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.

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.

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.

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.

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.

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.

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.