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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.

Efavirenz induces and inhibits multiple drug‐metabolizing enzymes, contributing to significant drug–drug interactions. This study quantified the impact of multiple doses of efavirenz on CYP3A activity via midazolam metabolism using a population pharmacokinetic approach. Healthy volunteers received 1 mg midazolam orally in two sessions: first with a single 600 mg efavirenz dose and then after chronic efavirenz dosing (600 mg/day for 17 days). Midazolam and 1′‐hydroxymidazolam were quantified via LC–MS/MS, and CYP2B6, CYP3A4, and CYP3A5 genotypes were assessed using TaqMan assays. Seventy‐two volunteers (n = 72) completed sampling after the initial dose, and 58 completed both occasions. Non‐linear mixed effects modeling was performed using the stochastic approximation expectation maximization estimation method in NONMEM. The base pharmacokinetic model employed was a two‐compartment model with first‐order absorption and first‐order elimination, incorporating proportional error terms for midazolam and 1′‐hydroxymidazolam. Covariate analysis utilized a full model approach to assess the impact of CYP3A4 and CYP3A5 genotypes, self‐reported sex, and multiple doses of efavirenz as covariates affecting the formation clearance of 1‐OH midazolam. CYP3A5 expressors exhibited a 1.27‐fold increase in midazolam clearance compared to non‐expressors, while CYP3A4 intermediate metabolizers showed a 0.94‐fold decrease relative to normal metabolizers. Clearance was also 1.30‐fold higher in females compared to males. Multiple doses of efavirenz increased midazolam clearance by 1.92‐fold (95% CI 1.65–2.28) after accounting for inter‐individual variability caused by other covariates. Furthermore, Ka and bioavailability (F) increased with repeated efavirenz exposure. In conclusion, this population pharmacokinetic analysis effectively quantified the specific induction effect of multiple doses of efavirenz on CYP3A compared to a single dose of efavirenz. Trial Registration: ClinicalTrials.gov identifier: NCT00668395

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.

A precision health initiative was implemented across a multi-hospital health system, wherein a panel of genetic variants was tested and utilized in the clinical care of chronic kidney disease (CKD) patients. Pharmacogenomic predictors of antihypertensive response and genomic predictors of CKD were provided to clinicians caring for nephrology patients. To assess clinician knowledge, attitudes, and willingness to act on genetic testing results, a Likert-scale survey was sent to and self-administered by these nephrology providers (N = 76). Most respondents agreed that utilizing pharmacogenomic-guided antihypertensive prescribing is valuable (4.0 ± 0.7 on a scale of 1 to 5, where 5 indicates strong agreement). However, the respondents also expressed reluctance to use genetic testing for CKD risk stratification due to a perceived lack of supporting evidence (3.2 ± 0.9). Exploratory sub-group analyses associated this reluctance with negative responses to both knowledge and attitude discipline questions, thus suggesting reduced exposure to and comfort with genetic information. Given the evolving nature of genomic implementation in clinical care, further education is warranted to help overcome these perception barriers.

Precision medicine involves using information on the effect of DNA sequence variations, together with environmental and lifestyle exposures, to improve disease prevention and treatment. Advanced practice nurses can enhance patient outcomes by adopting a precision medicine approach to patient care, which includes effective implementation of pharmacogenetic test results. However, lack of familiarity with genetic test results is often a barrier to its implementation. To aid advanced practice nurses in the interpretation and application of the test results in patient care, this discussion illustrates the benefit of pharmacogenetic testing through a case-based learning mechanism. •Advanced practice nurses are crucial for the effective implementation of precision medicine.•Drug-gene and drug-drug interactions present patient-specific challenges.•Two sets of guidelines assist with interpretation and patient recommendations.•With sufficient knowledge and experience, advanced practice nurses can apply pharmacogenetic results.

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. However, comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measured dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We established a comprehensive and spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we noted distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3 , KLF6 , and KLF10 regulated the transition between health and injury, while in thick ascending limb cells this transition was regulated by NR2F1 . Further, combined perturbation of ELF3 , KLF6 , and KLF10 distinguished two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks.

Abstract Despite important advances in studying experimental and clinical acute kidney injury (AKI), the pathogenesis of this disease remains incompletely understood. Single cell sequencing studies have closed this knowledge gap by characterizing the transcriptomic signature of different cell types within the kidney. However, the spatial distribution of injury can be regional and affect cells heterogeneously. We first optimized coordination of spatial transcriptomics and single nuclear sequencing datasets, mapping 30 dominant cell types to a human nephrectomy sample. The predicted cell type spots corresponded with the underlying hematoxylin and eosin histopathology. To study the implications of acute kidney injury on the distribution of transcript expression, we then characterized the spatial transcriptomic signature of two murine AKI models: ischemia reperfusion injury (IRI) and cecal ligation puncture (CLP). Localized regions of reduced overall expression were found associated with tissue injury pathways. Using single cell sequencing, we deconvoluted the signature of each spatial transcriptomic spot, identifying patterns of colocalization between immune and epithelial cells. As expected, neutrophils infiltrated the renal medullary outer stripe in the ischemia model. Atf3 was identified as a chemotactic factor in S3 proximal tubule cells. 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 can aid in uncovering the mechanisms driving immune cell infiltration and allow detection of relevant subpopulations in single cell sequencing. The complementarity of these technologies facilitates the development of a transcriptomic kidney atlas in health and disease. Competing Interest Statement The authors have declared no competing interest.

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.