Pierre C. Dagher

Acute kidney injury (AKI) is managed as a single clinical entity despite substantial biological heterogeneity. We established a cross-species framework to define etiology-specific molecular programs and determine how these programs relate to molecular heterogeneity and pathobiology in human AKI. We performed parallel sequencing of totalRNA (mRNA) and miRNA (kidney, whole blood) in 3 murine models (mild AKI: ischemia-reperfusion injury, IRI; cecal-ligation and puncture, CLP; endotoxemia, LPS); kidney mRNA and miRNA clearly distinguished the models, blood mRNA maintained model-specific separation, and blood miRNA was less distinct. IRI was characterized by epithelial injury/repair, and extracellular matrix remodeling (i.e., miR-125b-1-3p, Vim, Sprr2g ); CLP, broader inflammatory, metabolic programs (i.e., miR-692, Pfkfb3, Cdo1 ); LPS, inflammatory signaling (i.e., miR-155-5p, Irf7, Gbp3 ). We then compared these signatures to human whole blood RNAseq. Human blood samples varied along a shared gene axis that correlated to differing degrees with all three murine models, consistent with the heterogeneous nature of clinical AKI rather than discrete assignment to a single experimental etiology. Orthologs were linked to immune processes (i.e., SIGLEC14, DAAM2, CD177, DEFA3 ), and the presence of tubular casts in human biopsies, reflecting that mild murine AKI models capture etiology-specific biology relevant to severe human AKI. Integration of injury etiology, severity, and time may provide a framework for developing circulating biomarkers that may resolve the biological heterogeneity of human AKI.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia causes substantial morbidity, but tissue reservoirs that permit bacterial persistence remain poorly defined. Using intravenous USA300 MRSA infection in mice, multiplexed imaging, dual-species transcriptomics, host and bacterial genetics, and cell-based assays, we uncovered the renal inner medulla, the site of urine concentration, as an MRSA reservoir. In this hyperosmotic niche, MRSA evaded immune detection and co-opted tissue polyamines to sustain growth, enabling subsequent spread toward the renal cortex. Neutrophil recruitment to the inner medulla was profoundly delayed owing to osmotic inhibition of immune cell migration. Disruption of medullary osmolality with the loop diuretic furosemide accelerated neutrophil infiltration, limited bacterial spread, and improved renal outcomes. Mechanistically, polyamines promoted MRSA persistence through both extracellular and intracellular actions that depended on local osmolality and pH. In the medullary milieu, polyamines primarily associated with the bacterial surface and stabilized membranes against osmotic stress. Intracellular polyamines enhanced translation of guaB (inosine monophosphate dehydrogenase), the rate-limiting enzyme in bacterial de novo purine biosynthesis. The polyamine-catabolizing gene speG (spermidine acetyltransferase), uniquely present in epidemic USA300 MRSA strains, mitigated polyamine toxicity and conferred a selective advantage in the polyamine-rich kidney. These findings revealed the inner medulla as a physiologically immune-restricted MRSA reservoir and supported modulation of medullary osmolality and bacterial polyamine metabolism as candidate adjunctive strategies for limiting renal persistence and dissemination during MRSA bacteremia.

Integrating heterogeneous spatial multi-omics data to uncover underlying biological and pathological mechanisms remains a major challenge, while existing approaches generally lack mathematically justified distances for comparing complex spatial tissues. We introduce Spatial Optimal Transport (SpaOT), a spatial multi-omics integration framework based on a theoretically grounded semi-metric formulation of unbalanced optimal transport. Utilizing a total-variation-relaxed Fused Partial Gromov-Wasserstein strategy, SpaOT establishes a mathematically consistent distance geometry that enables stable biological comparison, representative tissue barycenters, and robust downstream analyses while accommodating differences in cellular composition, measurement technologies, spatial resolution, and noise. Across multiple studies with diverse spatial omics, SpaOT enables robust and accurate alignment, integration, and stratifications across samples, resolutions, and omics modalities in complex disease-related biological systems. These capabilities facilitate the identification of biologically meaningful cellular organization, molecular signatures, and spatial relationships, establishing SpaOT as a general foundation for spatial multi-omics integration and comparative analysis.

