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Sepsis is a dynamic state that progresses at variable rates and has life-threatening consequences. Staging patients along the sepsis timeline requires a thorough knowledge of the evolution of cellular and molecular events at the tissue level. Here, we investigated the kidney, an organ central to the pathophysiology of sepsis. Single-cell RNA-sequencing in a murine endotoxemia model revealed the involvement of various cell populations to be temporally organized and highly orchestrated. Endothelial and stromal cells were the first responders. At later time points, epithelial cells upregulated immune-related pathways while concomitantly downregulating physiological functions such as solute homeostasis. Sixteen hours after endotoxin, there was global cell–cell communication failure and organ shutdown. Despite this apparent organ paralysis, upstream regulatory analysis showed significant activity in pathways involved in healing and recovery. This rigorous spatial and temporal definition of murine endotoxemia will uncover precise biomarkers and targets that can help stage and treat human sepsis.

Single-cell sequencing studies have characterized the transcriptomic signature of cell types within the kidney. However, the spatial distribution of acute kidney injury (AKI) is regional and affects cells heterogeneously. We first optimized coordination of spatial transcriptomics and single-nuclear sequencing data sets, mapping 30 dominant cell types to a human nephrectomy. The predicted cell-type spots corresponded with the underlying histopathology. To study the implications of AKI on transcript expression, we then characterized the spatial transcriptomic signature of 2 murine AKI models: ischemia/reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were associated with injury pathways. Using single-cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. Neutrophils infiltrated the renal medulla in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubules. In the CLP model, infiltrating macrophages dominated the outer cortical signature, and Mdk was identified as a corresponding chemotactic factor. The regional distribution of these immune cells was validated with multiplexed CO-Detection by indEXing (CODEX) immunofluorescence. Spatial transcriptomic sequencing complemented single-cell sequencing by uncovering mechanisms driving immune cell infiltration and detection of relevant cell subpopulations.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and adenosine deaminase isoform switching. We found that A-to-I editing of antizyme inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I-edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

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.

Apoptosis in ischemic renal injury: Roles of GTP depletion and p53. Apoptosis is increasingly recognized as a major mode of cell demise after ischemic injury to the kidney. The mediators of apoptotic cell death are many and include changes in intracellular pH, calcium, free radicals, ceramide, and adenosine triphosphate (ATP) depletion. Recently, we identified guanosine triphosphate (GTP) depletion as an independent trigger for apoptotic death after chemical anoxia in vitro. We further demonstrated that GTP salvage with guanosine inhibits tubular cell apoptosis after ischemic injury in vivo. This inhibition of apoptosis was accompanied by a significant protective effect on renal function. We also showed that p53 is the mediator of apoptosis in the setting of GTP depletion and ischemic injury. Indeed, salvage of GTP with guanosine prevented the ischemia-induced increase in p53 protein. Further, pifithrin-alpha, a potent and specific inhibitor of p53, inhibited apoptosis and protected renal function with a profile similar to that seen with guanosine. Finally, the protective effects of pifithrin-alpha involved both down-regulation of the transcriptional activation of Bax and a direct inhibition of p53 translocation to mitochondria. We propose that GTP depletion and activation of p53 are major inducers of apoptotic cell death after ischemic renal injury. In this setting, guanosine and pifithrin-alpha are potent inhibitors of apoptosis and are thus potentially useful in preventing and ameliorating functional injury to the ischemic kidney.

STAT3 is a transcriptional regulator that plays an important role in coordinating inflammation and immunity. In addition, there is a growing appreciation of the role STAT3 signaling plays in response to organ injury following diverse insults. Acute kidney injury (AKI) from ischemia-reperfusion injury is a common clinical entity with devastating consequences, and the recognition that endothelial alterations contribute to kidney dysfunction in this setting is of growing interest. Consequently, we used a mouse with a genetic deletion of Stat3 restricted to the endothelium to examine the role of STAT3 signaling in the pathophysiology of ischemic AKI. In a mouse model of ischemic AKI, the loss of endothelial STAT3 signaling significantly exacerbated kidney dysfunction, morphologic injury, and proximal tubular oxidative stress. The increased severity of ischemic AKI was associated with more robust endothelial-leukocyte adhesion and increased tissue accumulation of F4/80+ macrophages. Moreover, important proximal tubular adaptive mechanisms to injury were diminished in association with decreased tissue mRNA levels of the epithelial cell survival cytokine IL-22. In aggregate, these findings suggest that the endothelial STAT3 signaling plays an important role in limiting kidney dysfunction in ischemic AKI and that selective pharmacologic activation of endothelial STAT3 signaling could serve as a potential therapeutic target.

