Blaise Robin

Key Points: Normalization methods affected the performance of gene-based diagnostic classifiers for kidney transplant rejection across a large multicenter cohort. Complex normalization reduced classifier performance by overcorrecting signal, while simpler methods preserved rejection signature and key robustness. By identifying optimal normalization methods, this work advances a standardized preprocessing framework for molecular diagnostics in transplantation. Background: The Banff 2022 classification endorses intragraft gene expression profiling using the Banff Human Organ Transplant (B-HOT) consensus gene panel for rejection diagnosis. However, lack of standardized analytical pipelines, including data normalization, limits clinical implementation, with its effect on diagnostic performance yet to be determined. Methods: We evaluated ten normalization methods in 868 kidney allograft biopsies from nine European and North American centers, all Banff-graded and B-HOT–profiled on nCounter, comprising derivation ( n =441), internal ( n =186), and external ( n =241) validation cohorts. Each method was assessed through its downstream impact on ( 1 ) gene count stability, ( 2 ) differential expression and cross-platform concordance with RNA sequencing (RNA-seq) data, and ( 3 ) discrimination and calibration of predictive models for antibody-mediated rejection (AMR) and T -cell–mediated rejection (TCMR). Results: Most methods improved count stability and showed high concordance with RNA-seq for overall gene expression. They also produced robust differential expression signatures consistent with those detected by RNA-seq, except for RUVSeq and RCRNorm , which identified fewer differentially expressed genes and showed lower concordance. In the overall validation cohort ( n =427), diagnostic performance was consistently high across nSolver -based approaches, nanostringr , NanoStringDiff , MetaNorm , and RCRNormFast (AMR, area under the ROC curve [AUROC], 0.88–0.91; area under precision-recall curves [AUPRC], 0.86–0.89; TCMR AUROC, 0.90–0.92; AUPRC, 0.78–0.83). Performance declined with RCRNorm (AMR AUROC/AUPRC, 0.55/0.41; TCMR, 0.53/0.18) and, for TCMR, with RUVSeq (AUROC, 0.84–0.85; AUPRC, 0.64–0.65). Calibration was satisfactory for most methods, except for RCRNorm and for TCMR models after RUVSeq . Conclusions: Normalization choice significantly impacted gene expression profiles and diagnostic classifier performance. Most methods, including nSolver-based pipelines, achieved robust discrimination for both AMR and TCMR. Complex methods, including RCRNorm, and RUVSeq for TCMR, reduced performance, with simpler approaches consistently outperforming them for B-HOT–based molecular diagnostics.

Background and Aims: Tissular gene expression profiling applicable to formalin-fixed, paraffin-embedded (FFPE) endomyocardial biopsies (EMBs) may refine the diagnosis of cardiac rejection while being easily implemented in clinical practice. This study aimed to develop and validate the first FFPE-based molecular diagnostic system dedicated to heart transplant rejection. Methods: An international study was conducted (NCT06436027), establishing a deeply phenotyped cohort of heart transplant recipients. EMBs were graded according to international classifications, and gene expression was analysed on FFPE-EMBs using the Banff Human Organ Transplant Panel. Molecular classifiers for antibody-mediated rejection (AMR) and acute cellular rejection (ACR) were developed, with discrimination and calibration assessed in internal and external validation cohorts. Results: A total of 671 biopsies were included, with 591 in the main cohort (AMR: n = 188, ACR: n = 289, matched non-rejection: n = 114); this was split into a derivation set (n = 475) and an internal validation set (n = 116). The external validation cohort comprised 80 biopsies (AMR: n = 20, ACR: n = 32, non-rejection: n = 28). AMR was associated with significant transcripts related to the interferon-gamma pathway, endothelial activation, and monocyte-macrophage recruitment, while ACR was characterized by transcripts related to T-cell receptor signalling, CD3 receptor activation, and CD28 signalling. Molecular ACR and AMR models accurately identified rejection in the validation cohorts (internal: ROC-AUC: AMR = 0.812, ACR = 0.849; external: ROC-AUC: AMR = 0.822, ACR = 0.815) and were strongly associated with pathologic severity. Calibration was adequate. An automated report was developed to enhance the clinical applicability of these classifiers. Conclusions: A novel FFPE-based molecular diagnostic system accurately identified cardiac allograft rejection. This tool is readily applicable in clinical practice as a companion to pathology and has the potential to refine the diagnosis of rejection.

The development of new high-throughput technologies in genomics and then in transcriptomics has modified clinical approach in nephrology. At the interface between high-throughput technologies (microarray, new generation sequencing «NGS») and few mRNA analysis (reverse transcriptase quantitative PCR [RT-qPCR]), the nCounter® of NanoString® offers a new and complementary approach. Capable of analyzing formalin-fixed paraffin-embedded samples, this technology is a credible candidate for implanting transcriptomics in clinical routine.