Advances in tissue clearing and lightsheet microscopy enable mesoscale imaging of intact and convoluted tubular networks, yet analytical tools to map tubule continuity and assess injury patterns within and across tubules are limited. Here, we introduce TubuleMAP, a semi-automated pipeline for 3D tubule tracking and reconstruction that adapts to various morphological and staining patterns, leverages parallel processing of terabyte-scale data for large-scale analysis of tubular networks, and uses a napari interface for human oversight. Using TubuleMAP, we reconstruct 1,000 intact mouse nephrons in ~1-millimeter-thick kidney slab with ~400-fold higher throughput and ≤1% human effort compared to prior approaches. These reconstructions enable analysis of mesoscale nephron organization, quantitative profiling of pathologic morphologies, whole-nephron cytometry, and identification of rare morphologies at unprecedented scales. We demonstrate generalizability by reconstructing all seminiferous tubules in a mouse testis within a day. TubuleMAP is released as an open-source Python package.

Key Points: Used multiplex protein imaging to elucidate the intratubular cast components and the associated tubular alterations. Identified (Prominin-1/CD133), a dedifferentiation marker, as a major constituent of intratubular casts. Protein components within casts were associated with the injury of the surrounding tubular epithelium. Background: Kidney intratubular casts are frequently observed in the distal nephron segments of the kidney and have long been regarded as a sign of kidney disease. However, the composition and pathologic significance of intratubular casts have remained understudied. Methods: We leveraged Hematoxylin and Eosin (H&E) staining to identify intratubular casts along with concurrent codetection by indexing multiplexed spatial protein imaging on human kidney biopsy sections from the Kidney Precision Medicine Project. We also conducted immunoblotting of Prominin-1 (PROM1/CD133) in urine and assessed its levels from publicly available urinary proteomics datasets of the Kidney Precision Medicine Project consortium. Results: We analyzed 493 intratubular casts across 42 individuals with kidney disease or healthy controls. We identified PROM1 and insulin-like growth factor binding protein 7 as major constituents of casts (positive staining in 89.0% and 39.1%, respectively). Staining for uromodulin, an established cast component, was present in 86.6%. These components showed variable patterns across disease states. Intratubular casts were predominantly detected in the distal nephron segments, and their presence was associated with a marked loss of sodium-chloride cotransporter and aquaporin-2 expression in the cast-containing tubular epithelium, suggesting underlying injury. The loss of these transporters correlated with protein components within casts, and the presence of intracast PROM1 showed the strongest association, with an odds ratio of 26.7 (95% confidence interval, 13.1 to 54.7). Urinary PROM1 secretion was confirmed by immunoblotting and was greater in patients with AKI compared with healthy controls ( P = 0.01). Conclusions: We identified PROM1, a dedifferentiation and injury marker expressed in epithelial cells, as a novel major constituent of intratubular casts. Our studies suggest that protein composition signature within casts varies with disease state and is associated with tubular injury in distal nephron segments. Our study also suggests that urinary PROM1 may have potential as a biomarker for AKI. Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3//www.asn-online.org/media/podcast/JASN/2026_10_22_KTS_October2026.mp3

Building upon a foundational Human Kidney resource, we present a comprehensive multi-modal atlas that defines spatially resolved versus unresolved repair states and mechanisms in human kidney disease. Homeostatic interactions between injured kidney epithelium and its surrounding milieu determine successful repair outcomes, while pathogenic signaling promotes unresolved inflammation and fibrosis leading to chronic disease. We integrated multiple single-cell and spatial modalities across ∼700 samples from >350 patients (∼250 research biopsies), analyzing ∼1.7 million cells alongside complementary mouse multi-omic profiles spanning acute-to-chronic injury and aging (>300,000 cells) and spatial transcriptomic analysis of >150 human biopsies. This cross-species atlas delineates functional pathways and druggable targets across the nephron and defines gene regulatory networks and chromatin landscapes governing tubular, fibroblast, and immune cell transitions from injury to either recovery or failed repair states. We identified distinct cellular states associated with specific pathological features that show dynamic distributions between acute kidney injury (AKI) and chronic kidney disease (CKD), organized within unique spatial niches that reveal progression mechanisms from early injury to unresolved disease. Gene regulatory analyses prioritized key transcription factor activities (SOX4, SOX9, NFKB1, REL, KLFs) and their target networks establishing disease states and tissue microenvironments. These regulatory programs were directly linked to clinical outcomes, identifying molecular signatures of recovery and secreted biomarkers predictive of AKI-to-CKD progression, providing a key resource for therapeutic development and precision medicine approaches in kidney disease.