In response to viral pathogens, the host upregulates antiviral genes that suppress translation of viral mRNAs. However, induction of such antiviral responses may not be exclusive to viruses, as the pathways lie at the intersection of broad inflammatory networks that can also be induced by bacterial pathogens. Using a model of Gram-negative sepsis, we show that propagation of kidney damage initiated by a bacterial origin ultimately involves antiviral responses that result in host translation shutdown. We determined that activation of the eukaryotic translation initiation factor 2-α kinase 2/eukaryotic translation initiation factor 2α (Eif2ak2/Eif2α) axis is the key mediator of translation initiation block in late-phase sepsis. Reversal of this axis mitigated kidney injury. Furthermore, temporal profiling of the kidney translatome revealed that multiple genes involved in formation of the initiation complex were translationally altered during bacterial sepsis. Collectively, our findings imply that translation shutdown is indifferent to the specific initiating pathogen and is an important determinant of tissue injury in sepsis.

Diabetic kidney disease (DKD) remains the leading cause of end-stage kidney disease despite decades of study. Alterations in the glomerulus and kidney tubules both contribute to the pathogenesis of DKD although the majority of investigative efforts have focused on the glomerulus. We sought to examine the differential expression signature of human DKD in the glomerulus and proximal tubule and corroborate our findings in the db/db mouse model of diabetes. A transcriptogram network analysis of RNAseq data from laser microdissected (LMD) human glomerulus and proximal tubule of DKD and reference nephrectomy samples revealed enriched pathways including rhodopsin-like receptors, olfactory signaling, and ribosome (protein translation) in the proximal tubule of human DKD biopsy samples. The translation pathway was also enriched in the glomerulus. Increased translation in diabetic kidneys was validated using polyribosomal profiling in the db/db mouse model of diabetes. Using single nuclear RNA sequencing (snRNAseq) of kidneys from db/db mice, we prioritized additional pathways identified in human DKD. The top overlapping pathway identified in the murine snRNAseq proximal tubule clusters and the human LMD proximal tubule compartment was carboxylic acid catabolism. Using ultra-performance liquid chromatography–mass spectrometry, the fatty acid catabolism pathway was also found to be dysregulated in the db/db mouse model. The Acetyl-CoA metabolite was down-regulated in db/db mice, aligning with the human differential expression of the genes ACOX1 and ACACB. In summary, our findings demonstrate that proximal tubular alterations in protein translation and carboxylic acid catabolism are key features in both human and murine DKD.

Mycophenolic acid (MPA) is an immunosuppressant commonly used to prevent renal transplant rejection and treat glomerulonephritis. MPA inhibits IMPDH2 within stimulated lymphocytes, reducing guanosine synthesis. Despite the widespread use of MPA, interindividual variability in response remains with rates of allograft rejection up to 15% and approximately half of individuals fail to achieve complete remission to lupus nephritis. We sought to identify contributors to interindividual variability in MPA response, hypothesizing that the HPRT1 salvage guanosine synthesis contributes to variability. MPA sensitivity was measured in 40 healthy individuals using an ex vivo lymphocyte viability assay. Measurement of candidate gene expression (n ± 40) and single‐cell RNA‐sequencing (n ± 6) in lymphocytes was performed at baseline, poststimulation, and post‐MPA treatment. After stimulation, HPRT1 expression was 2.1‐fold higher in resistant individuals compared with sensitive individuals (P ± 0.049). Knockdown of HPRT1 increased MPA sensitivity (12%; P ± 0.003), consistent with higher expression levels in resistant individuals. Sensitive individuals had higher IMPDH2 expression and 132% greater stimulation. In lymphocyte subpopulations, differentially expressed genes between sensitive and resistant individuals included KLF2 and LTB. Knockdown of KLF2 and LTB aligned with the predicted direction of effect on proliferation. In sensitive individuals, more frequent receptor‐ligand interactions were observed after stimulation (P ± 0.0004), but fewer interactions remained after MPA treatment (P ± 0.0014). These data identify a polygenic transcriptomic signature in lymphocyte subpopulations predictive of MPA response. The degree of lymphocyte stimulation, HPRT1, KLF2, and LTB expression may serve as markers of MPA efficacy.

Cannabidiol (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.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.In a Phase 1 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/nucleus RNA sequencing, cytokine assays, and flow cytometry. Steady-state plasma concentrations of CBD were quantified via tandem mass spectrometry.METHODSIn a Phase 1 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/nucleus RNA sequencing, 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), which correlated with CBD plasma concentrations (R = 0.77, P-value = 0.01). Cannabidiol 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-cannabidiol cytokine assays revealed elevated proinflammatory IL-6 protein levels and anti-inflammatory IL-10 levels, with reduced TNF-α, LTA, and IL-2. In flow cytometry, the proportion of TEM and TEMRA increased post-cannabidiol with tacrolimus.RESULTSWe identified an increased proportion of T effector memory (TEM) cells post-cannabidiol (22% increase), which correlated with CBD plasma concentrations (R = 0.77, P-value = 0.01). Cannabidiol 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-cannabidiol cytokine assays revealed elevated proinflammatory IL-6 protein levels and anti-inflammatory IL-10 levels, with reduced TNF-α, LTA, and IL-2. In flow cytometry, the proportion of TEM and TEMRA increased post-cannabidiol with tacrolimus.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.CONCLUSIONCannabidiol 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.