Introduction The maintenance of a healthy epithelial-endothelial juxtaposition requires crosstalk within glomerular cellular niches. Here, we sought to understand the spatially anchored regulation and transition of endothelial and mesangial cells from health to injury in DKD. Methods From 132 human kidney samples, an integrated multiomics approach was leveraged to identify cellular niches, cell-cell communication, cell injury trajectories, and regulatory transcription factor networks in glomerular capillary endothelial (EC-GC) and mesangial cells. Data were culled from single nucleus RNA, ATAC-sequencing and four orthogonal spatial transcriptomic technologies for correlation with histopathological and clinical trial data. Results We identified a cellular niche in diabetic glomeruli enriched in a proliferative endothelial cell subtype (prEC) and altered vascular smooth muscle cells (VSMCs). Cellular communication within this niche maintained pro-angiogenic signaling with loss of anti-angiogenic factors. We identified a transcription factor network of MEF2C, MEF2A, and TRPS1 which regulated SEMA6A and PLXNA2, a receptor-ligand pair opposing angiogenesis. In silico knockout of the transcription factor network accelerated the transition from healthy EC-GCs toward a degenerative injured endothelial phenotype, with concomitant disruption of EC-GC and prEC expression patterns. Glomeruli enriched in the prEC niche had histologic evidence of neovascularization. MEF2C activity was increased in diabetic glomeruli with nodular mesangial sclerosis. The gene regulatory network (GRN) of MEF2C was dysregulated in EC-GCs of patients with DKD, but sodium glucose transporter-2 inhibitor (SGLT2i) treatment reversed the MEF2C GRN effects of DKD. Conclusions The MEF2C, MEF2A, and TRPS1 transcription factor network carefully balances the fate of the EC-GC in DKD. When the transcription factor network is “on” or over-expressed in DKD, EC-GCs may progress to a prEC state, while transcription factor suppression leads to cell death. SGLT2i therapy may restore the balance of MEF2C activity.

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 PointsUsed 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.BackgroundKidney 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.MethodsWe 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.ResultsWe 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).ConclusionsWe 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.

Chronic kidney disease (CKD) is a substantial global health problem with devastating impacts on patients’ morbidity and mortality. Kidney fibrosis, specifically tubulointerstitial fibrosis, is considered the final common pathway in the progression of virtually all forms of CKD. Peritubular capillary rarefaction, which refers to a decrease in peritubular capillary density leading to hypoxic and ischemic conditions, has long been recognized as a hallmark pathologic feature of tubulointerstitial fibrosis and a pivotal biological alteration leading to CKD progression. Conversely, recent literature has challenged this paradigm by proposing that tubulointerstitial fibrosis and CKD progression are closely associated with the upregulation of pro-angiogenic pathways. As such, peritubular capillary rarefaction may be a consequence rather than a cause of tubulointerstitial fibrosis. Furthermore, a growing body of evidence suggests that the microenvironment of the kidney vasculature, which may be referred to as the ‘vascular niche’, is a dynamic entity that regulates vascular homeostasis, key molecular signaling pathways and inflammation. In this review, we detail how the vascular niche may modify the course of various kidney diseases by influencing cell differentiation and the immune response. Understanding the complex interplay between the cellular and molecular components of the vascular niche may eventually lead to the identification of novel therapeutic targets to limit tubulointerstitial fibrosis and halt CKD progression. This could potentially involve modulating the secretion of angiocrine factors, regulating immune cell activity within the vascular niche, or interfering with the transformation of endothelial cells and pericytes into myofibroblasts, which are key players in kidney fibrogenesis.

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.

BACKGROUNDCannabidiol (CBD) is increasingly used for pain management, including in transplant recipients with limited analgesic options. Its immunomodulatory effects in humans are not well defined at a single-cell level at CBD steady state with concomitant tacrolimus treatment.METHODSIn a phase I ex vivo study, peripheral blood mononuclear cells from 23 participants who received oral CBD (Epidiolex) up to 5 mg/kg twice daily for 11 days were collected before CBD (pre-CBD) and at steady state (post-CBD). Lymphocytes were isolated and stimulated with anti-CD3/CD28 antibodies, with or without tacrolimus (5 ng/mL). Pharmacodynamic responses were assessed using CellTiter-Glo proliferation, single-cell and single-nucleus RNA sequencing, cytokine assays, and flow cytometry. Steady-state plasma concentrations of CBD were quantified via tandem mass spectrometry.RESULTSWe identified an increased proportion of T effector memory (TEM) cells post-CBD (22% increase), which correlated with CBD plasma concentrations (R = 0.77, P = 0.01). CBD reduced proliferation of T (37% decrease) and CD70hi B (17% decrease) lymphocytes with additive immunosuppressive effects to tacrolimus. Single-cell RNA sequencing revealed reduced IL2 and TNF signaling and altered receptor-ligand networks in TEM cells. Post-CBD cytokine assays revealed elevated proinflammatory IL-6 protein levels and antiinflammatory IL-10 levels, with reduced TNF-α, LTA, and IL-2. In flow cytometry, the proportion of TEM and TEMRA cells increased post-CBD with tacrolimus.CONCLUSIONCBD exerts mixed immunomodulatory effects in humans, combining antiproliferative and pro- and antiinflammatory responses. Understanding the clinical safety of CBD use is important given the paucity of pain control options available for immunocompromised transplant populations.TRIAL REGISTRATIONClinicalTrials.gov NCT05490511FUNDINGNIH/National Center for Complementary and Integrative Health (R01AT011463); NIH/National Institute of General Medical Sciences (NIGMS) (R35GM145383); Intramural Research Program of the NIH; NIH/NIGMS (T32GM008425).