Background Renal intratubular casts are frequently observed in the distal nephron segments of the kidney and have long been regarded as a sign of renal disease. However, the composition and pathological significance of intratubular casts have remained understudied. Methods We leveraged Hematoxylin and Eosin (H&E) staining to identify intratubular casts along with concurrent Co-detection by indexing (CODEX) multiplexed spatial protein imaging on human kidney biopsy sections from the Kidney Precision Medicine Project (KPMP). We also conducted immunoblotting of Prominin-1 (PROM1) in urine and assessed its levels from publicly available urinary proteomics datasets of the KPMP consortium. Results We analyzed 424 intratubular casts across 33 individuals with kidney disease or healthy controls. We identified PROM1 and IGFBP7 as major constituents of casts (positive staining in 90.1% and 35.6%, respectively). Staining for UMOD, an established cast component, was present in 86.1%. These components exhibited distinct alterations depending on the disease state. Intratubular casts were predominantly detected in the distal nephron segments, and their presence was associated with a marked loss of NCC and AQP2 expression in the cast-containing tubular epithelium, suggesting underlying injury. The loss of these membrane transporters correlated with protein components within casts, and the presence of intra-cast PROM1 showed the strongest association, with an odds ratio of 30.8 (95% confidence interval: 13.4-71.0). Urinary PROM1 secretion was confirmed by immunoblotting and was increased in patients with acute kidney injury (AKI) compared to healthy controls (p = 0.01). Conclusions We identified PROM1, a dedifferentiation and injury marker expressed in epithelial cells, as a novel major constituent of intratubular casts. Our studies suggest that protein composition signature within casts varies with disease state and is associated with tubular injury in distal nephron segments. Our study also suggests that urinary PROM1 may serve as a biomarker for AKI. Key Points ✓ Utilized CODEX multiplex protein imaging to elucidate the intratubular cast components and the associated tubular alterations. ✓ Identified PROM1, a dedifferentiation marker, as a major constituent of intratubular casts. ✓ Protein components within casts were altered by disease state and were associated with the injury of the surrounding tubular epithelium.

The organizational principles of nephronal segments are based on anatomical and physiological attributes that are linked to the homeostatic functions of the kidney. Recent molecular approaches have uncovered layers of deeper signatures and states in tubular cells that arise at various time points on the disease trajectory. Here, we introduce an analytical pipeline of multiplexed spatial protein imaging integrated with RNA expression to characterize proximal tubular subpopulations and neighborhoods in human kidney tissue. We demonstrate that, in reference tissue, a large proportion of S1 proximal tubular epithelial cells expresses thymus antigen 1 (THY1), a mesenchymal stromal and stem cell marker that regulates differentiation. Kidney disease is associated with loss of THY1 and transition toward expression of prominin 1 (PROM1), another stem cell marker recently linked to failed repair. Our data support a model in which the interplay between THY1 and PROM1 expression in proximal tubules associates with their regenerative potential and marks the timeline of disease progression.

Cannabidiol is widely available and often used for pain management. Individuals with kidney disease or renal allografts have limited analgesia options. We conducted a Phase 1 human study to compare the peripheral immune cell distribution before (pre-cannabidiol) and after exposure to cannabidiol at steady state (post-cannabidiol). This ex vivo study included specimens from 23 participants who received oral cannabidiol (up to 5 mg/kg twice daily) for 11 days. Lymphocytes were isolated and stimulated with anti-CD3/CD28 antibodies, with or without tacrolimus. Pharmacodynamic responses were assessed via CellTiter-Glo® proliferation, scRNA-seq, cytokine assays, and flow cytometry. Steady-state plasma concentrations of CBD were quantified via tandem mass spectrometry. We identified an increased proportion of T effector memory (TEM) cells post-cannabidiol (22% increase, P-value of 3.2 x 10 ^-32 ), which correlated with CBD plasma concentrations ( Pearson Corr= 0.77, P-value < 0.01 ). Post-cannabidiol cytokine assays revealed elevated proinflammatory IL-6 protein levels and anti-inflammatory IL-10 levels ( adjusted P-values < 0.0001 ). Cannabidiol reduced overall T and B lymphocyte proliferation with additive immunosuppressive effects to tacrolimus. In flow cytometry, the proportion of TEM and TEMRA increased post-cannabidiol with tacrolimus ( P-values < 0.05 ). Cannabidiol exhibits mixed immunomodulatory effects with pro- and anti-inflammatory signals. Understanding the clinical safety of cannabidiol use is important given the paucity of pain control options available for immunocompromised transplant populations.