The integration of single cell transcriptome and chromatin accessibility datasets enables a deeper understanding of cell heterogeneity. We performed single nucleus ATAC (snATAC-seq) and RNA (snRNA-seq) sequencing to generate paired, cell-type-specific chromatin accessibility and transcriptional profiles of the adult human kidney. We demonstrate that snATAC-seq is comparable to snRNA-seq in the assignment of cell identity and can further refine our understanding of functional heterogeneity in the nephron. The majority of differentially accessible chromatin regions are localized to promoters and a significant proportion are closely associated with differentially expressed genes. Cell-type-specific enrichment of transcription factor binding motifs implicates the activation of NF-κB that promotes VCAM1 expression and drives transition between a subpopulation of proximal tubule epithelial cells. Our multi-omics approach improves the ability to detect unique cell states within the kidney and redefines cellular heterogeneity in the proximal tubule and thick ascending limb.

Background Single cell sequencing technologies have advanced our understanding of kidney biology and disease but the loss of spatial information in these datasets hinders our interpretation of intercellular communication networks and regional gene expression patterns. New spatial transcriptomic sequencing platforms make it possible to measure the topography of gene expression at genome depth. Methods We optimized and validated a female bilateral ischemia reperfusion injury model. Using the 10X Genomics Visium Spatial Gene Expression solution, we generated spatial maps of gene expression across the injury and repair time course, and applied two open-source computational tools, Giotto and SPOTlight, to increase resolution and measure cell-cell interaction dynamics. Results An ischemia time of 34 minutes in a female murine model resulted in comparable injury to 22 minutes for males. We report a total of 16,856 unique genes mapped across injury and repair time course. Giotto, a computational toolbox for spatial data analysis, enabled increased resolution mapping of genes and cell types. Using a seeded non-negative matrix regression (SPOTlight) to deconvolute the dynamic landscape of cell-cell interactions, we find that injured proximal tubule cells are characterized by increasing macrophage and lymphocyte interactions even at 6 weeks after injury, potentially reflecting the AKI to CKD transition. Conclusions In this transcriptomic atlas, we defined region-specific and injury-induced loss of differentiation markers and their re-expression during repair, as well as region-specific injury and repair transcriptional responses. Lastly, we created a data visualization web application for the scientific community to explore these results (http://humphreyslab.com/SingleCell/; login: humphreyslab_visium password: irivisium).

Hematopoietic stem cells (HSCs) are maintained in a hypoxic niche to limit oxidative stress. Although iron elicits oxidative stress, the importance of iron homeostasis in HSCs has been unknown. Here we show that iron regulation by the F-box protein FBXL5 is required for HSC self-renewal. Conditional deletion of Fbxl5 in mouse HSCs results in cellular iron overload and a reduced cell number. Bone marrow transplantation reveals that FBXL5-deficient HSCs are unable to reconstitute the hematopoietic system of irradiated recipients as a result of stem cell exhaustion. Transcriptomic analysis shows abnormal activation of oxidative stress responses and the cell cycle in FBXL5-deficient mouse HSCs as well as downregulation of FBXL5 expression in HSCs of patients with myelodysplastic syndrome. Suppression of iron regulatory protein 2 (IRP2) accumulation in FBXL5-deficient mouse HSCs restores stem cell function, implicating IRP2 as a potential therapeutic target for human hematopoietic diseases associated with FBXL5 downregulation.

Hepatic iron overload is a risk factor for progression of hepatocellular carcinoma (HCC), although the molecular mechanisms underlying this association have remained unclear. We now show that the iron-sensing ubiquitin ligase FBXL5 is a previously unrecognized oncosuppressor in liver carcinogenesis in mice. Hepatocellular iron overload elicited by FBXL5 ablation gave rise to oxidative stress, tissue damage, inflammation, and compensatory proliferation of hepatocytes and to consequent promotion of liver carcinogenesis induced by exposure to a chemical carcinogen. The tumor-promoting outcome of FBXL5 deficiency in the liver was also found to be effective in a model of virus-induced HCC. FBXL5-deficient mice thus constitute the first genetically engineered mouse model of liver carcinogenesis promoted by iron overload. In addition, dysregulation of FBXL5-mediated cellular iron homeostasis was found to be associated with poor prognosis in human HCC, suggesting that FBXL5 plays a key role in defense against hepatocarcinogenesis.

Despite the growing number of major surgical procedures being performed for patients on maintenance hemodialysis, few reports focus on the management and outcome of such patients, especially those undergoing major abdominal surgery. We conducted a retrospective review of 30 patients on maintenance hemodialysis who underwent abdominal surgery, 20 of whom underwent an elective operation and 10, an emergency operation. The indications of elective surgery included gastrointestinal cancer, biliary tract disease, and abdominal aortic aneurysm, while those for emergency surgery mainly involved gastrointestinal perforation or bleeding. There were no statistically significant differences between the elective group and the emergency group regarding either the mean time on hemodialysis or the preoperative clinical data. The morbidity and mortality rates were 15% and 10%, respectively, for the patients who underwent elective surgery and 50% and 70%, respectively, for those who underwent emergency surgery (P < 0.01 and P < 0.05, respectively). Those patients with more than a 2-year history of hemodialysis had a significantly higher mortality rate following abdominal surgery than those with less than a 2-year history (P < 0.01). Thus, the morbidity and mortality rates of patients on maintenance hemodialysis who require major abdominal surgery are significantly high, which reinforces the need to further improve the intensive perioperative management of such patients.

Glioma-associated oncogene homolog-1 (Gli1)-positive resident mesenchymal stem cell-like cells are the predominant source of kidney myofibroblasts in fibrosis, but investigating Gli1-positive myofibroblast progenitor activation is hampered by the difficulty of isolating and propagating primary cultures of these cells. Using a genetic strategy with positive and negative selection, we isolated Kidney-Gli1 (KGli1) cells that maintain expression of appropriate mesenchymal stem cell-like cell markers, respond to hedgehog pathway activation, and display robust myofibroblast differentiation upon treatment with transforming growth factor-β (TGF-β). Coculture of KGli1 cells with endothelium stabilizes capillary formation. Single-cell RNA sequencing (scRNA-seq) analysis during differentiation identified autocrine ligand-receptor pair upregulation and a strong focal adhesion pathway signal. This led us to test the serum response factor inhibitor CCG-203971 that potently inhibited TGF-β-induced pericyte-to-myofibroblast transition. scRNA-seq also identified the unexpected upregulation of nerve growth factor (NGF), which we confirmed in two mouse kidney fibrosis models. The Ngf receptor Ntrk1 is expressed in tubular epithelium in vivo, suggesting a novel interstitial-to-tubule paracrine signaling axis. Thus, KGli1 cells accurately model myofibroblast activation in vitro, and the development of this cell line provides a new tool to study resident mesenchymal stem cell-like progenitors in health and disease.