubular atrophy (IFTA) in histologically normal kidney biopsies. METHODS: Computational modeling was done on tissue qPCR based gene expression measurements for the 11 CRM genes in 146 independent renal allografts from 122 unique patients with AR (n = 54) and no-AR (n = 92). 24 demographically matched patients with no-AR had 6 and 24 month paired protocol biopsies; all had histologically normal 6 month biopsies, and 12 had evidence of progressive IFTA (pIFTA) on their 24 month biopsies. Results were correlated with demographic, clinical and pathology variables. RESULTS: The 11 gene qPCR based tissue CRM score (tCRM) was significantly increased in AR (5.68 ± 0.91) when compared to STA (1.29 ± 0.28; p < 0.001) and pIFTA (7.94 ± 2.278 versus 2.28 ± 0.66; p = 0.04), with greatest significance for CXCL9 and CXCL10 in AR (p <0.001) and CD6 (p<0.01), CXCL9 (p<0.05), and LCK (p<0.01) in pIFTA. tCRM was a significant independent correlate of biopsy confirmed AR (p < 0.001; AUC of 0.900; 95% CI = 0.705-903). Gene expression modeling of 6 month biopsies across 7/11 genes (CD6, INPP5D, ISG20, NKG7, PSMB9, RUNX3, and TAP1) significantly (p = 0.037) predicted the development of pIFTA at 24 months. CONCLUSIONS: Genome-wide tissue gene expression data mining has supported the development of a tCRM-qPCR based assay for evaluating graft immune inflammation. The tCRM score quantifies injury in AR and stratifies patients at increased risk of future pIFTA prior to any perturbation of graft function or histology.

Transcriptional regulation of the human P450 oxidoreductase gene: hormonal regulation and influence of promoter polymorphisms.

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon. Common human POR polymorphisms at -208 and -173 had little influence on transcription, but the polymorphism at -152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between -249 and -261 and binding of thyroid hormone receptor-ß (TRß) at -240/-245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRß, and estrogen receptor-α were bound between -374 and -149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T(3) exerts major tropic effects via TRß, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T(3) also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRß and Smad3/4 in its expression and indicating that the common polymorphism at -152 may play a role in genetic variation in steroid biosynthesis and drug metabolism.