The mechanisms driving the development of extracapillary lesions in focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (CGN) remain poorly understood. A key question is how parietal epithelial cells (PECs) invade glomerular capillaries, thereby promoting injury and kidney failure. Here we show that expression of the tetraspanin CD9 increases markedly in PECs in mouse models of CGN and FSGS, and in kidneys from individuals diagnosed with these diseases. Cd9 gene targeting in PECs prevents glomerular damage in CGN and FSGS mouse models. Mechanistically, CD9 deficiency prevents the oriented migration of PECs into the glomerular tuft and their acquisition of CD44 and β1 integrin expression. These findings highlight a critical role for de novo expression of CD9 as a common pathogenic switch driving the PEC phenotype in CGN and FSGS, while offering a potential therapeutic avenue to treat these conditions.

Objective.Indirect evidence suggests a role for heme oxygenase-1 (HO-1) in limiting diabetic vasculopathy. The goal of this study was to assess the role of HO-1 in the development of microvascular lesions within glomeruli during diabetes mellitus using a mouse model with specific alteration of theHmox1gene.Approach and Results.The effects ofHmox1haploinsufficiency were studied as a means of assessing the intrinsic contribution of HO-1 in the development of renal microvascular lesions during diabetes. Renal function and histology were analyzed 10 weeks after diabetes induction with streptozotocin. DiabeticHmox1^+/−mice showed higher levels of albuminuria and blood urea compared to their wild-type diabetic littermates. More severe glomerular microvascular lesions were also observed in the diabeticHmox1^+/−mice. This was associated with a renal increase in the expression of the oxidative stress marker, nitrotyrosine.Conclusions.GeneticHmox1partial deficiency is sufficient to sensitize mice to the development of diabetic glomerular microvascular lesions. HO-1 exerts antioxidant effects in the kidney during diabetes mellitus. These have protective effects on the development of glomerular endothelial injury.

FSGS, the most common primary glomerular disorder causing ESRD, is a complex disease that is only partially understood. Progressive sclerosis is a hallmark of FSGS, and genetic tracing studies have shown that parietal epithelial cells participate in the formation of sclerotic lesions. The loss of podocytes triggers a focal activation of parietal epithelial cells, which subsequently form cellular adhesions with the capillary tuft. However, in the absence of intrinsic podocyte alterations, the origin of the pathogenic signal that triggers parietal epithelial cell recruitment remains elusive. In this study, investigation of the role of the endothelial PAS domain-containing protein 1 (EPAS1), a regulatory α subunit of the hypoxia-inducible factor complex, during angiotensin II–induced hypertensive nephropathy provided novel insights into FSGS pathogenesis in the absence of a primary podocyte abnormality. We infused angiotensin II into endothelial-selective Epas1 knockout mice and their littermate controls. Although the groups presented with identical high BP, endothelial-specific Epas1 gene deletion accentuated albuminuria with severe podocyte lesions and recruitment of pathogenic parietal glomerular epithelial cells. These lesions and dysfunction of the glomerular filtration barrier were associated with FSGS in endothelial Epas1 -deficient mice only. These results indicate that endothelial EPAS1 has a global protective role during glomerular hypertensive injuries without influencing the hypertensive effect of angiotensin II. Furthermore, these findings provide proof of principle that endothelial-derived signaling can trigger FSGS and illustrate the potential importance of the EPAS1 endothelial transcription factor in secondary FSGS.

Follicle-stimulating hormone receptor (FSHR) is present on endothelial cells of blood vessels and endometrial glands of the proliferative and secretory endometrium. So far, the expression of FSHR in endometriosis has not been studied. We evaluated FSHR expression in 194 tissue specimens representing 3 relevant types of endometriosis: rectovaginal endometriotic nodules, ovarian endometriotic cysts, and peritoneal endometriotic implants. Specimens of normal endometrium were used as controls. Archival formalin-fixed and paraffin-embedded material was analyzed immunohistochemically with a highly specific monoclonal antihuman FSHR antibody using the peroxidase method. A robust vascular FSHR expression was found in all 194 patients, irrespective of the endometriosis lesion location. Follicle-stimulating hormone receptor was not detected in normal host tissues located more than 5 mm from the lesions. The endometriotic lymphatic vessels do not express FSHR. The density of FSHR-positive vessels in patients with rectovaginal endometriotic nodules was 46.0 ± 5.7 vessels/mm². Similar values were obtained for ovarian endometriotic cysts and peritoneal endometriosis. The density of FSHR-positive vessels associated with the core of rectovaginal endometriotic nodules was 2-fold higher than that of the perilesional, adjacent normal host tissue (64.2 ± 8.2 vs 27.2 ± 3.2 vessels/mm², respectively). Expression of FSHR was also detected either in endometriotic glandular epithelial cells, endometriotic stromal cells, or in both cell types (23%, 25%, and 21% of patients, respectively). Normal endometrium expressed FSHR predominately in basalis, in a cellular distribution dependent on hormonal environment. In conclusion, our data suggest novel FSHR expression in endometriotic lesions, qualitatively and quantitatively different from that of normal endometrium.