Sepsis-associated AKI is a complex pathologic state driven by dynamic interactions between the host and microbes. The rapid progression and the absence of a molecular clock that stages the disease timeline make precise therapeutic interventions highly challenging. In this review, we aim to refine the timeline of sepsis-associated AKI by dissecting key molecular events that drive disease progression and may inform therapeutic strategies. AKI, initiated by microbes or infection mimicry, involves the rapid and simultaneous activation of inflammatory and anti-inflammatory pathways. This energy-intensive response is further fueled by the loss of distinction between self and nonself, leading to excessive antiviral responses mediated by self-derived nucleic acids. The resulting metabolic burden overwhelms cellular functions, triggering the integrated stress response and profound translation shutdown. Although this shutdown response may be necessary for energy preservation and for priming endogenous recovery mechanisms, prolonged inhibition of translation represents a maladaptive feature of septic AKI. Despite these challenges, the kidney exhibits remarkable resilience. Recovery relies on metabolic flexibility and stress-adaptive mechanisms, such as enhanced polyamine biosynthesis and RNA editing. Meanwhile, microbes also demonstrate metabolic adaptability, enabling them to evade host defenses and exploit the host environment. Understanding this dynamic interplay along the timeline of septic AKI is essential for developing rational therapeutic strategies.

Fibroblast growth factor 23 (FGF23) via its coreceptor αKlotho (KL) provides critical control of phosphate metabolism, which is altered in both rare and very common syndromes. However, the spatial-temporal mechanisms dictating kidney FGF23 functions remain poorly understood. Thus, developing approaches to modify specific FGF23-dictated pathways has proven problematic. Herein, wild type mice were injected with rFGF23 for one, four and 12h and kidney FGF23 bioactivity was determined at single cell resolution. Computational analysis identified distinct epithelial, endothelial, stromal, and immune cell clusters, with differential expressional analysis uniquely tracking FGF23 bioactivity at each time point. FGF23 actions were sex independent but critically relied upon constitutive KL expression mapped within proximal tubule (segments S1-S3) and distal convoluted tub/connecting tubule cell sub-populations. Temporal KL-dependent FGF23 responses drove unique and transient cellular identities, including genes in key MAPK-signaling and vitamin D-metabolic pathways via early- (transcription factor AP-1-related) and late-phase (initiation factor EIF2 signaling) transcriptional regulons. Combining ATACseq/RNAseq data from a cell line stably expressing KL with the in vivo scRNAseq pinpointed genomic accessibility changes in MAPK-dependent genes, including the identification of FGF23-dependent early growth factor-1 distal enhancers. Finally, we identified unexpected crosstalk between FGF23-mediated MAPK signaling and pro inflammatory TNF receptor activation via transcription factor NF-κB, which blocked FGF23 bioactivity in vitro and in vivo. Collectively, our findings have uncovered novel pathways at the single cell level that likely influence FGF23-dependent disease mechanisms.

In the kidney, cells of thick ascending limb of the loop of Henle (TAL) are resistant to ischemic injury, despite high energy demands. This adaptive metabolic response is not fully understood even though the integrity of TAL cells is essential for recovery from acute kidney injury (AKI). TAL cells uniquely express uromodulin, the most abundant protein secreted in healthy urine. Here, we demonstrate that alternative splicing generates a conserved intracellular isoform of uromodulin, which contributes to metabolic adaptation of TAL cells. This splice variant was induced by oxidative stress and was upregulated by AKI that is associated with recovery, but not by severe AKI and chronic kidney disease (CKD). This intracellular variant was targeted to the mitochondria, increased NAD+ and ATP levels, and protected TAL cells from hypoxic injury. Augmentation of this variant using antisense oligonucleotides after severe AKI improved the course of injury. These findings underscore an important role of condition-specific alternative splicing in adaptive energy metabolism to hypoxic stress. Enhancing this protective splice variant in TAL cells could become a therapeutic intervention for AKI.

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.

This protocol describes the method for preparation and staining of FFPE kidney tissue on slides for Akoya Phenocycler-Fusion multiplex staining using barcoded antibodies. Included are the protocols for tissue preparation, sectioning, deparaffinization, antigen retrieval and tissue staining. Purpose: Prepare FFPE tissue and perform multiplex antibody staining for Akoya Phenocycler-Fusion Imaging.

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.